United States 

Department of 

Agriculture

Forest Service

Pacific Southwest 

Research Station

General Technical 

Report

PSW-GTR-221

May 2009

Phytophthoras in Forests 

and Natural Ecosystems 

Proceedings of the Fourth Meeting of the 

International Union of Forest Research 

Organizations (IUFRO) Working Party 

S07.02.09

August 26–31, 2007, Monterey, California DEPAR TMENT OF AGRICULT URE
Technical Coordinators 

Ellen Michaels Goheen is a plant pathologist, USDA Forest Service, Forest Health 

Protection, Southwest Oregon Forest Insect and Disease Service Center, 2606 Old 

Stage Road, Central Point, OR 97502 (email address: egoheen@fs.fed.us)

Susan J. Frankel is Sudden Oak Death Research Program Manager, USDA Forest 

Service, Pacific Southwest Region Research Station, 800 Buchanan Street, West 

Annex Building, Albany, CA 94710-0011 (email address: sfrankel@fs.fed.us)

Cover and chapter heading illustrations by Charles M. Aquilina, Vallejo, CA. 

Thanks also to Stephanie Tencer, Paul Reeser, Eunsung Oh, and Elizabeth Fichtner 

for providing photographs used on the cover and chapter pages. 

Papers were provided by the authors in camera-ready form for printing. Authors are 

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Phytophthoras in Forests and 

Natural Ecosystems 

Proceedings of the Fourth Meeting of the 

International Union of Forest Research 

Organizations (IUFRO) Working Party S07.02.09

August 26–31, 2007, Monterey, California

Ellen Michaels Goheen and Susan J. Frankel,

Technical Coordinators

Financial and other support from:

USDA Forest Service, Pacific Southwest Research Station

USDA Forest Service, Pacific Southwest Region

Oregon State University

University of California, Davis

California Oak Mortality Task Force 

U.S. Department of Agriculture, Forest Service

Pacific Southwest Research Station

Albany, CA

General Technical Report PSW-GTR-221

May 2009
Abstract

Goheen, E.M.; Frankel, S.J., tech. coords. 2009. Proceedings of the fourth 

meeting of the International Union of Forest Research Organizations (IUFRO) 

Working Party S07.02.09: Phytophthoras in forests and natural ecosystems. 

Gen. Tech. Rep. PSW-GTR-221. Albany, CA: U.S. Department of Agriculture, 

Forest Service, Pacific Southwest Research Station. 334 p. 

The fourth meeting of the International Union of Forest Research Organizations 

(IUFRO) Working Party S07.02.09, Phytophthoras in Forests and Natural 

Ecosystems provided a forum for current research on Phytophthora species 

worldwide. Seventy-eight submissions describing papers and posters on recent 

developments in Phytophthora diseases of trees and natural ecosystems in 

Europe, Australasia, and the Americas are included. Research topics covered 

are Phytophthora biodiversity, ecology, epidemiology, management, and hostpathogen interactions.

Keywords: Phytophthora species, forest tree diseases.

Organizing Committee

Janice M. Alexander, University of California, Cooperative Extension, 

Marin County; and the California Oak Mortality Task Force

Elizabeth J. Fichtner, University of California, Davis

Susan J. Frankel, United States Department of Agriculture, Forest Service, 

Pacific Southwest Research Station

Ellen Michaels Goheen, United States Department of Agriculture, Forest 

Service, Southwest Oregon Forest Insect and Disease Service Center, Central 

Point, OR

Everett M. Hansen, Oregon State University

Daniel Hüberli, Murdoch University, Centre for Phytophthora Science and 

Management, Western Australia

Katharine M. Palmieri, University of California, Berkeley, and the California 

Oak Mortality Task Force

David M. Rizzo, University of California, Davis
Contents

1 Regional Updates

3 Progress in Understanding Phytophthora Diseases of Trees in Europe 

2004–2007

Thomas Jung, Andrea Vannini, and Clive M. Brasier

25 Phytophthora Research and Management in Australasia

Giles E.St.J. Hardy 

35 Phytophthora in Forests and Natural Ecosystems of the Americas

David Rizzo and Elizabeth Fichtner

45 Phytophthora Biodiversity

47 Phytophthora kernoviae in New Zealand

	 T.D. Ramsfield, M.A. Dick, R.E. Beever, I.J. Horner, M.J. McAlonan,

and C.F. Hill

54 The Search for Phytophthora Centres of Origin: Phytophthora Species in 

Mountain Ecosystems in Nepal

Andrea Vannini, Anna Brown, Clive Brasier, and Anna Maria Vettraino

56 Natural Hybrids of Resident and Introduced Phytophthora Species 

Proliferating on Multiple New Hosts in Europe

Willem Man in ’t Veld, Arthur de Cock, and Richard Summerbell

61 New Hypotheses on the Origin of the Interspecific Hybrid Oomycete 

Phytophthora alni

	 	 Renaud Ioos, Axelle Andrieux, Benoît Marçais, and Pascal Frey

Conference attendees, Asilomar Conference Center, Monterey, CA.
63 Population Structure of the Emerging Plant Pathogen Phytophthora ramorum

 on the West Coast of the United States

 S. Prospero, E.M. Hansen, N.J. Grünwald, J. Britt, and L.M. Winton

64 The Decline of Austrocedrus Forests in Patagonia (Mal del Ciprés): 

 Another Phytophthora-Caused Forest Disease

 Alina G. Greslebin and Everett M. Hansen

74 Kauri (Agathis australis) Under Threat From Phytophthora?

	 	 Ross E. Beever, Nick W. Waipara, Tod D. Ramsfield, Margaret A. Dick,

 and Ian J. Horner

86 Pythiaceous Fine Feeder Root Pathogens Associated With Eucalyptus 

 gomphocephala (Tuart) Decline in Southwest Western Australia

 Peter M. Scott, Bryan L. Shearer, Paul A. Barber, and Giles E.St.J. Hardy

87 Phytophthora Species Associated With Tanoak Stem Cankers in 

 Southwestern Oregon

 Paul Reeser, Wendy Sutton, and Everett Hansen

91 Monitoring Occurrence and Distribution of Phytophthora Species in Forest 

 Streams in North Carolina Using Baiting and Filtration Methods

 Jaesoon Hwang, Steven W. Oak, and Steven N. Jeffers

96 Phytophthora Species in Forest Trees in Hungary: A Genetic Approach

 Ferenc Lakatos and Ilona Szabó

101 Phytophthora Biodiversity: How Many Phytophthora Species Are There?

 Clive Brasier

117 Host-Pathogen Interactions

119 First Report of Ink Disease on Chestnut Caused by Phytophthora katsurae

 in Korea 

Eunsung Oh, Keum Chul Shin, Sang Hyun Lee, Kyung Hee Kim, and 

 Jong Kyu Lee

120 Development of Molecular Diagnostic Tools for the Detection of 

 Phytophthora cinnamomi From Cryptic Soil Samples in Southern Australia

 Nari M. Williams, Emer O’Gara, Giles E.St.J. Hardy, and Phillip A. O’Brien

121 Host-Pathogen Interaction of Phytophthora citricola and P. pseudosyringae

 With European Beech (Fagus sylvatica)

 Julia Koehl, Roberto Portz, Vera Martin, Angela Simonsmeier, and 

 Wolfgang Oßwald

123 Investigation of the Effects of Phytophthora alni subsp. alni Collar 

 Inoculation on CO2 Uptake, Transpiration, and Carbohydrate Content 

 of Alnus glutinosa Saplings

 Julia Koehl, Frank Fleischmann, Rainer Matyssek, and Wolfgang Oßwald
124 Phytophthora ramorum Tissue Colonization Studied With 

 Fluorescence Microscopy

 Marko Riedel, Stefan Wagner, Monika Götz, Lassaad Belbahri, 

 Francois Lefort, and Sabine Werres

131 Spread of Infection Within Tanoak (Lithocarpus densiflorus) Trees 

 Inoculated With Phytophthora ramorum

 Jennifer Parke, Bradley Collins, Gerri Buckles, Everett Hansen, and 

 Barb Lachenbruch

133 Phytophthora Ecology and Epidemiology

135 Studying Ink Disease Distribution Patterns on Heterogeneous Landscape 

 by Means of Remote Sensing Activities and GIS Technology

 Andrea Vannini, Giuseppina Natili, and Anna Maria Vettraino

136 Modelling the Potential Distribution of Phytophthora alni Root and 

 Collar Rot of Alders in Bavaria and Preliminary Application of the 

 Model to Estimate the U.S. Susceptibility to P. alni

 Thomas Jung, Marla Downing, Vernon Thomas, Markus Blaschke, 

	 	 Michael F. Tuffly, and Robin Reich

137 Susceptibility of Some Italian Ornamental and Forestry Species to 

 Phytophthora ramorum

 Anna Maria Vettraino, Barbara Ceccarelli, and Andrea Vannini

140 Widespread Phytophthora Infestations of Nurseries in Germany and 

 Austria and Their Role as Primary Pathway of Phytophthora Diseases 

 of Trees

 Thomas Jung, Jörg Schumacher, Sindy Leonhard, Günter Hartmann, 

 and Thomas Cech

142 Survival and Chlamydospore Production of Phytophthora ramorum in 

 California Bay Laurel Leaves

 Elizabeth J. Fichtner, David M. Rizzo, Shannon C. Lynch, Jennifer 

 Davidson, Gerri Buckles, and Jennifer Parke

151 Survival of Phytophthora cinnamomi in Soil After Prescribed Fire in a 

 Southern Appalachian Mountain Forest

 Inga M. McLaughlin, Steven N. Jeffers, and Thomas A. Waldrop

155 Persistence of Phytophthora lateralis After Wildfire: Preliminary 

 Monitoring Results From the 2002 Biscuit Fire

 Frank Betlejewski

158 Operational Program to Develop Resistance to Phytophthora lateralis in 

 Port-Orford-Cedar (Chamaecyparis lawsoniana): the First Ten Years

 Richard A. Sniezko, Leslie J. Elliott, Everett M. Hansen, Frank Betlejewski, 

 Donald J. Goheen, Kirk Casavan, Scott E. Kolpak, Charles L. Frank, 

 Peter A. Angwin, and Jim Hamlin
161 Management

163 Managing Phytophthora Species in the Forest: A Perspective

 Donald J. Goheen

169 Port-Orford-Cedar Family Variation in Resistance to Phytopthora lateralis: 

 Greenhouse Test of 125 Families

 Richard A. Sniezko, Everett M. Hansen, Angelia Kegley, Scott E. Kolpak, 

 and Paul Reeser

170 Phytophthora ramorum in Oregon Forests: Six Years of Detection, 

 Eradication, and Disease Spread

 Alan Kanaskie, Everett M. Hansen, Ellen Michaels Goheen, Michael 

 McWilliams, Paul Reeser, and Wendy Sutton

173 Persistence of Phytopthtora ramorum After Eradication Treatments in 

 Oregon Tanoak Forests 

 Ellen Michaels Goheen, Everett Hansen, Alan Kanaskie, Wendy Sutton, 

 and Paul Reeser

177 Management of Phytophthora kernoviae and P. ramorum in 

 Southwest England

 Joan F. Webber

184 Australasia is at High Risk of a Phytophthora ramorum Epidemic

 Daniel Hüberli, Kylie Ireland, Ian Smith, Bernard Dell, Michael Ormsby, 

 David Rizzo, Matteo Garbelotto, and Giles E.St.J. Hardy

188 Screening for Resistance to Phytophthora cinnamomi in Hybrid Seedlings 

 of American Chestnut

 Steven N. Jeffers, Joseph B. James, and Paul H. Sisco

195 Field Evaluation of Cork Oak Seedling Viability in a Soil Naturally 

 Infested With Phytophthora cinnamomi 

 A.C. Moreira, J. Albuquerque, E. Melo, J.M.S. Martins, R. Tapias, 

 and A. Cravador

204 National Best Practice in the Management of Phytophthora cinnamomi

 for Biodiversity Conservation in Australia

 Emer O’Gara, Kay Howard, Tim Rudman, and Giles E St.J. Hardy

205 Development of U.K./EU/EPPO Pest Risk Analyses for Phytophthora 

 kernoviae, P. ramorum, and P. lateralis

 Claire E. Sansford

218 Containment and Eradication of Phytophthora cinnamomi in Native 

 Vegetation in South-Western Australia and Tasmania 

 William A. Dunstan, Timothy Rudman, Bryan L. Shearer, Nicole A. Moore, 

 Bernard Dell, Colin Crane, Sarah Barrett, and Giles E.St.J. Hardy
227 Does Abiotic Stress on a Plant Influence Phosphite Protection to 

 Phytophthora cinnamomi?

 Daniel Hüberli, Trudy Paap, Nicole A. Moore, Karyn Gower, Nathalie, 

 Long, Sarah Barrett, Greg Freebury, Ted Spadek, Bernard Dell, and 

 Giles E.St.J. Hardy

230 In Vitro Activity of Plant Crude Extracts and In Situ Protective Effect of 

 Phlomis purpurea Against Phytophthora cinnamomi

 Dina Neves, Paula Caetano, Marília Horta, Nelson Sousa, Lídia Dionísio, 

 Naresh Magan, Alfredo Cravador

246 Investigating the Potential of Disease Control Practices for Integrated 

 Management of Phytophthora kernoviae on Mature Magnolias in the 

 United Kingdom

 S. Denman, S.A. Kirk, and A.Whybrow

248 Population Genetic Analyses of Four Phytophthora Species Recently 

 Described in California Forests Reveal They Are All Introduced

 M. Garbelotto, R. Linzer, W. Monahan, and S. Bergemann

249 IUFRO Fourth Meeting of Working Party 7.02.09, Phytophthoras in 

 Forest and Natural Ecosystems: Meeting Summary

 Susan J. Frankel

251 Posters

253 White Oak Decline and Mortality in Southern Ohio

 Yilmaz Balci, Robert P. Long, Selin Balci, Daniel Balser, and 

 William L. MacDonald

254 Detection and Diagnosis of Phytophthora ramorum With Specific 

 Hybridization Real Time PCR Probes

 Lassaad Belbahri, Gautier Calmin, Stephan Wagner, Eduardo 

 Moralejo, Steve Woodward, Tomasz Oszako, and François Lefort

255 Occurrence of Novel Phytophthora Taxa in Hungarian Alder Forests

 Lassaad Belbahri, József Bakonyi, Zoltán Nagy, Gautier Calmin, 

 András Koltay, Steven Woodward, and François Lefort

256 Phytophthora polonica sp. novo Isolated From Polish Alder Stands 

 in Decline

 Lassaad Belbahri, Tomasz Oszako, Gautier Calmin, Eduardo Moralejo, 

 J.A Garcia, E. Descals, and François Lefort

257 Production of Viable Oospores by Phytophthora ramorum

 Xavier Boutet and Anne Chandelier

261 Phytophthora cinnamomi Populations on Quercus Forests From Spain 

 and Portugal

 P. Caetano, A. Ávila, M.E. Sánchez, A. Trapero, and A.C. Coelho
270 Genetic Transformation of Phytophthora ramorum With the Jellyfish 

 GFP Gene

 Gautier Calmin, Marko Riedel, Lassaad Belbahri, Stefan Wagner, 

 Sabine Werres, and François Lefort

273 Phytophthora pseudosyringae Found on European Beech and 

 Hornbeam Trees in the United Kingdom

 S. Denman, J. Rose, and B. Slippers

281 Root Associations of Phytophthora ramorum and Phytophthora kernoviae

 in U.K. Woodlands

 Elizabeth J. Fichtner, David M. Rizzo, Susan A. Kirk, Alistair Whybrow, 

 and Joan Webber

282 “Mal del Ciprés” Disease: Analysis of the Association Between Aerial 

 Symptoms and Vitality of Trees

 María Pía Floria and Alina G. Greslebin

284 The Phytophthora Species Known as “Pg chlamydo”

 Everett Hansen, Paul Reeser, and Wendy Sutton

288 Virulence Behaviour and Gene Expression Patterns of a Phytophthora 

 cinnamomi-Cinnamomin Silenced Transformant

 Marília Horta, Paula Caetano, Ana C. Coelho, Dina Neves, Nelson Sousa, 

 and Alfredo Cravador

289 The Spatial and Temporal Dynamics of Phytophthora lateralis-Related 

 Mortality on Chamaecyparis lawsoniana After Ten-Plus Years on Two 

 Field Sites

 Scott E. Kolpak, Everett M. Hansen, Richard A. Sniezko, and Wendy Sutton

291 Adaptability and Susceptibility of Oaks to Phytophthora quercina Under 

 Drought and Nitrogen Stress

 Sindy Leonhard, Jörg Schumacher, and Alfred Wulf

292 Prevalence and Development of Disease on Coast Redwood Seedlings 

 Caused by Phytophthora ramorum

 Sunny Lucas, Jennifer Parke, and Yana Valachovic

296 Vegetation Dynamics and Impacts by Phytophthora ramorum in 

 Redwood-Tanoak Forests in California

 Shannon C. Lynch, Richard C. Cobb, and David M. Rizzo

303 Epidemiology of the Alder Phytophthora

 Benoit Marçais, Claude Husson, Jaime Aguayo, and Fabrice Elegbede

304 Monitoring Phytophthora ramorum Distribution in Streams Within 

 Coastal California Watersheds

 S.K. Murphy, C. Lee, Y. Valachovic, W. Mark, A. Jirka, D.R.Owen, 

 and D.M. Rizzo
305 Real Time PCR Protocols for Environmental Monitoring of Phytophthora 

 alni and Its Three Subspecies

 Tomasz Oszako, Lassaad Belbahri, Gautier Calmin, and François Lefort

306 Stand Structure and Local Distribution of Phytophthora ramorum in 

 Oregon Forests

 Ebba Peterson, Molly Botts, and Everett Hansen

310 Physiological Changes and Gene Expression on European Beech 

 (Fagus sylvatica) Infected With Phytophthora citricola

 Roberto L. Portz, Julia Koehl, Frank Fleischmann, and Wolfgang Oßwald

311 Characterizing the Community of Phytophthora Species in an 

 Oregon Forest Stream

 Philippe Remigi, Wendy Sutton, Paul Reeser, and Everett Hansen

315 Phytophthora ramorum Tissue Colonization Studied With Fluorescence 

 Microscopy

 Marko Riedel, Stefan Wagner, Monika Götz, Lassaad Belbahri, 

 François Lefort, and Sabine Werres

316 Phytophthora Species in Finland

 Anna Rytkönen, Arja Lilja, Päivi Parikka, Asko Hannukkala, Mirkka 

 Kokkola, and Jarkko Hantula

319 Screening Wild Cherry (Prunus avium) Micropropagated Clones for 

 Resistance to Four Phytophthora Species

 Alberto Santini, Luisa Ghelardini, Anna De Rogatis, Serena Guerri, 

 and Stefano Vedele

320 Involvement of Phytophthora Species in the Decline of Beech (Fagus 

 sylvatica) in the Southern Part of Belgium

 S. Schmitz, J. Zini, and A. Chandelier

324 Phosphite Application as an Explorative Tool for Understanding and 

 Controlling Eucalyptus gomphocephala (Tuart) Decline in Southwest 

 Western Australia

 Peter M. Scott, Harry T. Eslick, Paul A. Barber, Michael C. Calver, 

 Giles E.St.J. Hardy, and Bryan L. Shearer

325 An Evaluation of Stream Monitoring Techniques for Surveys for 

 Phytophthora Species in Victoria, Australia

 Benjamin Smith, Ian W. Smith, Rodney H. Jones, and James Cunnington

326 Molecular Testing Uncovers New Phytophthora Taxa From Natural 

 Ecosystems in Western Australia

 M.J.C. Stukely, J.L. Webster, J.A. Ciampini, T.I. Burgess, D. White, 

 W.A. Dunstan, and G.E.St.J. Hardy
327 Long-Term Storage of Phytophthora Cultures in Water

Wendy Sutton, Paul Reeser, and Everett Hansen

331 Occurrence and Impact of Phytophthora Species in Forest 

Trees in Hungary

	 	 Ilona Szabó and Ferenc Lakatos
Updates

1
2
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 3 

Progress in Understanding Phytophthora

Diseases of Trees in Europe 2004–20071

Thomas Jung,2 Andrea Vannini,3 and Clive M. Brasier4

Abstract 

Much new information on Phytophthora diseases of trees in Europe has been produced since 

the 2004 meeting and is reviewed here. Topics covered include updates on P. alni induced 

dieback of alders, deciduous and mediterranean oak decline, decline and dieback of European 

beech and ink disease of chstnut; the nursery-plantation pathway; the status of P. ramorum

and P. kernoviae in Europe; an upsurge in Phytophthora activity on Tilia, Acer and Betula

species; and the current status of the remarkable array of new Phytophthora taxa recovered 

from forest and natural ecosystems in Europe over the past decade. 

Introduction 

Knowledge of Phytophthora diseases of trees is growing rapidly. This is timely 

considering the increasing threat posed by invasive Phytophthoras as a consequence 

of the growing international trade in living plants. Recent developments in Europe 

include advances in the taxonomy and phylogeny of European forest Phytophthoras; 

new insight into the phylogeny of the Phytophthora alni hybrid complex; modelling 

of alder dieback in France and Bavaria; surveys of alder dieback and first records of 

P. alni in Poland, the Czech Republic, Switzerland and Vienna; ecophysiological 

studies on the Phytophthora diseases of black alder and European beech (Koehl and 

others this volume); Phytophthora surveys in mature beech stands across Europe 

indicating that Phytophthora infection is driving European beech decline; evidence 

for the spread of Phytophthora species within tree xylem; a Phytophthora survey in 

oak stands in Sweden and the development of a conceptual model for oak decline; 

insights into ink disease of chestnut in Southern Europe; upsurge of Phytophthora 

activity in maple, linden, horse chestnut and birch stands in Germany and 

Switzerland; Phytophthora surveys in forest stands in Hungary; extensive 

Phytophthora surveys in nurseries and plantations in Germany and Austria providing 

further evidence for the critical role of nurseries as the pathway of Phytophthora 

spread; the detection of multiple Phytophthora species in streams and soils in 

Scotland; and phosphite trials in mature beech, oak, maple and linden stands in 

Bavaria and Austria. Developments on P. ramorum and P. kernoviae include 

publication of the main outcome of the “RAPRA” project (see http://rapra.csl. 

gov.uk); new information on the current distribution of the pathogens in Europe; 

increasing knowledge on environmental factors affecting spread and infection of 

P. ramorum in Europe including a comparison between Californian and European 

 

1 A version of this paper was prexented at the Fourth IUFRO Phytophthoras in Forests and Natural Ecosystems, 

August 26–31, 2007, Monterey, California. 

2 Phytophthora Research and Consultancy, www.tree-diseases.com, Thomastrasse 75, D-83098 Brannenburg, 

Germany. 

3

 Dipartimento di Protezione delle Piante, Universita` degli Studi della Tuscia, Viterbo, Italy. 

4

 Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, U.K. 

Corresponding author: competence@tree-diseases.com. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 4 

climates and a detailed risk analysis for Italy; new information on the susceptibility 

of a wide range of woody species; evidence for asymptomatic infection and 

sporulation of P. ramorum and P. kernoviae on leaves and fruits; evidence for 

infection of intact bark by P. ramorum and P. kernoviae via zoospores; and further 

insights into the breeding behaviour of P. ramorum. 

Taxonomy and Phylogeny of New Phytophthora 

Taxa in European Forests

It has become customary to update the taxonomic status of the remarkable array of 

new Phytophthora taxa recovered from trees in Europe over the past decade (Brasier 

and Jung 2003, 2006). Since the IUFRO 2004 meeting, three known axa have been 

officially described as new species while five new Phytophthora taxa have been 

found in the course of different surveys of declining alder stands in Hungary and 

Poland (Belbahri and others and Lakatos and Szabo, this volume) enhancing the 

number of new Phytophthora taxa recovered from European forests since the mid 

1990s to 23 (table 1). 

Based on their ITS sequences all three newly described species fall outside the main 

Phytophthora ITS cluster described by Cooke and others (2000). P. polonica sp. nov. 

belongs to ITS clade 9 with P. insolita being its nearest relative; whereas P. 

kernoviae sp. nov. and P. gallica sp. nov. together with P. boehmeriae fall into ITS 

clade 10. Interestingly, in both clades the ancestral species are soilborne with 

persistent sporangia, i.e. P. polonica and P. insolita in clade 9 and P. gallica in clade 

10, while the phylogenetically “younger” species have an airborne lifestyle and 

caducous sporangia, i.e. P. captiosa and P. fallax in clade 9 and P. kernoviae and 

P. boehmeriae in clade 10. This supports the hypothesis (Brasier 1983; Cooke and 

others 2000) for evolutionary development from a soilborne to an aerial lifestyle 

within the genus Phytophthora. 

Some of these new species and taxa have recently been recorded from new hosts as 

well as from new countries or even continents (Table 1). For instance, the oak fine 

root pathogen P. quercina has recently been found widespread in US oak forests 

(Schwingle and others 2007a) while P. kernoviae and P. europaea were recovered 

from Cherimoya and Iris, respectively, in New Zealand (Beever, Jung unpublished). 

P. pseudosyringae was isolated from hornbeam (Carpinus) in the U.K. and Romania 

(Denman and others this volume; T. Jung and D. Chira unpublished). P. taxon 

riversoil has been found on fallen alder leaves in Spain (A. Moralajo, unpublished) 

and may shortly be described as P. ripicola sp. nov. New host and country records 

of P. ramorum will be reviewed later in this article. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 5 

Table 1—New Phytophthora taxa on trees and shrubs in Europe (new taxa 

and developments since 2004 are highlighted in bold) 

New taxon Host, ecology, distribution, 

identified by and date 

Taxonomic status 

in 2007 

P. italica sp. nov. Myrtle, Italy. I.P.V., Palermo, 1990s Cacciola and others 

1996 

P. inundata sp. nov. Trees/shrubs (Aesculus, Salix, 

Olea), after flooding Europe, South 

America. FR, U.K., 1970s 

Brasier and others 

2003 

P. quercina sp. nov. Oak fine roots, ubiquitous across 

Europe and Turkey, Eastern US. T. 

Jung, 1990s 

Jung and others 1999 

P. ramorum sp. nov. ‘sudden oak death’, Rhododendron, 

Viburnum, beech, red oaks, Europe, 

USA. BBA, Germany, 1990s 

Werres and others 

2001 

P. uliginosa sp. nov. Oak forest soils, beech bark, 

Germany, Poland. T. Jung, 1990s 

Jung and others 2002 

P. psychrophila Oak and beech soils rare, 

Germany, Austria, France. T. Jung, 

1990s 

Jung and others 2002 

P. europaea sp. nov. Oak forest soils, France, Germany, 

Eastern USA, Iris New Zealand. 

INRA Nancy / T. Jung, 1990s 

Jung and others 2002 

P. pseudosyringae sp. 

nov. 

Roots and bark of oaks, beech, 

hornbeam and alders; Germany, 

France, Italy, U.K., Romania, 

California, Oregon. INRA Nancy / T. 

Jung, 1990s 

Jung and others 2003 

P. alni sp. nov Alder, swarm of species hybrids, 

Europe. Forest Research, U.K., 

1990s 

Brasier and others 

2004 

P. hedraiandra sp. 

nov.

Roots, collar and leaves of 

Viburnum. The Netherlands, Italy, 

Spain. Soil of Juglans nigra, 

Hungary. Nurseries, US. Plant 

Protection Service, Wageningen, 

NL, 2001 

De Cock and 

Lévesque 2004 

P. kernoviae sp. nov. Bark of beech and oak; leaves and 

shoots of Rhododendron, Cornwall, 

U.K., and Cherimoya, New 

Zealand. FR/CSL, U.K., 2003 

Brasier and others 

2005 

P. polonica sp. nov. Soils of alder stands, Poland. 

University of Jussy, Switzerland, 

IMEDEA, Spain, Forest Research 

Institute, Poland, 2004 

Belbahri and others 

2006 

P. gallica sp. nov. Soil around oak roots and reed 

beds. Germany, France. T. Jung 

1990s 

Jung and Nechwatal 

2008 
GENERAL TECHNICAL REPORT PSW-GTR-221

 6 

Table 1 (continued). New Phytophthora taxa on trees and shrubs in Europe 

(new taxa and developments since 2004 are highlighted in bold) 

New taxon Host, ecology, distribution, 

identified by and date 

Taxonomic status 

in 2007 

P. taxon Pgchlamydo Prunus, Douglas fir, beech, nursery 

soils, wet sites Europe, North 

America, Argentina. Forest 

Research, U.K., 1970s 

P. taxon Riversoil Forest/riverbank soil U.K., FR, U.K., 

1990s. Fallen alder leaves, Spain. 

IMEDEA 2006

P. taxon Oaksoil Oak soil, rare, France. INRA, 

Nancy, 1990s 

P. taxon Forestsoil Forest soil (oak, Carpinus etc.) rare, 

France, Hungary. INRA, Nancy, 

1990s 

P. taxon Salixsoil Soil around roots of Salix and

Viburnum, alder debris, reed beds 

and streams. U.K., Germany, Italy, 

Denmark.. Forest Research, U.K., 

1970s 

Informally designated 

(Brasier and others 

2003) 

Phytophthora sp. 1 Soil of Alnus glutinosa + Juglans 

nigra stands; Hungary, University of 

West Hungary 

Phytophthora sp. 2 

Phytophthora sp. 3 

Soil of Alnus glutinosa stands. 

Hungary, University of West 

Hungary 

Lakatos & Szabo, this 

volume

P. sp. hungarica 

P. sp. sylvatica 

Soil and roots of Alnus glutinosa

stands. Hungary, Poland. University 

of Jussy, CH, Hungarian Academy 

of Sciences and Hungarian Forest 

Research Institute 

Informally designated 

at GenBank 

Surveys of Phytophthora Populations in Natural 

Ecosystems in Scotland 

Molecular methods are increasingly used for routine surveys of pathogens in plant 

tissue. They also have great potential for monitoring diversity of Phytophthora

populations in natural habitats. In 2006, water and soil samples were taken from 

streams in five nature reserves in Scotland and analysed for the presence of 

Phytophthora species using a method that includes filtration, DNA extraction, nested 

PCR of the ITS 1, cloning and sequencing (Silvia Scibetta, David Cooke and Santina 

Cacciola unpublished, see Brasier this volume). Seventeen different Phytophthora

species were detected in the water samples and only five species in adjacent soil 

samples. Some water samples contained up to five different Phytophthora species. 

The results of the Scottish stream survey are in good aggreement with those of stream 

surveys in the Eastern and Western US (Remigi and others and Hwang and others,

this volume) and Australia (Smith and others, this volume) all showing rich and 

diverse Phytophthora populations in watercourses. In Europe, the use of (cheap) 

unfiltered surface water for irrigation of parks, gardens and nursery fields is still 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 7 

widespread. Clearly this practice is likely to result in the infestation of the irrigated 

soils with Phytophthora species. 

New Insights Into the Evolution of the P. alni 

Hybrid Complex 

Based on morphological studies, the near tetraploid structure of P. alni ssp. alni

(PAA), the intermediate chromosome numbers of the other two subspecies P. alni

ssp. uniformis (PAU) and P. alni ssp. multiformis (PAM) and on AFLP and ITS DNA 

sequence analyses Brasier and others (1999, 2004) suggested that the progenitors of

PAA were most probably P. cambivora and an unknown taxon close to P. fragariae 

and that PAU and PAM are most probably a result of recombination and 

chromosome losses in PAA. This hypothesis fits well with the observed phenotypic 

variation, the developmental instability, and the intermediate chromosome numbers, 

but fits not totally with the observed pattern of ITS polymorphisms and is not well 

supported by the AFLP data. Recently, Ioos and others (2006 and this volume) 

investigated four nuclear DNA genes (ASF-like, GPA1, RAS-YPT, TRP1), two 

mitochondrial DNA genes (cox1, nadh1) of a European-wide range of isolates of 

PAA, PAM, PAU, P. cambivora, P. fragariae and other Phytophthora species. Their 

results strongly indicate that (1) PAU and PAM or PAU- and PAM-like ancestors 

were most probably the progenitors of PAA, (2) PAM may also be the result of an 

ancient reticulation, and (3) that several hybridisation events must have occurred. It 

was also demonstrated that P. fragariae was not involved in the evolution of the 

hybrids and that P. cambivora was unlikely to be a direct progenitor of the hybrids, 

although it could possibly be a parental species of a PAU-like ancestor. A study of 

the expression of the elicitin genes of PAA, PAM, and PAU isolates and 

microsatellite patterns in PAA and PAM by Ioos and others (this volume) further 

supports these hypotheses. Due to the recent finding of ‘PAU-like’ isolates in streams 

without visible alder damages in remote areas of Alaska (Lori Trummer and Charles 

G. Shaw, personal communication) a further elucidation of the hybridisation events 

that led to the P. alni hybrid complex may be expected. 

It was suggested by Brasier and others (1999, 2004) that the hybridisation events 

occurred in a nursery somewhere in Europe. Their view that nursery environments 

generally present suitable conditions for hybridisations are supported by the 

widespread finding of natural hybrids between P. cactorum and P. hedraiandra in 

nurseries and planted stands of Rhododendron (Man In’ t Veld and others this 

volume). The hybrids proliferate in several European countries and seem to 

outcompete P. cactorum on Rhododendron. 

Modelling Decline and Dieback of Alders Caused by 

Phytophthora alni 

The serious root and collar rot of alders caused by P. alni continues to spread across 

Europe. In northeast France and Bavaria, where the disease has reached epidemic 

levels, two models were recently developed. The model of Thoirain and others 

(2006) was based on data from 78 plots located along 35 rivers in NE France. It 

demonstrated that the watercourse type, i.e. width and slope of the river and speed of 

water flow, mean summer temperatures of the water and the soil texture of the river 
GENERAL TECHNICAL REPORT PSW-GTR-221

 8 

banks were the most important factors effecting disease incidence along the rivers. 

Infection rate and mortality of alders increased when river width, summer 

temperature of river water and clay content of the riverbank were higher and the 

slope of the river bed and water flow rate were lower. Interestingly, the total oxidised 

nitrogen concentration was found to be related to disease prevalence, as suggested 

earlier (Gibbs and others 1999). Jung and others (see abstract in this volume) tested 

three different models for describing the distribution of the alder disease on forest 

sites in Bavaria. The models were based on data on the establishment type and 

flooding conditions of 434 sample points of which 307 points were infested by P. 

alni; on GIS datasets on soil texture, aspect, slope and landform; and on a Vegetation 

Index calculated from satellite imagery. While both the spatial (only GIS datasets) 

and non-spatial models (only establishment type and flooding condition) were mora 

accurate at predicting infected sites than disease-free sites, the combined model 

predicted both healthy and diseased sites with high accuracy (85 percent and 86.6 

percent). The French and the Bavarian models can now be used for assessing the risk 

of P. alni-induced decline and dieback of alders on riparian and forest sites, 

respectively, in other regions. The Bavarian variables together with additional 

datasets on drainage, streams, climate, distribution of alder species, distribution of 

wholesale and retail nurseries and urban settlements are currently being applied in a 

preliminary Multi-Criteria model to assess regions of the USA with alder forests 

potentially susceptible to to P. alni. 

Alder Surveys and New Country Records for 

Phytophthora alni

In Poland, a statewide disease survey has been conducted and alder dieback caused 

by P. alni has been found to be widespread along more than 30 river systems (Leszek 

Orlikowski, personal communication). In the Czech Republic alder mortality has 

been widespread since severe flooding in 2002 and P. alni has been recovered from 

more than 60 alder stands in Bohemia (Cerny and others 2007). In 2007, PAU was 

also isolated from collar necroses of young black alders in a forest plantation in 

Moravia (T. Jung and L. Jankowski, unpublished results). 

Recently, P. alni (PAA) was recovered for the first time from collar rot tissue of 

riparian black alders in Switzerland (Ottmar Holdenrieder, personal communication). 

In 2005 a survey of Phytophthora root and collar rot was conducted along 26 rivers 

and streams in the wider city of Vienna, Austria. Typical Phytophthora symptoms 

were found along 16 rivers. P.alni (PAA and PAM) was recovered from collar rot 

tissues of alders growing along 4 streams with high alder mortality while in seven 

watercourses P. citricola and P. gonapodyides were found causing a scattered 

dieback of individual alder trees. Interestingly, P. alni was only found in river 

systems with infested alder plantations established in their catchments a few years 

ago (table 2). This confirms the results of Jung and Blaschke (2004) that the planting 

of infested nursery stock is the major pathway of P. alni spread and the primary 

cause of the Phytophthora root and collar rot epidemic of alders. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 9 

Table 2—Survey of root and collar rot of alders along rivers and streams in 

the city of Vienna (T. Jung and T. Cech, unpublished results) 

Phytophthora species 1 Number of 

streams with 

young alder 

plantations in 

catchment 

Total Infested PAA PAM CIT GON P. sp. 

Present 14 7 2 2 3 0 4 

Absent 12 4 0 0 3 1 1 

Total 26 16 2 2 6 1 5 

1

 PAA = P. alni ssp. alni, PAM = P. alni ssp. multiformis, CIT = P. citricola, 

GON = P. gonapodyides, P. sp. = typical collar rot symptoms, but isolation failed 

because the necroses were old and dry. 

Decline and Dieback of European Beech 

In our last review (Brasier and Jung 2006) we presented evidence from disease 

surveys in Southern and Northern Germany (Hartmann and others 2006, Jung 2006, 

Jung and others 2005), Austria, Southern and Central Italy (Belisario and others 

2005, Diana and others 2006, Motta and others 2003), Sweden and the U.K. (Brown 

and Brasier 2007) that an upsurge of Phytophthora activity on European beech has 

occurred. Between 2004 and 2007 most of these surveys have continued and a new 

survey has been conducted in Belgium (Schmitz and others this volume). New 

disease records have also come from Slovenia (Munda and others 2007), Switzerland 

(T. Jung unpublished), Romania (T. Jung and D. Chira unpublished), the Czech 

Republic (T. Jung and L. Jankowski unpublished) and the Netherlands (W. Man In’ t 

Veld unpublished, http://rapra.csl.gov.uk). In total 11 Phytophthora species have 

been found to date in declining beech forests across Europe with P. cambivora and P. 

citricola having the widest distribution and P. ramorum infections of beech being 

restricted to southwest England and the Netherlands (Table 3).

In Bavaria, Southern Germany, where beech stands have been severely declining 

since the abnormally wet and and extremely dry years of 2002 and 2003 respectively, 

more than 300 declining beech trees in 112 stands were sampled. Phytophthora

species were recovered from 81 percent of the trees in 93 percent of the stands (Jung, 

in press). In this study evidence was also presented that Phytophthora species, in 

particular P. citricola, may be involved in the aetiology of Beech Bark Disease in 

mature stands. 

In Belgium a severe decline and dieback of beech stands has occurred since the late 

1990s. Various hypotheses on the causes of the decline have been presented 

including frost events and bark beetle attacks. Recently, P. cambivora has been 

isolated from bark cankers in 19 out of 49 stands investigated (Schmitz and others 

this volume). 
GENERAL TECHNICAL REPORT PSW-GTR-221

 10 

Collectively, the above studies provide accumulating evidence that interactions 

between introduced Phytophthora species, either well established or still invasive, 

together with climatic extremes, are a significant factor in the widespread decline and 

dieback of beech stands across Europe (Jung, in press). 

Table 3—Phytophthora species associated with decline and mortality of 

beech in Europe 

Phytophthora spp.1 

Country / region 

CAC CAM CIT GON KER PSE RAM SYR CHLA ULI PSY 

North Germany X X 

South Germany X X X X X X X X X 

Austria X X 

Switzerland X X 

The Netherlands X X 

U.K. X X X X X X 

Belgium X 

Italy X X X 

Slovenia X 

Czech Republic X 

Romania X 

Turkey X 

1 CAC = P. cactorum, CAM = P. cambivora, CIT = P. citricola, GON = P. 

gonapodyides, KER = P. kernoviae, PSE = P. pseudosyringae, RAM = P. ramorum, 

SYR = P. syringae, CHLA = Phytophthora taxon ‘Pg chlamydo’, ULI = P. uliginosa, 

PSY = P. psychrophila. 

Colonisation of Tree Xylem by Phytophthora Species 

The recovery of non-aerial Phytophthora species with persistent sporangia, i.e. P. 

cambivora, P. citricola and P. gonapodyides, from aerial stem lesions that tend to be 

aligned along the stem raised the question on how these pathogens are able colonise 

the stems, sometimes reaching up to 20 m stem height (Brown and others 2006; 

Brown and Brasier 2006; Jung 2006, Jung and others 2005). Between 2004 and 2006, 

in southwest England, Brown and Brasier (2007) investigated stems of 53 beech, oak 

and maple trees with aerial stem lesions in detail. They found that individual ‘major’ 

lesions and adjacent ‘island’ lesions in the phloem were connected by underlying 

strips or intermittent pits of discoloured xylem in line with the wood grain. Isolations 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 11 

were made from 81 wood panels taken from below phloem lesions and 81 percent 

yielded Phytophthora spp. (P. ramorum, P. kernoviae, P. citricola, P. cambivora and 

P. gonapodyides). In 66 cases both a wood panel and an overlying phloem panel 

were sampled, and Phytophthora spp. were isolated from both xylem and phloem in 

56 percent, from xylem only in 23 percent and from phloem only in 8 percent of 

cases. These results show that Phytophthora species are able to survive and spread 

within tree xylem; that it is important to attempt to isolate from discoloured xlem 

panels as well as from infected phloem, since the former may give a higher isolation 

success rate; and that debarking of infected stems, such as P. ramorum-infected 

stems, for quarantine purposes may be insufficient to prevent spread of Phytophthora

species. The authors also suggest that apparent linear spread of a Phytophthora over 

longer distances along a stem might be due to spores being transported on embolisms 

within cavitated xylem vessels. 

Involvement of Phytophthora Species in European 

Oak Declines 

Numerous studies have been carried out on the causes of the widespread oak declines 

in western, central and southern Europe (Balci and Halmschlager 2003a, b; Brasier 

and others 1993; Gallego and others 1999; Hansen and Delatour 1999; Jung and 

others 1999, 2000; Vettraino and others 2002). Another study was recently carried 

out at the northern limit of the distribution of Quercus robur in Southern Sweden. 

Thirty two oak stands were investigated and P. quercina was recovered from almost 

50 percent of the declining stands but only from 11 percent of the healthy stands 

(Jönsson and others 2005). The calculation of odds ratio showed that the probability 

of a stand to be in decline is more than four times higher if P. quercina is present. 

These findings are in close accordance with results from earlier studies in southern 

Germany and Italy (Jung and others 2000; Vettraino and others 2002) and again 

indicate that Phytophthora species are strongly involved in European oak decline. 

Following an analysis of many climatic and site factors within the Swedish oak 

stands, and accepting the experimental evidence that P. quercina is able to cause 

substantial fine root loss on oaks in a sandy acid forest soil (Jönsson 2004), a 

conceptual model for oak decline in Sweden has been developed (Jönsson 2006). 

The model addresses the overall complexity of oak decline and includes 

Phytophthora infection levels, microbial activity, secondary pathogens, site 

and climatic factors and ecophysiological processes. 

Since the IUFRO 2004 meeting, considerable research has also been undertaken on 

Mediterranean oak decline associated with P. cinnamomi. Moreira and Martins 

(2005) investigated 56 cork and holm oak stands (Q. suber and Q. ilex) in Portugal 

and recovered P. cinnamomi from oaks and from 56 percent of the associated shrub 

flora at 27 sites demonstrating that like in the Jarrah forest in Western Australia the 

pathogen may represent a threat at ecosystem level in Europe, too. An analysis of 

various site factors indicated that P. cinnamomi induced oak decline was favoured by 

shallow soils with low fertility and low mineral nutrient levels, by a south-facing 

aspect, and by slopes and depressions. In another study on the survival and viability 

of cork oak seedlings in an oak stand naturally infested by P. cinnamomi, Moreira 

and others (this volume) found that the maternity of the progeny significantly 

influenced both acorn germination and seedling survival, resulting in a distinct 
GENERAL TECHNICAL REPORT PSW-GTR-221

 12 

susceptibility to P. cinnamomi among half-siblings which could be the basis for a 

resistance screening program. 

Investigations by M. Horta and colleagues in Portugal into the biochemical 

mechanisms of pathogenesis in P. cinnamomi have continued. Horta and others (this 

volume) demonstrated that the silencing of the beta-cinnamomin coding gene by 

genetic transformation of protoplasts led to a decreased virulence of the transformed 

P. cinnamomi isolate against cork oak seedlings. This result indicates that this 

elicitin gene might be involved in pathogenesis, a feature which could potentially 

have wide implications for disease control via resistance breeding or via genetic 

manipulation of the pathogen. Another control option for P. cinnamomi in 

mediterranean oak forests might come from the experiments of Neves and others 

(this volume), also in Portugal, who showed that the understory plant Phlomis 

purpurea produces substances that inhibit mycelial growth of P. cinnamomi in vitro. 

In a greenhouse trial the presence of Phlomis purpurea significantly protected holm 

oak seedlings from infections by P. cinnamomi. In an interesting AFLP analysis of a 

wide collection of P. cinnamomi isolates from declining holm and cork oak trees 

from the southwest region of the Iberian Peninsula, Caetano and others (this volume) 

demonstrated the presence of two clearly distinct populations, a Portuguese 

population which is spreading into south-western Spain and a Spanish population. 

Finally, P. quercina was recovered for the first time alongside P. cinnamomi from 

rhizosphere soil of declining cork oaks in Portugal (T. Jung unpublished). 

Ink Disease of Chestnut in Southern Europe 

Interesting results are also reported from several projects on ink disease of chestnut 

(Castanea sativa) caused by P. cambivora and P. cinnamomi. Fonseca and others 

(2004) investigated the relationship among the occurrence of ink disease, edaphic 

factors and management practices, and found that the probability of a stand having 

ink disease increases if the compaction and organic matter content of the soil are 

increased and manuring is the usual silvicultural practice. 

Variation in susceptibility to P. cambivora of 23 populations of European chestnut 

growing at 10 locations in Italy, Greece, France, Spain and the U.K. was studied by 

three research groups using a root-inoculation test on seedlings of half-sib families 

(Robin and others 2006). The results showed significant variation in resistance to ink 

disease. One or more resistant trees were found in 15 populations but only three 

families showed resistance comparable to the resistant control clone. Variation in 

resistance of chestnut clones to P. cinnamomi was also shown by Miranda-Fontaìna 

and others (2007). In this study at least 2 C. sativa clones showed remarkable 

resistance to root rot and stem lesions caused by P. cinnamomi. 

Portuguese and Italian research groups (Martins and others 2007, Vannini and others 

this volume) demonstrated the practical use of remote sensing and the application of 

geostatistical methods to data from proximal and remote sensing for monitoring and 

epidemiological studies on ink disease. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 13 

Upsurge of Phytophthora Activity on Several 

Broadleaved Tree Species in Central Europe 

Over the past few years a severe and widespread dieback and decline of mature trees 

of linden (Tilia cordata, T. platyphyllos, T. x europaea), maple (Acer platanoides, A. 

pseudoplatanus, A. campestre), birch (Betula pendula, B. pubescens) and horse 

chestnut (Aesculus hippocastanum) has is occurred in western and southern parts of 

Germany and Switzerland. Symptoms include an increased crown transparency, 

dieback of branches, small-sized and often yellowish foliage, extensive fine root 

losses, dieback of tap roots and cankers on suberised roots. In some stands collar rot 

and aerial stem lesions were found on maples, horse chestnuts and more rarely on 

linden trees. Dieback and mortality rates are most pronounced in old parks and 

forests (>100 years). A survey of rhizosphere soil samples and, if present, of bark 

lesions from 125 trees in 41 stands showed almost 100 percent infestation by in total 

7 Phytophthora species (T. Jung unpublished). P. citricola and P. cactorum were 

most frequently associated with the dieback (table 4). 

These results extend the picture coming from research on the association of 

Phytophthora species with the current declines and diebacks of oak, beech, chestnut 

and alders in Europe and indicate that similar infestation levels are occurring on 

these other broadleaved tree species. 

Table 4—Association of Phytophthora species with decline and dieback of 

linden, maple, birch and horse chestnut trees in Germany and Switzerland 

(T. Jung unpublished results) 

No. of stands (trees) with Phytophthora spp. 1 Tree 

species 

Total 

no. of 

stands 

(trees)

No. 

infected 

stands 

(trees)

CAC CAM CIT GON PSEU RAS SYR 

Tilia spp. 15 

(61) 

15 

(61) 

12 

(49) 

1 

(15) 

9 

(31) 

1 

(10) 

1 

(1) 

 1 

(2) 

Acer spp. 11 

(34) 

11 

(31) 

 1 

(2) 

10 

(27) 

3 

(4) 

 3 

(3) 

Betula

spp. 

5 

(13) 

5 

(13) 

2 

(4) 

 3 

(9) 

2 

(2) 

 1 

(2) 

Aesculus 

hippocast. 

10 

(17) 

9 

(15) 

5 

(6) 

 7 

(13) 

 1 

(1) 

Total 

stands 

trees 

41 

125 

40 

(98%) 

120 

(96%) 

19 

(46%) 

59 

(47%) 

2 

(5%) 

17 

(14%)

29 

(71%)

80 

(64%)

6 

(15%)

(16) 

1 

(2%) 

1 

(1%) 

1 

(2%) 

2 

(2%) 

5 

(12%) 

6 

(5%) 

1 CAC = P. cactorum, CAM = P. cambivora, CIT = P. citricola, GON = P. 

gonapodyides, PSE = P. pseudosyringae, RAS = P. taxon ‘raspberry’, SYR = P. 

syringae. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 14 

Spread of Phytophthoras From Nurseries Into 

Forests and Plantations 

Evidence had accumulated from meeting to meeting that Phytophthoras are spreading 

from infested nursery stock into forests. The first firm evidence that an epidemic in a 

natural ecosystem has been caused by distribution of infested nursery stock came 

from the investigation of riparian and forest alder ecosystems in Germany and 

elsewhere in Europe (Gibbs and others 1999, 2003; Jung and Blaschke 2004). At the 

2004 IUFRO workshop Orlikowski and others (2006) presented results from a survey 

of 30 horticultural and forest nurseries in Poland showing regular infestation with a 

range of 9 different Phytophthora species. At the same meeting Hartmann and others 

(2006) and Jung (2006) reported widespread infestation of nursery beech fields in 

Germany. 

Over the last few years four research groups in Germany and Austria have intensely 

surveyed local nursery fields and plantations erey (see abstract of Jung and others in 

this volume). In total 102 fields of alder (Alnus), beech (Fagus), linden (Tilia), maple 

(Acer), oak (Quercus) and horse chestnut (Aesculus) in 60 forest and horticultural 

nurseries in Germany and Austria were sampled. 64 fields (63 percent) in 48 

nurseries (80 percent) were found to be infested with atotal of 13 Phytophthora

species, among them the most aggressive ones to the respective tree species. Each 

nursery field yielded on average 2.1 Phytophthora species. In some cases up to 

fivedifferent Phytophthora species were recovered from a single field and even from 

the rhizosphere of an individual plant. In addition, 26 8–15 year-old plantations of 

oak, beech, linden and maple were screened and 24 (92.3 percent) were infested with 

in total 11 Phytophthora species, among them again the most aggressive ones to the 

respective tree species. Each plantation contained on average 1.7 Phytophthora

species. Together with the finding of P. alni in 362 alder plantations of up to 21 years 

of age on non-flooded sites in Bavaria (Jung and Blaschke 2004), there is now strong 

evidence that the planting of infested nursery stock has a major role in the spread of 

Phytophthora diseases of trees into forests and natural ecosystems in central Europe. 

In southern Europe also, a series of Phytophthora problems has recently been 

reported in nurseries (e.g. (Alvarez and others 2007, Belisario and others 2006, 

Moralejo and others 2007, Munda and others 2007, Nipoti and others 2005, Pane and 

others 2005). Likewise, recent nursery surveys in the US have also demonstrated 

high infestation levels (Ferguson and Jeffers 1999, Mac Donald and others 1994, 

Schwingle and others 2007b). 

What factors are responsible for these high infestation levels, beyond the wider 

problem of the international movement of plants? We suggest the following. First, 

over the past two decades there has been a progressive development across the 

European nursery sector to pyramid sales: fewer and fewer large units are producing 

the nursery stock which is bought in and sold by the majority of small nurseries. As a 

result, once a new Phytophthora pathogen enters the nursery production sector the 

intensive horizontal and vertical movement of plants between nurseries has the 

potential to spread it quickly and efficiently. This, together with the continuous 

production of nursery stock received from other nurseries, may be the prime reason 

for the observed accumulation of Phytophthora species in nursery fields. A second 

pathway by which Phytophthora species are entering the nurseries is likely to be the 

use of unfiltered surface water for irrigation. Thus, it was recently shown from stream 

surveys in Europe and in the Americas and Australia that watercourses are also 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 15 

infested with an array of Phytophthora species (see Remigi and others, Hwang and 

others, Smith and others, and Brasier in this volume). Third, regular use of fungistatic 

chemicals, in particular phosphites, in nurseries may be resulting in infected hosts on 

which the expression of disease symptoms is supressed. These may then be 

outplanted into forests because the infestation is invisible to the buyers of the plants. 

The apparent scale of the nursery problem indicates that the system of growing and 

distributing nursery stock in Europe needs to be overhauled. One proposal is for a 

significant reduction in the international movement of rooted plants to prevent the 

initial introduction and spread of exotic Phytophthoras (see Brasier 2008). In addition 

as a containment measure for existing nursery infestations, we would suggest 

development of a “Code of ‘Good Practice for Nurseries” along the lines already 

proposed by the EU Concerted Action project on the alder Phytophthora (Gibbs and 

others 2003). Viz. the production of nursery plants in steam sterilised soil mixture in 

containers without soil contact; the use of tap water, filtered well water or sterilised 

surface water for irrigation; the avoidance of fungistatic chemicals;and the exclusive 

purchase by retail nurseries of nursery stock from production nurseries that produce 

plants according to the recommended standards. 

Recent Research on P. ramorum and P. kernoviae 

in Europe

Results of the multitude of experiments and surveys performed within the frame of 

the part EU-funded ‘RAPRA’ project on P. ramorum (http://rapra.csl.gov.uk) under 

the guidance of Joan Webber (FR, U.K.) have substantially increased our knowledge 

and understanding of pathogenicity, host range, distribution, and survival of P. 

ramorum and of the biological and climatic conditions favouring disease outbreaks. 

Major outcomes include the following. 

Distribution and Host Range of P. ramorum in Europe 

In considering the distribution of P. ramorum in Europe a clear distinction must be 

made between the nursery environment and field situations and between foliar as 

opposed to stem infections of trees. 

Up to IUFRO 2007 P. ramorum has been found in nurseries of 16 European 

countries: Belgium, Czech Republic, Denmark, Finland, France, Germany, Ireland, 

Italy, the Netherlands, Norway, Poland, Slovenia, Spain, Sweden, Switzerland and 

the U.K. In the U.K. alone, 580 outbreaks have been recorded from nurseries and 

plant retail outlets. Most records came from Rhododendron, Viburnum, Camellia and 

Pieris. 

Outside nurseries P. ramorum has been recovered from 160 locations in the U.K. 

while elsewhere in Europe (the Netherlands, Germany, Ireland, Norway, Slovenia 

and Switzerland) the number of findings varies widely but is generally much lower 

than in the U.K. Most records outside nurseries are coming from Rhododendron, but 

leaf and shoot infections are also recorded from Quercus ilex, Castanea sativa, 

Fraxinus excelsior and Q. cerris as well as from ornamental species of the genera 

Drimys, Magnolia, Michelia, Cinnamomum and even Eucalyptus, mainly from 
GENERAL TECHNICAL REPORT PSW-GTR-221

 16 

Cornwall, southwest England. Only in England and the Netherlands, has P. ramorum 

been found causing bleeding stem lesions on a range of tree species (table 5). 

Table 5—Records of P. ramorum from stem lesions on trees in Europe 

Country Tree species Family Number of 

trees 

England Aesculus hippocastanum Hippocastanaeae 1 

England Acer pseudoplatanus Aceraceae 1 

England Castanea sativa Fagaceae 1 

England Fagus sylvatica Fagaceae 6 

Netherlands F. sylvatica Fagaceae 6 

England Nothofagus obliqua Fagaceae 2 

England Quercus acuta Fagaceae 1 

England Q. cerris Fagaceae 5 

England Q. falcata Fagaceae 1 

England Q. petraea Fagaceae 1 

Netherlands Q. rubra Fagaceae 8 

England Schima sp. Theaceae 2 

Total 35 

Data from RAPRA database on naturally infected hosts, http://rapra.csl.gov.uk. 

Table from Webber 2008. 

The outcome of susceptibility tests with P. ramorum on more than 260 species and 

reports of 140 naturally infected host species in Europe are given in two large 

databases on the ‘RAPRA’ website. 

Distribution and Host Range of P. kernoviae in Europe 

In Europe in 2007 P. kernoviae was still exclusively found in the U.K. Up to July 

2007, 40 outbreaks had been recorded from woodlands and gardens in Cornwall 

Southwest England and Wales. The pathogen was also found in two U.K. nurseries. 

Two outbreaks in Northern England (1 nursery, 1 single rhododendron in a managed 

garden) have been eradicated. Until July 2007 P. kernoviae has been recorded from 

more than 20 tree species and many ornamentals. The major hosts in the U.K. are 

Rhododendron and European beech. Complete host lists and additional information 

on P. kernoviae are published on the Web site http://www.defra.gov.uk/planth/ 

pkernovii2.htm. 

Factors Affecting Epidemiology of P. ramorum 

in Europe 

From U.K. field and experimental data and from modelling there is now clear 

evidence that in Europe, as in California and Oregon, P. ramorum needs a susceptible 

foliar host for inoculum buildup and suitable climatic conditions in order to infect—

and initiate lesions on—stems of trees. To date stem infections have occurred only in 

woodlands where Rhododendron ponticum, itself an invasive species in the U.K., is a 

significant understorey species. It was shown that due to its high susceptibility and 

the ability to support high levels of sporulation, R. ponticum plays a key role in 

escape of P. ramorum into natural and semi-natural environments and the subsequent 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 17 

spread to trees (Denman and others 2006, Webber 2008). Other tree and shrub 

species that are abundant in the understorey of some forest types in Europe, i.e. 

Vaccinium spp., Quercus ilex, Rhamnus alaternus, Viburnum tinus and Arbutus 

unedo, were also shown to support abundant sporulation and might enable the 

spread of P. ramorum to trees in areas such as in Iberia. 

In an extensive susceptibility test with 69 woody and herbal species belonging to 24 

families and 3 vegetation zones in Italy using both a leaf dip inoculation method and 

a wounded detached stem test, P. ramorum caused leaf symptoms and stem lesion on 

80 percent and 100 percent respectively, of the tested plant species (Vettraino and 

others this volume). Generally, species belonging to mediterranean macchia 

vegetation were less susceptible than those belonging to cold- and warm-temperate 

vegetation. However, all the plant species tested supported a high rate of pathogen 

sporulation, even on asymptomatic leaves. The risk of establishment and spread of P. 

ramorum in Italy, based on distribution and susceptibility of potential hosts and foliar 

hosts for pathogen sporulation has been predicted and mapped, and 40 percent of the 

Italian woodland was found being suitable. In Sardinia three areas of ca 33.000 

hectares have been identified as being at high risk. 

A climate matching model (CLIMEX) was used by Robert Baker (CSL) to compare 

southern Oregon with Europe, and the comparison then revised according to 

Meentemeyer and others (2004). The model identified the west of the U.K., Ireland 

and north-west parts of France, Spain and Portugal as regions with the closest ecoclimate matching in relation to P. ramorum. This is a similar result to CLIMEX 

modelling of the potential activity of P. cinnamomi on trees in Europe (Brasier 

and Scott 1994). 

Potential for P. ramorum and P. kernoviae to Enter 

Unwounded Bark and Cause Lesions 

Generally, there is good agreement between wound inoculation tests with P. 

ramorum and P. kernoviae on potential host species and data on host susceptibility in 

the field. However, there are some discrepancies in particular with Quercus robur

and Acer pseudoplatanus indicating that resistance might operate at least at two 

levels: resistance to initial zoospore penetration on the bark surface and resistance to 

phloem invasion after initial infection. This conclusion was supported by several log 

inoculation experiments conducted by Brasier and Brown (2007). 

In two in vitro experiments with freshly cut logs of five host species P. ramorum

zoospores could penetrate unwounded bark—and cause lesions—on Fagus sylvatica, 

Castanea sativa, Quercus rubra and Picea sitchensis. However, bark of Q. robur was 

penetrated but very few lesions developed. In another experiment freshly cut logs of 

A. pseudoplatanus, F. sylvatica and Q. robur were placed below Rhododendron trees 

with multiple natural foliage infections by P. ramorum or P. kernoviae. Again, P. 

ramorum could infect bark of F. sylvatica and Q. robur but not of A. pseudoplatanus, 

and caused substantial lesions on F. sylvatica but not on Q. robur. P. kernoviae could 

penetrate unwounded bark and cause lesions on Fagus but not on Acer and Quercus. 

These results are consistent with lesion occurrence on standing trees in the field. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 18 

New Insights Into the Breeding System of P. ramorum

In mating studies between A1 and A2 isolates of P. ramorum a high frequency (ca 57 

percent) of gametangia were abnormally developed or contained visibly aborted 

oospores, and an even higher proportion were classified as non-viable following vital 

staining (Brasier and others 2007). Nevertheless, there is clearly a potential for 

genetic recombination of P. ramorum via the sexual stage, and the recent discovery 

of at least three isolates in Belgium which appear to be of European lineage but of 

A2 mating type is alarming (Webber 2008). 

Dissemination of Phytophthora spp. via People 

From 2004–2007 ca 400 samples were collected from walkers’ boots in the P. 

kernoviae Management Zone in Cornwall, southwest England and baited using apple 

traps. Phytophthora spp. were recovered from more than 30 percent of the samples. 

The most frequently isolated species was P. citricola, but 10–15 percent of the 

samples contained either P. ramorum or P. kernoviae (Webber and Rose 2008). 

Phytophthora recovery followed a seasonal pattern with spikes in June–July and 

October–November. These results indicate a high potential for spread of the species 

in infested soil via movement of humans, animals and machinery. 

Asymptomatic Infection and Sporulation of P. ramorum

and P. kernoviae

In infection trials asymptomatic infection and sporulation of P. ramorum and P. 

kernoviae on leaves and fruits of Crataegus monogyna, Laurus nobilis, Q. ilex, Rosa 

sempervivens and Smilax aspera was demonstrated and endured at least 8 and 10 

days, respectively (Denman and others 2008). Also Vettraino and others (this 

volume) found that P. ramorum was able to sporulate on asymptomatic leaves of 

plants from Mediterranean macchia. This asymptomatic sporulation of P. ramorum

and P. kernoviae raises serious questions about international plant quarantine 

protocols which typically rely on visual inspection of plants. 

Management of Phytophthora Diseases of Trees in 

Europe 

There is much valuable long term expertise on the management of Phytophthora

diseases of trees in Australia (P. cinnamomi / Jarrah dieback) and the US (P. 

lateralis/Port-Orford cedar dieback). At previous meetings of this IUFRO group this 

has been represented by many talks, posters and exciting excursions. Steadily 

increasing knowledge of the ecology and epidemiology of different Phytophthora

species, pathways, dissemination mechanisms, aetiology, host ranges and efficiency 

of direct control measures is leading to the first integrated management plans for 

Phytophthora diseases of trees in Europe. In the U.K. Denman and others (this 

volume) investigated the potential of disease control practices for the integrated 

management of P. kernoviae on mature magnolias and developed simple and easily 

comprehensible decision systems for landowners. Also in the U.K., an area of about 

5.5 square miles in southern Cornwall with an extensive outbreak has been defined as 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 19 

the Phytophthora kernoviae Management Zone (PkMZ). Here an intensive integrated 

management process is being applied in an attempt to contain this invasive pathogen. 

In Germany, Austria and Italy extensive field trials were started in 2006 to test 

whether or not phosphite application is a useful tool for controlling Phytophthora

diseases of mature trees of beech, linden, oak and chestnut. In southern Germany 130 

mature trees of F. sylvatica, Q. robur, and T. cordata in five stands and in Austria 20 

mature trees of F. sylvatica in three stands are being sprayed annually at the stem 

base with 50 percent potassium phosphite amended with 2.5 percent of an 

organosilicatious surfactant in an attempt to prevent infection. In order to discover 

which application method has the highest efficacy, in southern Germany a 14 ha 

forest with mature (80–400 years old) trees of F. sylvatica, Q. robur and Q. petraea 

is being treated from helicopters with 50 percent potassium phosphite + 0.1 percent 

organosilicatious surfactant. While in central Italy chestnut stands in six areas with a 

total of 30–35 ha are being treated with stem injections of potassium phosphite. 

Preliminary results from all trials show considerable improvements of the crown 

conditions of treated trees as compared to untreated control trees (T. Jung and M. 

Blaschke unpublished results, T. Cech personal communication, A. Vannini 

unpublished results). 

Resistance screening can also be an option for the management of Phytophthora

diseases, as has been shown P. lateralis onPort-Orford cedar (Hansen and others 

2000, Sniezko this volume; Sniezko and Hansen 2003). As discussed earlier in this 

review, Robin and others (2006) found P. cambivora resistant trees in 15 chestnut 

populations from different European countries while Moreira and others (this 

volume) found that maternal progeny significantly influenced seedling survival in P. 

cinnamomi infested cork oak stands. Jung and Blaschke (2006) demonstrated that 

surviving alder trees in riparian ecosystems affected by the aggressive P. alni ssp. 

alni for more than 10 years often have a significantly lower susceptibility than 

declining alder trees. Such results are very promising and could yet be the basis of 

resistance screening programmes for chestnut and black alder in the future. 

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oak death 3rd science symposium. Gen. Tech. Rep. PSW-GTR-214. Albany, CA: U.S. 

Department of Agriculture, Forest Service, Pacific Southwest Research Station: 195–198. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 25 

Phytophthora Research and 

Management in Australasia1

Giles E.St.J. Hardy 2

Abstract 

This article provides a brief overview of the status of Phytophthora diseases in Australasia. 

Phytophthora cinnamomi remains the key species contributing to the decline of many 

wildland ecosystems throughout Australia. Recent molecular studies indicate that many new 

species exist in culture collections that need describing and their role as pathogens fully 

ascertained. An overview of the science, management and communication processes being 

undertaken to reduce the impact of P. cinnamomi is also covered for each Australian State and 

for New Zealand. Activities across the region vary considerably and reflect the amount of 

resources that are being provided, which are often minimal. 

Introduction 

In Australia, Phytophthora cinnamomi remains the major Phytophthora pathogen and 

continues to impact on the function and health of many wildland ecosystems. For 

example, in the South-West Botanical Province of Western Australia it impacts on 

over 3000 of 5710 described plant species (Shearer and others 2004). It is widespread 

throughout Australia and is located mainly in regions with higher than 600 mm 

rainfall (fig. 1). Unless significant advances are made in control, P. cinnamomi will 

continue to move autonomously and by inadvertent anthropogenic spread through 

many susceptible wildland plant communities for many decades to come. 

Phytopthora ramorum is listed as a Category 1 quarantine pest in Australia. The CRC 

National Plant Biosecurity and the Department of Environment, Water, Heritage and 

Arts are currently funding a Ph.D. research project (see Hüberli and others this 

proceedings) to determine the possible host range of Australian flora and their ability 

to support sporulation by P. ramorum. This work is being conducted in David 

Rizzo’s laboratory at the University of California, Davis. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26–31, 2007, Monterey, California. 2

 Centre for Phytophthora Science and Management, Murdoch University, Murdoch Western Australia 6150, 

Australia. 

Corresponding author: g.hardy@murdoch.edu.au. 
GENERAL TECHNICAL REPORT PSW-GTR-221

26 

Figure 12Phytophthora cinnamomi isolations and broad climatic envelope of 

P.Finnamomi susceptibility in Australia (O’Gara and others 2005). 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

27 

Western Australia 

New Phytophthora species 

Recently many isolates in the culture collections of the Centre for Phytophthora 

Science and Management and the Department of Environment and Conservation 

(DEC) have been sequenced using the ITS region of genomic rDNA (Cooke and 

others 2000) to confirm previous morphological identification and to determine what 

Phytophthora species are present in the State. This work indicates that there are at 

least 10 new undescribed species (fig. 2). 

Prior to the availability of molecular tools for use in diagnostics, a number of these 

isolates had been submitted to world culture collections (IMI and CBS) and allocated 

to already designated species based on morphological features. For example, many P. 

sp. 4 isolates had previously been designated as P. citricola, P. sp. 3 as P. drechsleri, 

P. sp. 1 as P. boehmeriae and P. sp. 10 as P. megasperma var. sojae (table 1). All 

new species were isolated from plants with disease symptoms or from the soil 

rhizosphere of plants expressing disease symptoms. Pathogenicity tests to confirm 

Koch’s Postulates are currently being undertaken. It is likely that all of these putative 

new species are pathogens; however, this needs to be confirmed. It will be interesting 

in the future to determine whether these Phytophthora species are endemic or 

introduced to Australia. This knowledge will help provide clear management and 

quarantine goals. However, irrespective of whether they are endemic or not, they 

should be managed to reduce their spread around the country. This will help reduce 

the chance of them being introduced to areas free of Phytophthora species and the 

risks of hybridization occurring. More importantly it is critical to keep in mind that 

global climate change may create more conducive conditions for many of these 

Phytophthora species to impact on wildlands in a similar fashion to P. cinnamomi.

For example, P. sp. 4 has a wider distribution than P. cinnamomi and has been 

associated with plant deaths on calcareous soils where P. cinnamomi does not cause 

disease. It is also associated with deaths of Eucalyptus gomphocephela (see Scott and 

others, this proceedings). 
GENERAL TECHNICAL REPORT PSW-GTR-221

28 

Figure 22Phytophthora species (in italics) that are present in Western Australia and 

where they sit with regards to Cooke’s ITS Clades (Cooke and others, 2000 ). P. 

sp. isolates (Psp1 – Psp10) remain undescribed (Tree Courtesy Treena Burgess). 

Phytophthora inundata 

P.sp. 6 

P.sp. 10 

P.sp. 7 

Phytophthora megasperma 

P.sp. 3 

P.sp. 8 

Phytophthora gonapodyides 

Phytophthora citricola 

Phytophthora inflata

P.sp. 4 

Phytophthora capsici

Phytophthora

Phytophthora

P.sp. 2 

Phytophthora nicotianae 

Phytophthora

Phytophthora

Phytophthora

P.sp. 1 

Phytophthora cinnamomi 

Phytophthora

Phytophthora drechsleri 

Phytophthora cryptogea 

P.sp. 5 

Phytophthora

Phytophthora

P.sp. 9 

Phytophthora

Phytophthora boehmeriae 

10

10

99 

100

74

100

99

99 100

67

100

78

61

10

93

97

100

97

100

82

85

100

100

94

100

98

100

100

7

100

10

100

10

100

100

100

100 9

100

100

100

95

94

87

100

10 95

91 100

100

100

B

Cooke’s

Group 6

Group 2

Group 1

Group 4

Group 7

Group 8

Group 9
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

29 

Table 12Examples of new Western Australian Phytophthora species based 

on molecular traits previously described as other Phytophthora species based 

on morphological traits (IMI = International Mycological Institute, U.K.; CBS = 

Centraalbureau voor Schimmelcultures, Netherlands). 

Isolate Morphological DNA 

DDS 1450 P. citricola (IMI 329674) P. sp. 4 

DCE 444 P. drechsleri (A1) 

(IMI 329666) 

P. sp. 3 

TCH 009 P. megasperma var. sojae 

(IMI 329669) 

P. sp. 10 

DDS 3884 P. boehmeriae (CBS 

100410) 

P. sp. 1 

P. sp. 10 previously described as P. megasperma var. sojae has been observed to 

have a high impact on many Proteaceous species in the Fitzgerald River National 

Park in the south-west of Western Australia, particularly after very wet rainfall 

events. Whilst, P. sp. 4 appears to have a wider distribution than P. cinnamomi as it is 

also associated with plant deaths on calcareous soils. Therefore, with the availability 

of molecular diagnostic tools there is no doubt more species will be found, and whilst 

P. cinnamomi remains the major Phytophthora pathogen in wildlands, it is critical 

that attention is also paid to new species as they are identified. 

Bell Track Infestation 

The Fitzgerald River National Park (FRNP) in the south-west of Western Australia is 

one of the World’s 25 Biodiversity ‘hotspots’ with large numbers of endemic flora 

and fauna. There is a 195 ha P. cinnamomi infestation in the park known as the ‘Bell 

Track Infestation’ that threatens in the immediate future some 6,000 ha of wildlands 

with many additional thousands of ha in the long-term if it breaks out of its existing 

catchment. The Department of Environment and Conservation have established two 

key goals to manage this infestation: first, to contain the further spread of P. 

cinnamomi, and second, to protect the unique biodiversity values of the FRNP from 

P. cinnamomi. In order to achieve this, extensive aerial mapping, digital elevation 

models and hydrological investigations have been conducted of the infestation 

together with extensive and intensive mapping of the pathogen through soil baiting 

and molecular diagnostics (see Anderson and others, this proceedings). Many 

thousands of soil samples have been baited to ensure accurate mapping of the 

pathogen. These activities have allowed detailed information to be gathered of stream 

paths, and where water will accumulate and be shed from given 50 and 100 year 

extreme rainfall events. Natural sumps were also identified. This information has in 

turn allowed for the planning of storage gully dams and flood diversion drains across 

catchments to be installed. These are intended to be able to manage extreme rainfall 

events and prevent the movement of P. cinnamomi infested water into adjacent 

disease-free catchments. The information has also facilitated the installation of a 13 

km fence around the infestation. This has been installed to prevent movement of the 

pathogen by native fauna such as kangaroos and to prevent any unintentional 

anthropogenic movement. In addition, approximately 2 km of high density 

polyethylene sheeting has been buried at points adjacent to uninfested catchments at 
GENERAL TECHNICAL REPORT PSW-GTR-221

30 

approximately 80 cm depth to act as a barrier to prevent root to root movement of the 

pathogen. Currently, there are discussions on whether to incorporate the eradication 

and containment methods developed by Dunstan and others (see this Proceedings) to 

reduce the impact of P. cinnamomi at Bell Track. 

The infestation at Bell Track has been treated regularly over the past decade with 

aerial applications of phosphite (phosphonate) (24 kg/ha a.i.) at low volume. These 

applications have been highly efficacious in reducing the impact and spread of P. 

cinnamomi at Bell Track. In addition, at Bell Track along approximately 30 ha of the 

immediate disease front and at two other infested sites the trunks of woody 

Proteaceous species have been sprayed to run-off with 30 percent a.i. phosphite 

together with Pulse. This has been done to address four questions; 1) does high 

intensity phosphite application prevent or reduce the rate of spread of the pathogen? 

2) does high intensity phosphite application reduce inoculum levels ahead of a 

disease front? 3) does high intensity phosphite application reduce mortality of 

susceptible plant species? and, 4) does high intensity phosphite application effect the 

epidemiology of the pathogen? 

Finally, due to the existing high impact of P. cinnamomi in the Bell Track and the 

loss of many of the deep rooted mid and upper story canopy species, many areas of 

the infestation have a changed hydrological balance and will be subject to massive 

surface water flow in a high rainfall event. Therefore, to re-establish natural 

hydrological balance across the infestation, DEC have put in place a revegetation 

program with an emphasis on deep rooted, long lived, greater than 0.5 m in height, 

rapidly growing, easily cultivated and P. cinnamomi resistant species that are local to 

the region. After extensive screening, Acacia saligna, Calothamnus quadrifidus and 

Eucalyptus pleurocarpa have been selected for establishment on the site. 

Mapping and Awareness 

In Western Australia, ‘Project Dieback’ funded by the National Heritage Trust 

through the Department of Environment, Water, Heritage and the Arts have made 

significant contributions to Phytophthora dieback issues in the State. In particular, 

they have put substantial effort into raising and obtaining community awareness and 

engagement, and towards mapping accurately the distribution of Phytophthora 

throughout the south-west of Western Australia. The mapping will provide managers 

and community groups with an accurate ‘Dieback’ occurrence map which will allow 

for assessment methodology for strategic regional planning over the next 5-10 years. 

Based on the mapping and knowledge of susceptible and threatened species and 

communities, key uninfested areas of wildlands that are considered protectable can 

be allocated resources to ensure that they remain protected. In addition, the mapping 

can be used to predict autonomous spread and to help determine where controls such 

as phosphite applications can be applied. ‘Project Dieback’ has also developed 

signage (fig. 3) that is being utilised throughout the region by government and local 

government agencies. Mining companies and other industries that manage activities 

in wildlands are also utilising the signage. This will ensure the community in general 

is not confused by different dieback signs on different land tenures. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

31 

Figure 32Signage now in place to clearly articulate to land users the ‘Phytophthora 

dieback’ status of an area (Courtesy Project Dieback and the Department of 

Environment and Conservation). 

Other Research in Western Australia 

A number of other research activities are in progress and these include: 1) Influence 

of physiological stresses (fire/waterlogging/drought) on phosphite applications and 

pathogen control (Hüberli and others), 2) Eradication and containment from infested 

sites in wildland ecosystems (Dunstan and others (see this proceedings), 3) Screening 

Australian native plants for susceptibility to P. ramorum (Ireland and others (see this 

proceedings), 4) Taxonomy of new Phytophthora species in Australia, 5) Role of fire 

and fire frequency and its impact on P. cinnamomi in wildland ecosystems, 6) To 

investigate the effects of phosphate and phosphite on P. cinnamomi control using 

Arabidiopsis thaliana, 7) Understanding the mechanisms by which phosphite affects 

the pathogenicity of P. cinnamomi, 8) New methods for measuring phosphite in 

planta, 9) Impacts of P. cinnamomi on biodiversity values in wildlands, 10) 

Microarray analysis of the effect of phosphite on Phytophthora cinnamomi. 
GENERAL TECHNICAL REPORT PSW-GTR-221

32 

Victoria 

Research 

Current research activities include examining the durability of resistance of 

susceptible eucalypts regenerating on ex-dieback sites. These if shown to be tolerant 

or resistant to P. cinnamomi have the potential to be utilised as potential seed sources 

for further restoration work on dieback sites. Surveys are being conducted in streams, 

rivers and other water bodies in natural ecosystems to determine what Phytophthora 

species are present in Victoria. Finally, an assessment of the risk posed by 

Phytophthora species to natural ecosystems is on-going. 

A large research project conducted out of D. Cahill’s laboratory is aimed at defining 

plant resistance against P. cinnamomi and the application of resistance to 

revegetation. This work aims to: 1) determine at the cellular, biochemical and 

molecular levels what constitutes resistance of native plants to P. cinnamomi, 2) 

identify the pathways within plants that regulate resistance and then explore the 

potential to manipulate these, 3) examine resistance in native stands affected by the 

pathogen, 4) use knowledge of resistance to implement field trials in sites that 

encompass a range of environments, and 5) utilise model systems such as 

Arabidiposis, Populus deltoids and P. tremula. 

Management 

In Victoria, P. cinnamomi is listed as a ‘potentially threatening process’ under the 

Flora and Fauna Guarantee Act 1988. A Strategic Management Plan to address the 

threat of P. cinnamomi on public land is in place. The key elements of the plan are 

(1) the protection of the most important assets at highest risk, (2) development of 

Regional Management Plans to zone areas by level of risk, (3) clear cross-agency and 

cross-tenure governance arrangements are being proposed, and (4) the development 

of ‘Best Practice Guidelines’. As part of these actions and to reduce the spread of the 

pathogen portable hygiene (soil wash-down) modules for operations such as fire or 

forestry operations are being developed. Footwear washing units have also been built 

and placed on walking tracks to reduce the spread of the pathogen. 

New South Wales 

Research 

Susceptibility trials have commenced on Eucalyptus imlayensis an endangered mallee 

of south-eastern New South Wales. Eradication of a P. cinnamomi front moving 

towards a Wollemi Pine (Wollemi nobilis) stand is being attempted. Wollemi Pine 

was discovered in 1994 and is a member of the Araucariaceae and less than 100 

mature trees are known to exist. It is susceptible to P. cinnamomi. Eradication of the 

pathogen is being attempted ahead of the front, a site of approximately 50 m, using 

soil drenches of fungicides and trunk injections of phosphite (phosphonate). 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

33 

Management 

A Statement of Intent is in preparation for P. cinnamomi, this is the first to be done in 

New South Wales under new legislation. A phosphite spray program has been 

instigated in an infested area of the South East Forests National Park to protect the 

habitat of the smoky mouse (Pseudomys fumeus), an endangered species. Many of 

the plants in its preferred habitat are susceptible to P. cinnamomi. Hygiene protocols 

are also in place to reduce infested soil movement between diseased and disease-free 

areas of its habitat. 

A survey and management plan have being conducted and developed, respectively, 

for the Sydney and Metropolitan and Hawkesbury Nepean Catchments. 

South Australia 

Management of Phytophthora in infested areas together with preventing disease-free 

areas becoming infested remain priorities. Research at the University of Adelaide is 

being conducted on understanding the impact of P. cinnamomi on native vegetation 

in South Australia with an aim to gain knowledge on the susceptibility of threatened 

plant species, patterns of Phytophthora spread and the role of soil microbiota in 

disease suppression. 

Tasmania 

In addition to the eradication trials of Dunstan and others (this Proceedings), 

eradication trials of infested sites are being proposed on a tin mine. Due to the 

changing of plant species names a reassessment of species susceptibility is in 

progress with the aim to upgrade the species susceptibility list. 

New Zealand 

A major collaborative project between Landcare Research, Ensis and HortResearch 

has been using ITS sequences to verify the Phytophthora species present in New 

Zealand and to develop a rapid DNA based identification scheme. This program will 

continue with on-going surveillance as part of routine diagnostic work. The presence 

of 21 species in the country has been confirmed with two newly described species, P. 

europaea and P. kernoviae being confirmed, with an additional two putatively new 

species to be described. P. inundata was also recorded as a new-to-New Zealand 

record in 2007. 

A number of other research projects are in progress. These include research on: the 

epidemiology and population dynamics of P. kernoviae (Ramsfield and others, see 

this Proceedings); the identity and epidemiology of Phytophthora taxon Agathis 

(Beever and others, see this Proceedings); the treatment of nursery-grown Nothofagus 

seedlings for planting into P. cinnamomi infested soils (Johnston and others see this 

Proceedings); the control of Phytophthora in forest nurseries through induced 

resistance (Dick and others, see this Proceedings). 
GENERAL TECHNICAL REPORT PSW-GTR-221

34 

Conclusion 

Many new species are being recorded in Australasia which is not surprising given the 

availability of molecular diagnostic tools. With increased activity in baiting streams 

and water sources (Ian Smith and Daniel Hüberli, pers com.) it is likely many more 

will be found. There have been examples of successful containment and eradication 

of P. cinnamomi from sites in Tasmania and Western Australia. There has been a 

very active increase in the Strategic Management of Phytophthora across Australia, 

together with a massive increase in community and agency involvement in the 

process. The National Resource Management regions across Australia have been 

incorporating Phytophthora into their management plans and developing ‘cross 

regional’ coordination. All of which are major advances in the last three years. 

Despite these activities there is an urgent need for adequate funding for management, 

communication and research to adequately manage P. cinnamomi in wildlands. A 

recent cost-benefit analysis study estimates that the Net Present Value of biodiversity 

assets at risk from Phytophthora dieback is approximately Aus $6.4 billion. Loss of 

carbon sequestration due to the death of woody plants is not included in this 

calculation. It is also estimated that an investment of Aus $58 million over 7 years 

will reduce losses by Aus $500 million. Therefore, reasonably modest investments 

into science, communication and management of Phytophthora diseases are required 

to derive considerable outcomes in managing biodiversity assets from Phytophthora 

dieback. 

Acknowledgments 

Many thanks to Keith McDougall (NSW), Rosalie Daniel (NSW), David Guest 

(NSW), Ian Smith (Victoria), David Cahill (Victoria), Renata Velzeboer (South 

Australia), Annabelle Bushell (WA), Joanna Young (WA), Greg Strelein (WA), Tim 

Rudman (Tasmania), Margaret Dick, Tod Ramsfield, Ross Beaver and Ian Horner 

(New Zealand) who kindly contributed information about Phytophthora from their 

respective States or New Zealand. Daniel Hüberli kindly reviewed this paper. 

Literature Cited 

Shearer, B.L.; Crane C.E.; Cochrane A. 2004. Quantification of the susceptibility of the 

native flora of the South-West Botanical Province, Western Australia, to Phytophthora 

cinnamomi. Australian Journal of Botany 52, 435–443. 

Cooke, D.E.L.; Drenth, A.; Duncan, J.M.; Wagels, G.; Brasier, C.M. 2000. A molecular 

phylogeny of Phytophthora and related oomycetes. Fungal Genetics and Biology 30, 17–32. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 35 

Phytophthora in Forests and Natural 

Ecosystems of the Americas1

David Rizzo2 and Elizabeth Fichtner2 

Abstract 

This article provides a brief overview of the status of Phytophthora diseases in the Americas. 

Although Phytophthora ramorum has stolen many of the recent headlines, much work has 

advanced on many other Phytophthoras ranging from old (e.g., P. cinnamomi, P. lateralis) to 

many recently described species such as P. siskiyouensis and P. austrocedrae. Phytophthora 

cinnamomi has been shown to threaten native vegetation in the rare Ione chaparral ecosystem 

in the western Sierra Nevada foothills of California. Resistance programs developed in 

Oregon have made significant advances in identifying resistant lines of Port-Orford cedar 

(Chamaecyparis lawsoniana). Phytophthora siskiyouensis, described from coastal Oregon and 

California streams has been found associated with urban tree decline in central California and 

in Australia. In Argentina, mortality of Austrocedrus chilensis in Patagonia “Mal del Cipre ́s” 

has been studied for many years and while several Phytophthora species were initially 

cultured from soils associated with declining A. chilensis forests, P. austrocedrae a new 

species, was isolated from necrotic lesions of stem and roots of A. chilensis. Research on all 

Phytophthora species has been conducted at multiple spatial and temporal scales and 

encompasses all parts of the host-pathogen interactions. 

Introduction 

Since the first meeting of the IUFRO Phytophthora group in 1999, our knowledge of 

the biology, diversity, ecology and epidemiology of Phytophthoras in forests of North 

America has greatly expanded. Although, Phytophthora ramorum has stolen many of 

the recent headlines, much work has advanced on many other Phytophthoras ranging 

from old (e.g., P. cinnamomi, P. lateralis) to many recently described species. 

Research has been conducted at multiple spatial and temporal scales and 

encompasses all parts of the host-pathogen interactions. The genome of P. ramorum

has been sequenced leading to a number of papers on comparative genomics with P. 

sojae and P. infestans. Field studies have begun to elucidate the epidemiology and 

management of Phytophthora from the plot to the landscape level. One of the most 

important developments is the increased emphasis on the connections between the 

ornamental horticultural industry and native ecosystems. At each meeting of the 

IUFRO working group, increasingly more presentations have discussed the role of 

nursery plants in the spread and establishment of Phytophthora in forests. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Department of Plant Pathology, University of California, Davis, CA 95616 USA. 

Corresponding author: dmrizzo@ucdavis.edu. 
GENERAL TECHNICAL REPORT PSW-GTR-221 

 36 

Monitoring for Phytophthoras in North America 

Following the finding of P. ramorum in California, there have been increased levels 

of monitoring of both nurseries and natural ecosystems throughout the United States, 

Canada and Mexico. Such monitoring efforts have been designed to support 

regulations and to enhance the application and efficacy of mitigation actions (e.g., 

eradication efforts) by enabling early pathogen detection. Monitoring has led to the 

discovery of a number of new species as well as increasing the geographic and host 

ranges of a number of previously described Phytophthora species. The diversity of 

Phytophthora in natural ecosystems is quite astounding and new species have often 

been found in forests and nurseries that have already been extensively examined. 

Table 1 illustrates examples of new species of Phytophthora associated with trees or 

shrubs that have been described since the first IUFRO Phytophthora meeting in 1999. 

One fascinating, and somewhat disconcerting, finding has been that several of these 

species were already known from other continents (P. pseudosyringae, P. europaea, 

P. hedraiandra, P. ramorum) at the time they were first observed in North America 

or were noted on another continent soon after they were first described in the 

Americas (P. siskiyouensis). 

Table 1. Examples of Phytophthora species that have been described in the 

Americas since the first IUFRO Phytophthora conference in 1999. Those 

marked with an asterisk (*) represent species that had previously been 

described from another continent or were noted on another continent soon 

after they were described in the Americas. 

Phytophthora 

species 

Habitat Location Reference 

P. ramorum* forests, 

nurseries 

CA, OR Rizzo and others 2005 

P. europaea* forests eastern US, 

CA 

Balci and others 2007; Rizzo, 

unpublished 

P. pseudosyringae* forests, 

nurseries 

CA, OR, 

NC 

Hwang and others 2007; 

Wickland and others 2008 

P. nemorosa forests, 

nurseries 

CA, OR Hansen and others 2003, 

Wickland and others 2008 

P. hedraiandra* nurseries MN, NC Schwingle and others 2007 

P. foliorum nurseries CA, TN Donahoo and others 2006 

P. austrocedrae forests Argentina Greslebin and others 2007 

P. siskiyouensis* forests, urban CA, OR Reeser and others 2007, 

Rooney-Latham and others 

2007, Smith and others 2006 

P. quercina-like forests eastern US Balci and others 2007, 

Schwingle and others 2007b 
 37 

In forests, broad scale sampling has been accomplished through soil (Balci and others 

2007, Schwingle and others 2007a) and stream monitoring (Hwang and others 2007, 

Wamishe and others 2007). Much of this has been accomplished via a national 

survey utilizing stream monitoring (Oak and others 2006). In the eastern United 

States, a number of Phytophthoras common to temperate forest regions of Europe 

have been found in soils and streams including P. cinnamomi and P. cambivora. P. 

pseudosyringae and P. europaea have only recently been described in Europe, but 

have turned out to be fairly common in eastern US forests. To date, P. 

pseudosyringae and P. europaea have only been observed in water courses or soils in 

the eastern US, but not from infected plant material. In contrast, both of these species 

have also been described from infected plant material in California and Oregon 

forests; P. pseudosyringae from cankers and foliar infections (see below) and P. 

europaea from infected foliage of bay laurel (Umbellularia californica). 

Phytophthora quercina was originally described in Europe in the mid-1990s in areas 

associated with oak decline (Jung and others 1999). Balci and others (2007) recently 

described a P. quercina-like taxon from soils in the central US; Schwingle and others 

(2007a) described a similar isolate from Missouri soils as P. quercina. At this time it 

is not clear of the final identity of these taxa (Balci, personal communication). New 

species descriptions of this and other taxa will most likely result from this work 

(Balci, personal communication). 

Because of quarantine regulations, a much greater effort has been made to identify 

Phytophthoras recovered in ornamental nurseries to the species level. Recent nursery 

surveys have turned up new species (e.g., P. foliorum) as well as a number of 

surprising species formerly unknown in the nursery industry. Yakabe (2007) 

conducted a survey of aerial Phytophthoras in California nurseries, finding many of 

the usual suspects such as P. cactorum, P. syringae, and P. citricola that have long 

been associated with ornamental plants. However, she also identified a number of 

species that have typically been associated only with forest plants and streams, 

including P. nemorosa, P. pseudosyringae, and Pg “chlamydo”. 

New Phytophthoras 

Three recently described species illustrate how much more we need to learn about 

Phytophthora biodiversity and biogeography. Reeser and others (2007) have 

described P. siskiyouensis from southwestern Oregon. This species was recovered 

from leaf baits while monitoring streams and soils for P. ramorum. It was also 

isolated from a blighted bay laurel shoot and tanoak cankers. Based on their research, 

Reeser and others considered P. siskiyouensis be native to coastal Oregon and 

possibly the Pacific Northwest. However, around the time it was described, P. 

siskiyouensis was isolated from Italian alder (Alnus cordata) in an urbanized area in 

Foster City, California (Rooney-Latham and others 2007). It was also isolated from 

Alnus glutinosa in a garden in Melbourne, Australia and reported as Phytophthora sp. 

at the 3rd IUFRO meeting (Smith and others 2006). Interestingly, P. siskiyouensis 

has not been isolated from alder in the area from which it was first described. 

 

Another previously unknown species, P. foliorum, was discovered during nursery 

surveys because its genomic DNA cross-reacted with the ITS-based diagnostic PCR 

primers used to screen plants for the presence of P. ramorum (Donahoo and others 

2006). Phytophthora foliorum was isolated from evergreen hybrid azalea leaves 
GENERAL TECHNICAL REPORT PSW-GTR-221 

 38 

collected in nurseries in both California and Tennessee. Sequence data indicate that 

P. foliorum fits into the same clade as P. ramorum, P. lateralis and P. hibernalis

(Donahoo and others 2006, Blair and others 2008). 

In Argentina, mortality of Austrocedrus chilensis in Patagonia ‘Mal del Cipre ́s’has 

been studied for many years and while several Phytophthora species were initially 

cultured from soils associated with declining A. chilensis forests, none were shown to 

have a clear relationship with tree death (Greslebin and others 2007). Eventually, a 

new species, P. austrocedrae, was isolated from necrotic lesions of stem and roots of 

A. chilensis. Once again, as with many of the other Phytophthoras discussed here, 

there appears to be strong linkages to nursery crops leading to the establishment of P. 

austrocedrae in the forest (Greslebin and Hansen, this volume). 

Phytophthora nemorosa and P. pseudosyringae 

While P. ramorum gets most of the attention, two of the most common 

Phytophthoras isolated from forest trees in coastal areas of California and Oregon are 

actually P. nemorosa and P. pseudosyringae. As with many of the species described 

above, they were initially found during surveys for P. ramorum. In California both 

species are primarily isolated from foliage of bay laurel and symptoms of P. 

nemorosa and P. pseudosyringae on all hosts are indistinguishable from the 

symptoms caused by P. ramorum (Wickland and others 2008). 

Phytophthora nemorosa is found from Monterey Co. in central California to Coos 

Co. in central Oregon. These sites are primarily distributed within 50 km of the 

Pacific Ocean. However, P. nemorosa has also been isolated from bay laurel foliage 

in Yosemite Valley in the central Sierra Nevada Mountains. Phytophthora nemorosa

is primarily found infecting trees in cooler, wetter coast redwood forests and less 

often in drier, warmer mixed-evergreen forest dominated by coast live oak. In 

California, the pathogen is most often isolated from bay laurel leaves, stems and 

twigs of tanoak, and leaf and small stem tissue of redwood. P. nemorosa has 

occasionally been isolated from bleeding cankers on coast live oak stems and one 

time each from necrotic branch tissue on a Douglas-fir (Pseudotsuga menzesii) 

sapling and from leaf tissue of hazelnut (Corylus cornuta), evergreen huckleberry 

(Vaccinium ovatum) and honeysuckle (Lonicera hispidula). 

Phytophthora pseudosyringae is found a bit further south than P. nemorosa and 

ranges from San Luis Obispo Co. to Curry Co. in southern Oregon. It also was 

isolated from bay laurel leaves in Yosemite Valley and in the northern Sierra Nevada 

Mountains (Butte County). In contrast to P. nemorosa, P. pseudosyringae is 

primarily isolated from hosts found in coast live oak woodlands in California and 

only occasionally found in more mesic coast redwood forests. Bay laurel is the most 

common host while infection of coast live oak stems is less frequent. Phytophthora 

pseudosyringae has been isolated a single time each from foliage of big leaf maple 

(Acer macrophyllum) and manzanita (Arctostaphylos sp.) 

Much more needs to be determined regarding the life histories of P. nemorosa and P. 

pseudosyringae including their complete host range, methods of dispersal, and 

summer survival in California’s Mediterranean climate. For example, in European 

oak and beech forests P. pseudosyringae is described as a soil inhabitant causing root 
 39 

and crown rot. Although this species was occasionally isolated from crown rot on 

coast live oak in California, it was primarily isolated from foliage on bay laurel. The 

soil phase of P. pseudosyringae in California forests needs additional investigation as 

do the aerial aspects of its biology in European forests. 

Phytophthora cinnamomi Update 

Phytophthora cinnamomi has long been important in forests, orchards and 

ornamental settings in the Americas and much of the history of this species has been 

reviewed in previous IUFRO proceedings (Hansen 1999, Goheen and Hansen 2006). 

Phytophthora cinnamomi continues to be important on oaks and chestnut in the 

southeastern USA, in southern California on coast live oak, and in oaks in forests of 

central and southern Mexico. For example, more recent studies on oak decline in 

Mexico have been carried out in five states of Mexico: Aguascalientes, Colima, 

Guanajuato, Jalisco and Nayarit. Based on their incidence, Phytophthora cinnamomi, 

Hypoxylon antropunctatum, and drought were considered the most important 

contributors to the decline and death of the oaks (Alvarado-Rosales and others 2007). 

Furthermore, P. cinnamomi was the most frequently recovered Phytophthora from 

soils surveyed in association with oak species in the northeast and mid-western 

United States (Balci and others 2007). 

Given California’s Mediterranean climate, it has been somewhat surprising that P. 

cinnamomi has not been reported more often from native ecosystems. Phytophthora

cinnamomi has long been known in California from fruit tree orchards, vineyards, 

Christmas tree plantings, and in ornamental settings. It was first reported on 

ornamental oaks (Quercus agrifolia, Q. suber) in the state in 1975 (Mircetich and 

others 1977). In 2001, P. cinnamomi was first shown to cause disease of oaks in 

natural oak woodlands of California (Garbelotto and others 2006), and recent studies 

have further elucidated the importance of P. cinnamomi in other California natural 

systems. 

Phytophthora cinnamomi has been shown to threaten native vegetation in the rare 

Ione chaparral ecosystem in the western Sierra Nevada foothills of CA (Swiecki and 

others 2003 2005). Phytophthora cinnamomi causes root and crown rot of Ione 

manzanita (Arctostaphylos myrtifolia), a rare plant limited to the unusually acidic 

“Ione” soils containing cement-like crusts of yellow iron-oxide, a characteristic 

typical of tropical soils. Phytophthora cinnamomi has caused large patches of ione 

manzanita mortality, and genetic evidence suggests that the pathogen has been 

introduced on multiple occasions, with subsequent spread emanating from the initial 

points of introduction. The long-term implications of these infestations on the ione 

manzanita are unknown, but the plant is currently considered threatened. 

In the San Francisco Bay area, P. cinnamomi has been found in coast live oak forests 

in areas impacted by P. ramorum (Swiecki, unpublished). The interesting aspect of 

this finding is that the main hosts appear to be madrone (Arbutus menziesii), 

manzanita (Arctostaphylos sp.) and bay laurel rather than the native oaks. P. 

cinnamomi has been isolated from the roots of declining bay laurel and madrone on 

the edge of the dieback zone (Swiecki and Fichtner, unpublished), but also from soils 

in areas outside the dieback zone. It is not yet known whether P. cinnamomi is widely 

distributed in these native forests, but only causing mortality after P. ramorum-
GENERAL TECHNICAL REPORT PSW-GTR-221 

 40 

related disturbance, or whether it was recently introduced. The linkage of P. 

ramorum and P. cinnamomi on individual sites has very important implications for 

forest dynamics and ecosystem restoration. Furthermore, the purchase of nurseryreared plants for laboratory inoculations has uncovered other, common 

Phytophthoras on madrone; highlighting the potential for introduction of other forest 

pathogens through revegetation programs (Fichtner, unpublished). 

Phytophthora ramorum Update 

So much has happened in P. ramorum research, management, and regulation over the 

past three years that it would take a review article to discuss all of the new findings 

and research directions. Many papers have been presented at this conference on P. 

ramorum and these proceedings include topics spanning pathogen biology and 

genetics to epidemiology and management. We will highlight several recent lines of 

research. 

Recent evidence of xylem infection of P. ramorum in naturally-infected trees 

suggests a reconsideration of the distinction between canker diseases and vascular 

wilts (Parke and others 2007; Brown and Brasier 2007). Historically, canker-causing 

Phytophthora species were generally believed to incite disease by killing the phloem; 

however, Parke and others (2007) have demonstrated that colonization of tanoak 

xylem by P. ramorum results in reduced water transport and may be a physiological 

explanation for the “sudden” death of tanoak under conditions of water stress. The 

group proposes that the colonization of non-living vessels offers a conduit for lateral 

movement of the pathogen within the host with protection from host defenses. 

The survival potential of P. ramorum in soils and soilless media and the potential for 

the pathogen to initiate root infections have been investigated both to enhance 

understanding of pathogen transmission pathways and assess the risk of 

establishment in new ecosystems. The pathogen can oversummer in forest soils in 

California, but survival is limited by soil-drying conditions (Fichtner and others 

2007). Chlamydospores have been shown to survive adverse temperature conditions 

for a few days, suggesting the potential for pathogen survival in many eastern United 

States ecosystems and highlighting the risk associated with potential introduction of 

the pathogen to these systems (Tooley and others 2008). Natural infection of tanoak 

roots by P. ramorum has been reported in infested California forests (Parke and 

others 2005), but the range of forest plant species supporting root infections and the 

prevalence of root infections is unknown. Shishkoff (2007) has demonstrated that P. 

ramorum can infect roots of multiple ornamental perennial trees and shrubs and also 

some species native to invaded California forests, including bay laurel. Phytophthora 

ramorum may persist for months in soilless potting media (Shishkoff 2007, 

Linderman and Davis, 2006), and infested potting media may serve as an inoculum 

source for root infections in container–grown plants (Parke and Lewis 2007). 

Chlamydospores have been shown to germinate to form sporangia in the vicinity of 

roots and serve as primary inoculum for root infections (Shishkoff 2007). 

The sequence of the P. ramorum genome was published in 2006 (Tyler and others 

2006). This data has lead to a number of comparative genomics papers looking at P. 

ramorum and P. sojae. More importantly from a sudden oak death perspective, the 

genome sequence has allowed for the development of microsatellite markers for use 

in P. ramorum population genetic studies (Ivors and others 2006, Prospero and others 
 41 

2007). Linkage of spatially-defined, population genetic analyses has allowed for 

indirect assessment of pathogen dispersal distances and the reconstruction of the 

spread of P. ramorum across landscapes (Mascheretti and others, in press; Hansen 

and others 2008). 

Extensive ground-based monitoring, aerial images, GIS technology, and modeling 

have also been put to use in epidemiological research. Through a comparison of 

recent and historical aerial images, Meentemeyer and others (2008) examined how 

changes in land-use (mostly fire suppresion) over the past 60 years have increased 

susceptible forest area at the expense of grassland and chaparral in Sonoma County, 

California. This increase in forest area has primarily been associated with increases in 

bay laurel, the main driver of P. ramorum epidemics in California mixed-evergreen 

forests. Other modeling papers have utilized biological and climate data from 

California and Oregon to model potential locations for invasion by P. ramorum in 

other locations in North America (e.g., Venette and Cohen 2006; Kelly and others 

2007). The combination of population genetic studies and landscape modeling will 

become increasingly important in designing management actions at spatial scales 

ranging from individual forest stands to regulatory actions at continental scales. 

Phytophthora lateralis Update 

Phytophthora lateralis has long been the primary Phytophthora species associated 

with extensive tree mortality forests in the Americas; it is still an ecologically and 

economically important disease. But we would like to end this Phytophthora update 

with some positive news. Resistance programs developed in Oregon have made 

significant advances in identifying resistant lines of Port-Orford cedar 

(Chamaecyparis lawsoniana) (Sniezko and colleagues, this volume). As a result of 

this research, Monrovia has recently released two P. lateralis- resistant varieties 

(‘Silberstar’ and ‘Golden King’) for ornamental plantings (see http://monrovia.com/). 

This is not the same as outplanting in native environments as part of forest restoration 

efforts. But it is still an excellent result and justifies continuing research efforts. 

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Biodiversity

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 47 

Phytophthora kernoviae in New Zealand1

T.D. Ramsfield,2 M.A. Dick,2 R.E. Beever,3 I.J. Horner,4 M.J. 

McAlonan,5 and C.F. Hill6

Abstract 

Phytophthora kernoviae was first recognised in New Zealand in 2005 by DNA sequencing of 

an isolate that had been recovered from diseased Annona cherimola (cherimoya or custard 

apple) in an abandoned orchard in Northland in 2002. Subsequent investigation has recovered 

P. kernoviae from the soil in Northland, Auckland, Bay of Plenty and Taupo regions. 

Similarity between P. kernoviae and descriptions of an undescribed Phytophthora sp. found 

previously in New Zealand indicates that the organism has been present here since at least 

1953. This, along with the geographic range of P. kernoviae, and a polymorphism in the ITS 

sequence, suggest that the pathogen has been present in New Zealand for an even longer time. 

Little is known of the ecological behaviour of the pathogen in New Zealand; disease has only 

been recorded on A. cherimola. 

Introduction 

Phytophthora spp. are some of the most invasive and serious plant pathogens and are 

of concern to regulatory agencies in many countries, including the New Zealand 

Ministry of Agriculture and Forestry (Biosecurity NZ). In a study in 2005-6, to 

clarify which species of Phytophthora are present in New Zealand, ITS DNA 

sequence data obtained from cultures that had been stored in culture collections 

maintained by Landcare Research (ICMP), NZ Forest Research Institute (NZFS) and 

HortResearch were compared with authentic sequence data from GenBank (Beever 

and others 2006). One of the findings from this study was the presence of an isolate 

in New Zealand that was similar to P. kernoviae. Phytophthora kernoviae has been 

recently described from Cornwall in the U.K. (Brasier and others 2005), and is a 

serious pathogen that has been causing significant disease of Fagus sylvatica

(European beech) and the exotic Rhododendron ponticum in woodlands since 2003. 

The pathogen is also capable of causing lesions on trunks of Quercus robur and 

Liriodendron tulipifera as well as the foliage of Magnolia spp. and Pieris spp. In 

Cornwall, the host range and pathogenesis process of P. kernoviae are similar to the 

invasive Phytophthora ramorum, but these two species are phylogenetically distant. 

In view of the disease caused by P. kernoviae in Cornwall on F. sylvatica, it was 

considered desirable to further investigate the presence of this pathogen in New 

Zealand, especially because of the importance of the indigenous Nothofagus spp., 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Sciona Forest Biosecurity and Protection, Private Bag 3020, Rotorua 3046, New Zealand. 3

 Landcare Research, Private Bag 92170, Auckland 1142, New Zealand. 

4

 HortResearch, Private Bag 1401, Havelock North 4157, New Zealand. 

5 Matapaua Bay, Whitianga, New Zealand. 

6

 Biosecurity New Zealand, Ministry of Agriculture and Forestry, PO Box 2095, Auckland 1140, New Zealand. 

Corresponding author: Tod.Ramsfield@scionresearch.com. 

a

Scion is the trading name of the New Zealand Forest Research Institute Ltd. 
GENERAL TECHNICAL REPORT PSW-GTR-221 

 48 

which are closely related to Fagus spp., in New Zealand forests. Herein we describe 

the results of follow-up investigations into the presence of P. kernoviae in New 

Zealand and provide evidence that suggests P. kernoviae has been in New Zealand 

for over 50 years. 

Initial Discovery 

The first isolate to be identified as P. kernoviae in New Zealand (ICMP 14761) was 

isolated in September 2002 from rotting fruit of Annona cherimola, growing in an 

abandoned orchard close to native shrublands and forest in Northland (Braithwaite 

and others 2007). At the time this isolate was obtained, P. kernoviae had yet to be 

described, and the isolate was identified as P. hibernalis based on morphological 

characteristics. The isolate was revived during the study by Beever and others (2006), 

in which the sequence data from the internal transcribed spacer (ITS) region of the 

ribosomal DNA repeat suggested the isolate was the newly described species P. 

kernoviae. Biosecurity New Zealand was notified and the affected cherimoya orchard 

was visited in December 2005 to investigate. It was found that trees in the orchard 

had shoot dieback and leaf lesions and mummified fruit were present. One isolate 

(ICMP 16306) collected in December 2005 from shoots with dieback had an identical 

ITS sequence to the isolate collected in 2002 and matched it morphologically. It was 

thus postulated that P. kernoviae was causing leaf, shoot and fruit disease of 

cherimoya in Northland. No other plant species at the site showed obvious symptoms 

of infection. 

Historical Presence of P. kernoviae in New Zealand 

Following the recognition of P. kernoviae on cherimoya, a further isolate of this 

species was identified in the study by Beever and others (2006) using ITS sequence 

data. Isolate ICMP 15082 had been baited from soil that was collected in kauri forest 

at Trounson Kauri Park (Northland), a native forest remnant, in 2003. This collection 

site is approximately 50 km from the affected cherimoya orchard. 

Perusal of early work in New Zealand led to the proposal that an undescribed 

Phytophthora species, that was the focus of a M. Sc. thesis by one of us (McAlonan), 

might also be P. kernoviae. This thesis (McAlonan 1970) describes a Phytophthora 

sp. (referred to as the “Tokoroa Phytophthora”), that was morphologically similar to 

P. kernoviae. It was recovered from under plantation Pinus radiata stands on pumice 

soils in the vicinity of Tokoroa, c.400 km south of the Northland sites. Although 

isolates from the thesis study were not available, the collection sites were listed in the 

thesis and these were revisited in 2006. Isolates matching the description of the 

“Tokoroa Phytophthora” were recovered by baiting soil that was collected from these 

sites. Surveys of the understorey vegetation showing possible disease symptoms of 

unknown cause at these collection sites have, to date, not recovered P. kernoviae. The 

“Tokoroa Phytophthora” was first recognized as distinct by FJ Newhook in 1953 

(McAlonan, 1970) and was likely the focus of a study of non-lethal root infection by 

an unnamed species of Phytophthora described as ‘widespread in pumice soil’ by 

Newhook (1961). 
 49 

Assessment of P. kernoviae in New Zealand 

The morphology of the recently collected isolates, ICMP 14761 from Northland and 

ICMP 16723 from Tokoroa was studied in detail and compared with the original 

description of P. kernoviae recorded in Brasier and others (2005) and the description 

of the “Tokoroa Phytophthora” by McAlonan (1970). The isolates were grown on 

carrot agar (CA) and pea extract agar (PEA) and compared with the descriptions 

recorded by Braiser and others (2005) and McAlonan (1970). 

In addition to cultural characteristics and morphology, DNA sequence data was 

generated for all isolates recovered from the Northland and Tokoroa sites. The ITS 

was PCR amplified and sequenced following the methods of Cooke and others 

(2000). Representatives of Phytophthora clades 1 – 8, as recognized by Cooke and 

others (2000), were selected and their ITS sequences were obtained from GenBank. 

ITS sequence data from other species of Phytophthora that are present within clades 

9 and 10 (Dick and others 2006), and also P. quininea, because of its relationship 

with P. richardiae as determined by Kroon and others (2004), were also included. 

The phylogenetic relationship of P. kernoviae to the other members of clades 9 and 

10 was assessed using a neighbour-joining analysis with the maximum composite 

likelihood model and 5000 bootstrap replications using MEGA version 4 (Tamura 

and others 2007), based on ITS sequence data that was aligned using Clustal W 

within MEGA 4. 

Results 

As indicated in Table 1, despite subtle differences in oogonia size, sporangial 

dimensions and pedicel length, other characteristics such as the low optimum 

temperature for growth and the lack of chlamydospores are uniform. All isolates were 

homothallic. Although there are some small differences in the descriptions of 

“Tokoroa Phytophthora” and P. kernoviae, some of these differences may have 

arisen through differences in culture technique. McAlonan (1970) grew his isolates 

on pea extract agar and soil extract agar, media that are less commonly used now. 

The New Zealand isolates were therefore grown on media used by McAlonan (1970) 

for comparison. The characteristic ‘mouse’ shaped sporangia of P. kernoviae,

illustrated by Brasier and others (2005), did not form on the media used by 

McAlonan (1970), who described the sporangia as pyriforme, but they comprised a 

considerable proportion of sporangia formed on carrot agar, the medium used by 

Brasier and others (2005). 
GENERAL TECHNICAL REPORT PSW-GTR-221 

 50 

Table 1. Morphological comparison of P. kernoviae as in Brasier and others 

(2005), “Tokoroa Phytophthora” as in McAlonan (1970), ICMP14761 and 

ICMP16723. 

 P. kernoviae 

(Brasier and 

others, 2005) 

P. kernoviae

NZ isolate 

ICMP 14761 

McAlonan (1970) Phytophthora

Thesis site 2006 

ICMP 16723 

Colonies 

In dark on carrot agar 

(CA) 

mycelium largely 

submerged with 

central aerial 

area 

mycelium largely 

submerged with 

central aerial 

area 

little or no aerial 

hyphae on 3 

media tested 

little aerial hyphae 

on 3 media 

Diurnal alternating rings 

of mycelium 

alternating rings 

of mycelium 

Not mentioned alternating rings of 

mycelium 

Growth rate in dark 3.8-4.6 mm/day 

@ 20°C on CA 

2.7-4 mm/day @ 

20°C on CA, 3.0 

mm/day @ 20°C 

on PEA 

3.1 mm/day @ 

18°C on PEA 

3.0 mm/day @ 

20°C on CA 

2.9 mm/day @ 

20°C on PEA 

2.7 mm/day @ 

18°C on PEA 

Optimum temperature 18°C 18°C 21°C 20°C 

Upper temperature limit ca 26°C ca 26°C ca 24°C ca 25°C 

Gametangia 

Diameter oogonia (μ) 21 – 28 20 – 26.5 24.5 – 28 23 – 26 

Diameter oospores (μ) 19 – 25 16.5 – 20 22 – 26 21 – 22 

Position oospore plerotic plerotic plerotic plerotic 

Antheridia amphigynous amphigynous amphigynous, 

occ. paragynous 

amphigynous, occ. 

paragynous 

Antheridial dimensions 10 – 14 x 9 – 12 8 – 14 x 8 – 12.5 10 – 11 x 10 – 

12 

9 – 10 x 10 – 11 

Sporangia 

 papillate and 

caducous 

papillate and 

caducous 

papillate and 

caducous 

papillate and 

caducous 

shape ovoid, limoniform 

to asymmetric 

ovoid, 

limoniform to 

asymmetric 

pyriforme ovoid, limoniform, 

pyriforme to 

asymmetric 

vacuole common common not mentioned common 

dimensions (μ) 34 – 52 x 19 – 

31 

32 – 48 x 16 – 

25 

25 – 37 x 17 – 

23 

28 – 34 x 22 – 24 

pedicel (μ) 5 – 19 10 – 22 4 – 7 7 – 10 

Chlamydospores None none none none 
 51 

When the ITS sequences were BLAST searched in GenBank, it was found that the 

isolates from New Zealand matched 812/813 base pairs with AY940661, the 

representative U.K. isolate. The difference was a one base substitution at position 

679. Phytophthora kernoviae isolate AY940661 (Brasier and others 2005) has 

adenine (A) at position 679, while all isolates from New Zealand have guanine (G) at 

that position except for two isolates from the Tokoroa sites which were polymorphic 

for adenine and guanine (A/G) at that position. We have not recovered an isolate of 

P. kernoviae from New Zealand that is monomorphic for adenine (A) at position 679. 

Our phylogenetic analysis confirmed the results of Brasier and others (2005) and 

Blair and others (2007) that P. boehmeriae is the closest relative of P. kernoviae (fig 

1). 

Figure 1-Neighbour joining analysis of ITS sequences from authentic 

Phytophthora spp. that represent clades 1-8 (sensu Cooke and others, 2000) 

and selected species that have been recorded in clades 9 and 10 to show the 

relationship of P. kernoviae to other Phytophthora spp. Bootstrap support 

values following 5000 replications are indicated at the nodes. 

Discussion 

Phytophthora kernoviae has resulted in extensive damage to woodlands of southwest England (Brasier and others 2005) and is a phytosanitary concern for many 

countries. Regulatory authorities in the U.K. have responded to P. kernoviae as an 

invasive organism and are attempting to eradicate it (Brasier and others 2005). The 

geographic origin of P. kernoviae is unknown; however, because of its low optimum 
GENERAL TECHNICAL REPORT PSW-GTR-221 

 52 

temperature and host range Brasier and others (2005) speculated that the organism 

may be native to the south-west China, the Himalayas or perhaps even Patagonia. 

The phylogenetic position of P. kernoviae in clade 9 / 10 with species known 

predominantly from the southern hemisphere may be further evidence for a southern 

hemisphere origin for this species. 

McAlonan (1970) studied the pathogenicity of the “Tokoroa Phytophthora” on Pinus 

radiata and found that the organism was “mildly pathogenic” to the roots of this 

conifer, causing less damage than P. cinnamomi or P. citricola. When he assessed the 

pathogenicity of the “Tokoroa Phytophthora” on Pseudotsuga menzesii, the roots 

remained healthy (McAlonan, 1970). The species of Phytophthora described as 

‘widespread in pumice soils’, and likely to have been P. kernoviae, was observed by 

Newhook (1961) to infect roots of P. radiata causing some root mortality and 

reduced growth, but infections were non-lethal and the root systems would have 

passed as healthy if there were not controls for comparison. He noted that 

Phytophthora spp. are an “important, but not easily recognized biotic factor of the 

environment” in New Zealand. 

We conclude that P. kernoviae is present in New Zealand, and that it has been here 

for more than 50 years because: its presence was first recorded here over 50 years 

ago, it is relatively widespread in the North Island, it is present in soil in native 

forests or recently converted native forests, and the polymorphism in the ITS 

sequence at position 679 indicates some genetic diversity in the New Zealand 

population. To date, we have not recovered an isolate that is a 100 percent match 

with the U.K. isolate and the difference in disease expression between the New 

Zealand and U.K. populations of this organism is striking. The phylogenetic position 

in clade 9, with other species that are present in the Southern Hemisphere, suggests 

that the species may have an origin in this region. 

Our study of P. kernoviae in New Zealand is ongoing and we are planning a survey 

coupled with a population genetic study that will assess the hypothesis that P. 

kernoviae is native to New Zealand. At the present time, we consider it premature to 

suggest that the pathogen is native to New Zealand. 

Acknowledgements 

The authors acknowledge funding from MAF Biosecurity New Zealand and the 

contributions of MAF BNZ staff Mark Braithwaite, Mark Bullians, George Gill and 

Heather Pearson. 

Literature Cited 

Beever, R.E.; Ramsfield, T.D.; Dick, M.A.; Park, D.; Fletcher, M.J.; Horner, I.J. 2006.

Molecular characterisation of New Zealand isolates of the fungus Phytophthora [MBS305]. 

Landcare Research Contract Report LC0506/155. Prepared for MAF Policy, New Zealand. 

43 p. 

Blair, J.E.; Coffey, M.D.; Park, S.; Geiser, D.M.; Kang, S. 2007. A multi-locus phylogeny 

for Phytophthora utilizing markers derived from complete genome sequences. Fungal 

Genetics and Biology. In Press. 
 53 

Braithwaite, M.; Bullians, M.S.; Pay, J.M.; Gill, G.S.C.; Hill, C.F. 2007. A disease survey 

of cherimoya orchards in Northland, New Zealand. Proceedings, 16th Biennial Australasian 

Palnt Patholgy Conference, Adelaide 24-27 September 2007. 

Brasier, C.M.; Beales, P.A.; Kirk, S.A.; Denman, S.; Rose, J. 2005. Phytophthora 

kernoviae sp. nov., an invasive pathogen causing bleeding stem lesions on forest trees and 

foliar necrosis of ornamentals in the U.K. Mycological Research 109: 853-859. 

Cooke, D.E.L.; Drenth, A.; Duncan, H.J.M.; Wagels, G., Brasier, C.M. 2000. A 

molecular phylogeny of Phytophthora and related oomycetes. Fungal Genetics and Biology 

30: 17-32. 

Dick, M.A.; Dobbie, K.; Cooke, D.E.L.; Brasier, C.M. 2006. Phytophthora captiosa sp. 

nov. and P. fallax sp. nov. causing crown dieback of Eucalyptus in New Zealand. 

Mycological Research 110: 393-404. 

Kroon, L.P.N.M.; Bakker F.T.; van den Bosch, G.B.M.; Bonants, P.J.M.; Flier, W.G. 

2004. Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear 

DNA sequences. Fungal Genetics and Biology 41: 766-782. 

McAlonan, M.J. 1970. An undescribed Phytophthora sp. recovered from beneath stands of 

Pinus radiata. Master of Science Thesis, Auckland University, New Zealand. 64 p. 

Newhook, F.J. 1961. Non-lethal pathological infection of roots. Nature 191: 615-616. 

Tamura, K.; Dudley, J.; Nei, M.; Kumar, S. 2007. MEGA4: Molecular Evolutionary 

Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596- 

1599. 
GENERAL TECHNICAL REPORT PSW-GTR-221 

 54 

The Search for Phytophthora Centres of 

Origin: Phytophthora Species in Mountain 

Ecosystems in Nepal.1

Andrea Vannini,2 Anna Brown,3 Clive Brasier,3 and 

Anna Maria Vettraino2

Abstract 

Asia is a centre of diversity for many plant genera that are hosts of invasive Phytophthora

species in other parts of the world. Examples of prominent invasives include P. ramorum and 

P. kernoviae, recently spreading in Europe and North America (Brasier and others, 2005; 

www.rapra.csl.gov.uk). One hypothesis is that these pathogens originated in a specific 

geographic area of in Asia, such as Yunnan or the Himalayas, and were then transferred via 

commercial movement of plants to other parts of the world. To investigate the presence of 

Phytophthoras in Himalayan forest ecosystems a preliminary expedition was undertaken in 

October 2005 to the forests of Western Nepal. During this expedition 47 soil samples were 

collected between Kolti (1396m) and Rara Lake (3500m) in the Bajura district. The samples 

came from 2 distinct ecological zones: the temperate forest, sub-tropical forest. Sixteen target 

broadleaved and coniferous trees and shrubs were sampled. The geographic co-ordinates of 

the samples were recorded by GPS and used to elaborate a survey map (fig.1) 

Figure 1- purple spots correspond to the geographic position of the samples 

collected between Kolti (a) and Rara Lake (b) 

A total of 39 Pythium and 89 Phytophthora isolates were obtained through soil baiting. A 

Phytophthora, with some morphological similarities to P. meadii, but also many differences, 

was associated with sub-tropical forest vegetation including Lithocarpus, Cupressus, Cornus, 

Carpinus and Castanopsis species (fig.2a). This taxon has been informally labelled ‘P. 

meadii-like’ while its properties are being investigated and may be a previously undescribed 

species. A second species, P. nicotianae, was found only in the sub-tropical agricultural zone 

around Ficus, Persea and Olea species (fig.2b). P. citricola was the most frequently isolated 

species (fig.2c), and was confined to the soil around temperate forest trees including Acer, 

Aesculus, Juglans, Ulmus and Viburnum species. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Department of Plant Pathology, University of Tuscia, via S. Camillo de Lellis,01100-Viterbo, Italy. 

3

 Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, U.K.

Corresponding author: vannini@unitus.it. 

a 

b
 55 

a b c

Figure 2-Isolates of P. meadii-like (a), P. nicotianae (b) and P. citricola (c) 

grew at 20°C on PDA for 1 week 

The distinct zoning of the two possibly endemic Himalayan forest Phytopthoras, P. citricola

and P. meadii-like, may lead to further understanding of the role of the genus in forest 

ecosystems. Whether P. meadii-like presents a threat to forest ecosystems in other parts of the 

world has yet to be determined. The discovery of P. nicotianae in association with native 

Olea cuspidata trees in Nepal may have significance for proposed commercial cultivation of 

European olive, O. europaea, in this region. 

Literature Cited 

Brasier, C.M.; Beales, P.A.; Kirk, S.A.; Denman, S.; Rose, J. 2005. Phytophthora

kernoviae sp. nov., and invasisve pathogen causing bleeding stem lesions on forest trees and 

foliar necrosis of ornamentals in the U.K. Mycological Research 109, 853-859. 
GENERAL TECHNICAL REPORT PSW-GTR-221 

 56 

Natural Hybrids of Resident and Introduced 

Phytophthora Species Proliferating on 

Multiple New Hosts in Europe1

Willem Man in ’t Veld,2 Arthur de Cock and Richard Summerbell 

Abstract 

In 1996 we encountered some atypical Phytophthora cactorum strains. They all possessed 

numerous abortive oospores, amphigynous and paragynous antheridia and had a maximum 

growing temperature of 350

 C, whereas P. cactorum itself generally has healthy oospores, 

paragynous antheridia only and a maximum growing temperature of 320

 C. At first we tried to 

identify these strains with isozyme analysis, since the isozyme alleles of Phytophthora 

cactorum are known (Oudemans & Coffey, 1991). Using the dimeric enzymes malate 

dehydrogenase (MDH) and malic enzyme (MDHP) the population was divided into two 

groups: the first group (I) had with both enzymes three-banded patterns, one band having the 

same mobility as the typical P. cactorum band. The second group (II) had the same threebanded patterns with MDHP as group I, but with MDH it generated only the typical P. 

cactorum band. Three-banded patterns with dimeric enzymes are the result of two different 

gene products, wich can combine in three different ways creating two different homo-dimeric 

bands and one hetero-dimeric band in between. This characteristic pattern usually indicates 

that outcrossing has occurred. Restriction enzyme analysis of the PCR amplified ITS region 

using Alu I also divided the atypical P. cactorum population into the same two groups (fig 1). 

Isolates of group I contained fragments of both P. nicotianae and P. cactorum whereas 

isolates of group II only generated the typical P. cactorum band. Isolates of group I were 

identified as hybrids of P. nicotianae and P. cactorum, also based on RAPD evidence (Man in 

’t Veld and others 1998). Isolates of group II were hypothesized to be the result of 

intercrossing of P. nicotianae x cactorum hybrids or backcrossings to P. cactorum. In order to 

collect more evidence for either hypothesis, mitochondrial DNA was digested with restriction 

enzymes (fig 2). Hybrids of P. nicotianae x cactorum contained mtDNA of P.nicotianae, 

confirming the parenthood of P.nicotianae. Isolates of group II, however, possessed neither 

mtDNA of P.nicotianae nor that of P.cactorum. Instead, an unknown mtDNA fragment 

profile was revealed. It was concluded that there was no connection of isolates of group II 

with P.nicotianae x cactorum hybrids. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Plant Protection Service, 15 Geertjesweg, 6700 HC, Wageningen, the Netherlands. 

Corresponding author: w.a.man.in.t.veld@minInv.nl. 
 57 

 1 2 3 4 5 6 7 8 9 10 11 

Fig.1. Restriction enzyme analysis (Alu 1) of the internal transcribed 

spacer sequence of the ribosomal DNA gene repeat (ITS). 

1, 2: Phytophthora cactorum

3, 4, 5: Phytophthora nicotianae

6, 7, 8, 9: group II (=Phytophthora hedraiandra x cactorum hybrids) 

10, 11: group I (=Phytophthora nicotianae x cactorum hybrids)
GENERAL TECHNICAL REPORT PSW-GTR-221 

 58 

Fig. 2. Restriction enzyme patterns of mitochondrial DNA of 

Phytophthora spp. digested with Hind II 

1, 2, 3: P. cactorum 

4,5: group II (= P. hedraiandra x cactorum hybrids) 

6, 7, 8: group I (= P. nicotianae x cactorum hybrids) 

9, 10: P. nicotianae

1 2 3 4 5 6 7 8 9 10 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

59 

After some years of no activity concerning the isolates of group II, we decided in 2004 to start 

up again the investigations. In a final attempt to elucidate the true identity of isolates of group 

II we sequenced the ITS region of these isolates. This was the key experiment since, in 

contrast to Phytophthora species, wich have generally homogeneous ITS sequences through 

concerted evolution, we discovered double bases at four distinct positions in the sequences of 

all isolates of group II. Furthermore, the double bases were exactly at those four positions 

were P. cactorum and the newly discovered P. hedraiandra contained different bases and they 

were even composed of those present in P. cactorum and P. hedraiandra (table 1). 

Table 12Features of Phytophthora species, variable positions in ITS 

sequences, Cytochrome oxidase I (Cox I) type and malic enzyme (Mdhp) 

alleles. 

Phytophthora 

species 

ITS variable 

positions 

Cox I type Isozyme alleles 

Mdhp 

 74 100 101 686 

cactorum G G T T cactorum AA 

hybrid (=group II) R R Y K cactorum AAB 

hybrid (=group II) R R Y K hedraiandra AAB 

hedraiandra A A C G hedraiandra BB 

It was hypothesized that isolates of group II possibly represented hybrids of P. cactorum and 

P. hedraiandra. Isozyme analysis was also in line with this hypothesis since the three banded 

MDHP pattern of isolates of group II was the result of the P. hedraiandra and the P. cactorum

MDHP alleles and P. hedraiandra and P. cactorum possessed the same MDH band, which 

explained the single band of group II isolates. In order to confirm the parenthood of the 

present hybrids we sequenced the maternally, inherited mitochondrial encoded cytochrome 

oxidase I (Cox I). Most hybrid strains appeared to have the Cox I gene of P. hedraiandra and 

in one strain Cox I of P. cactorum was found, confirming the parenthood of both presumed 

parents (Man in ’t Veld and others, 2007). Most hybrid strains were trisomic for the 

chromosome carrying the Mdhp locus. Two strains, having homozygous ITS sequences but 

containing isozyme alleles of both parents, were hypothesized to originate from intercrossings 

of the hybrids or from backcrossing or mitotic genome rearrangements. 

The present hybrids were discovered in the Netherlands, Belgium, Germany, Switserland and 

Slovenia altogether from six hosts (Allium cepa, Allium porrum, Idesia, Kalmia latifolia, 

Penstemon and Rhododendron). All hosts, except Rhododendron have not been reported as 

host of either parent. Since 1992 only the present hybrids were isolated from Rhododendron

indicating that the present hybrids are proliferating more successfully than P. cactorum on this 

host. 

Rhododendron is a common host for P. cactorum and P.hedraiandra. Two Phytophthora

species can not diverge in the same geographical area when they have a common host, which 

is in this case Rhododendron. Hence, since P. cactorum is well known in Europe for a long 

time, P. hedraiandra must have been introduced recently. In fact in the Netherlands the first 

P. hedraiandra strain was isolated in 2001. 

P. hedraiandra and P. ramorum are both considered to be recently introduced species from 

hitherto unknown origin, but they followed different strategics to survive. P. ramorum

proliferates predominantly clonally both in America and Europe presumably by means of its 

wind dispersed sporangia. In contrast, P. hedraiandra is hardly spreading; instead this species 

hybridized with the resident P. cactorum and the hybrids proliferated successfully, especially 

in the Netherlands (30 strains) and Belgium (40 strains). 
GENERAL TECHNICAL REPORT PSW-GTR-221

 60 

Literature Cited 

Man in 't Veld, W.A.; Veenbaas-Rijks, W.J.; Ilieva, E.; De Cock, A.W.A.M.; Bonants, 

P.J.M.; Pieters, R. 1998. Natural hybrids of Phytophthora nicotianae and Phytophthora 

cactorum demonstrated by isozyme analysis and random amplified polymorphic DNA. 

Phytopathology. 88: 922–929.

Man in 't Veld, W.A.; De Cock, A.W.A.M.; Summerbell, R.C. 2007. Natural hybrids of 

resident and introduced Phytophthora species proliferating on multiple new hosts. Eur J Plant 

Pathol. 117: 25–33.

Oudemans, P.; Coffey, M.D. 1991. Isozyme comparison within and among worldwide 

sources of three morphologically distinct species of Phytophthora. Mycol Res. 95: 19–30.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

61 

New Hypotheses on the Origin of 

the Interspecific Hybrid 

Oomycete Phytophthora alni1

Renaud Ioos,2,3 Axelle Andrieux,3 Benoît Marçais,3 

 and Pascal Frey3

Abstract 

An emergent disease of alder is caused by a complex of three taxa belonging to the genus 

Phytophthora (Oomycetes): P. alni subsp. alni (Paa), P. alni subsp. multiformis (Pam) and P. 

alni subsp. uniformis (Pau). Paa was previously hypothesized to be an allopolyploid hybrid 

between two genetically close species, P. cambivora and a taxon close to P. fragariae, 

whereas Pau and Pam were believed to be genetic breakdowns of Paa (Brasier and others 

1999). 

In order to examine these hypotheses, we studied the occurrence and the allelic distribution of 

four nuclear and two mitochondrial genes on a wide collection of P. alni and closely related 

species. Paa possesses three different alleles for each of the nuclear genes we studied, two of 

which are present in Pam as well, whereas the third matches the single allele present in Pau. 

Moreover, Paa displays common mtDNA patterns with both Pam and Pau. A combination of 

the data suggests that Paa may have been generated on several occasions by sexual 

hybridization between Pam and Pau, and that Pam may result itself of an ancient reticulation 

(Ioos and others 2006). 

In addition, we studied the expression of elicitin genes, a multigenic family specific to the 

genus Phytophthora. The cumulative patterns of Pau and Pam in regard with Paa confirmed 

our first results and demonstrated that for this allopolyploid taxa, distinct genomes may be coexpressed (Ioos and others 2007a). 

Last, in order to study the genetic variability of the different taxa, microsatellite markers were 

isolated in Paa and characterized (Ioos and others 2007b). These markers revealed a low 

genetic diversity in all three taxa. The cumulative microsatellite patterns observed in Paa

confirmed the hybridization scheme and confirmed that Paa was generated by several 

hybridization events. Genotypes resolved for Pam suggest that this taxon may also be 

allopolyploid. 

Altogether, our results clearly demonstrate that (i) P. cambivora and P. fragariae are not the 

progenitors of P. alni, and (ii) Paa was generated at several occasions by hybridization 

between Pam and Pau. This study raises new issues about the geographic origin, host range, 

ecology and aetiology of Pam and Pau. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Laboratoire National de la Protection des Végétaux, Unité de Mycologie Agricole et Forestière, Domaine de 

Pixérécourt, F54220 Malzéville, France. 3

 INRA, UMR IAM, Forest Pathology, F54280 Champenoux, France. 

Corresponding author:renaud.ioos@agriculture.gouv.fr. 
GENERAL TECHNICAL REPORT PSW-GTR-221

62 

Literature Cited 

Brasier, C.M.; Cooke, D.E.L.; Duncan, J.M. 1999. Origins of a new Phytophthora 

pathogen through interspecific hybridisation. Proceedings of the National Academy of 

Sciences of the USA 96: 5878–5883. 

Ioos, R.; Andrieux, A.; Marçais, B.; Frey, P. 2006. Genetic characterization of the natural 

hybrid species Phytophthora alni as inferred from nuclear and mitochondrial DNA analyses. 

Fungal Genetics and. Biology 43: 511-529. 

Ioos, R.; Panabières, F.; Industri, B.; Andrieux, A.; Frey, P. 2007a. Distribution and 

expression of elicitin genes in the interspecific hybrid homycete Phytophthora alni. Applied 

and Environmental Microbiology, 73(17):5587-5597. 

Ioos, R.; Barres, B.; Andrieux, A.; Frey, P. 2007b. Characterization of microsatellite 

markers in the interspecific hybrid Phytophthora alni ssp. alni, and cross-amplification with 

related taxa. Mol. Ecol. Notes 7, 133-137. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

63 

Population Structure of the Emerging Plant 

Pathogen Phytophthora ramorum on the 

West Coast of the United States1

S. Prospero,2 E.M. Hansen,3 N.J. Grünwald,3,4 

J. Britt3, and L.M. Winton5

Abstract 

Phytophthora ramorum is a devastating pathogen in native forests in California and 

southwestern Oregon and in nursery crops in California, Oregon and Washington. In this 

study we analyzed the population structure of P. ramorum in the west coast (CA, OR, and 

WA) of the United States by screening 579 isolates recovered from 2001 to 2005 at 10 

microsatellite loci. 

The overall P. ramorum population was composed of 73 multilocus genotypes. Based on 

allele patterns, 72 genotypes belonged to the North American clonal lineages NA1 or NA2 

(both A2 mating type) and one nursery genotype (OR and WA) belonged to the European 

clonal lineage (EU1, A1 mating type). Within the North American types, 71 genotypes 

clustered into the same clonal lineage (NA1), while one nursery genotype (CA and WA) 

belonged to NA2. None of the genotypes detected shared alleles of NA1 or NA2 and EU1, 

indicating that sexual reproduction is currently not occurring in the west coast population. 

Twenty out of 73 genotypes were present in more than one population. The lowest incidence 

of genotypes shared among populations was observed in the OR-forest population (7 out of 

30), which was dominated by a genotype not found elsewhere. Using assignment tests, a high 

percentage of the genotypes could not be assigned to the population from where they 

originated. The number of genotypes shared between two populations was not correlated with 

the minimal geographic distance between the two populations. The overall estimated FSTvalue among the four populations was 0.183 (95 percent confidence interval: 0-0.377). When 

using clone-corrected data sets, pairwise differentiation was significant only between the ORforest and the three other populations. Accordingly, genetic distances among OR-nursery, CA, 

and WA populations were very small. 

Our study demonstrates how the spread and the population structure of an invasive plant 

pathogen can be strongly affected by human activity. Accepting the hypothesis of a recent 

introduction of P. ramorum into western United States, the movement of infected nursery 

stock has probably played a major role in the rapid expansion of the geographical range of the 

pathogen over long distances. Thus, the current P. ramorum NA1 population is genetically 

similar from California to Washington. The dominance in southwestern Oregon of a genotype 

not found elsewhere may account for the significant differentiation between the OR-forest and 

the other three populations. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26 - 31, 2007, Monterey, California . 

2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA; Present address: 

INRA, UMR1202 Biodiversité Gènes et Communautés, Equipe de Pathologie Forestière, BP 81, 33883 Villenave 

d’Ornon Cedex, France.

3

 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA. 4

 USDA ARS, Horticultural Crops Research Laboratory, Corvallis, OR 97330, USA. 5

 USDA ARS, Subarctic Agricultural Research Unit, Fairbanks, AK 99775, USA. 

Corresponding author: sprosper@bordeaux.inra.fr. 
GENERAL TECHNICAL REPORT PSW-GTR-221

64 

The Decline of Austrocedrus Forests in 

Patagonia (Mal del Ciprés): Another 

Phytophthora-caused Forest Disease1

Alina G. Greslebin2 and Everett M. Hansen3

Abstract

Austrocedrus chilensis, an indigenous Cupressaceae of the Patagonian Andes forests, is 

suffering a disease that has been called “Mal del Ciprés” (MDC). This disease was first 

reported more than 50 years ago but, in spite of many studies, its causes remained unclear 

until recently. The disease begins in the root system, the distribution and pattern of spread of 

mortality in a stand is consistent with a soil-borne pathogen, and it is associated with 

seasonally poorly drained soils. Symptoms include defoliation, basal resinous exudates and 

red-brown necrotic lesions in the inner bark extending up the bole from killed roots. Brown 

cubic rots in roots and sapwood caused by wood-decomposer fungi are frequently- but not 

always- associated with dead or dying trees. These characteristics have led several workers to 

suggest that a Phytophthora species might be the causal agent of the disease. Several attempts 

to find a Phytophthora species responsible for the disease have been made. Five species were 

isolated from soil and/or associated streams: P. syringae, P. cambivora, P. gonapodyides and 

the undescribed taxa “Pgchlamydo” and “P. taxon raspberry” and another two species -P.

pseudotsugae and P. cactorum- were reported from soil and/or fine roots in a previous study. 

None of them showed a clear relationship with the disease. Isolations from the margins of the 

necrotic lesions in the inner bark using Phytophthora-selective media initially failed, but an 

ELISA test on necrotic phloem tissues was positive for Phytophthora, and subsequent DNA 

extraction from necrotic bark and amplification of ITS DNA using Phytophthora-specific 

primers was successful. Thus encouraged, isolation attempts were renewed and were finally 

successful. The isolated species was an undescribed taxon of Phytophthora that was formally 

named Phytophthora austrocedrae. It is homothallic with amphigynous antheridia and semipapillate sporangia, very slow growing with a maximum radial growth rate ranging from 1.0–

1.8 mm/day in V8A at optimal temperature (17.5C). ITS rDNA sequence places it near P. 

syringae in phylogenetic clade 8 of the genus. It was isolated from symptomatic trees in all 

localities affected by MDC throughout the range of the disease, showing that the pathogen is 

widely distributed. Pathogenicity tests fulfilled Koch’s postulates demonstrating it is the 

primary cause of the disease. This work presents the results of pathogenicity tests and their 

implications on the aetiology of the disease. A discussion of subjects that should be addressed 

in future work is also presented. 

Introduction

Austrocedrus chilensis (D. Don.) Pic. Serm. & Bizarri (Ciprés de la cordillera) is an 

endemic tree of the Cupressaceae of southern Argentina and Chile. Among the few 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Área de Protección Forestal, Centro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP), CC 14, 

9200, Esquel, Chubut, Argentina.

3 Department of Botany and Plant Pathology, Oregon State University, Cordley Hall 2082, Corvallis, OR 97331-

2902, USA. 

Corresponding author: agreslebin@ciefap.org.ar. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

65 

conifers inhabiting southern Argentina it has the largest distribution, covering ca. 

140,000 ha. (Bran and others 2002). It is a very valuable species because of it’s 

ecological role, the high quality of it’s wood and it’s scenic appeal. Throughout its 

range, A. chilensis suffers a lethal disease known as “Mal del Ciprés” (MDC). This 

disease was first detected 60 years ago and, since then, the affected area has been 

constantly increasing. Several studies on biotic and abiotic factors related to the 

disease have been done but the origin and causes of MDC remained unclear until 

recently. For a detailed description of the disease and background see Greslebin and 

others (2005). 

The symptomatology of the disease was traditionally described as a chlorosis, 

withering and subsequent progressive defoliation of the crown (Varsavsky and others 

1975, Havrylenko and others 1989, Rajchenberg and Cwielong 1993), and a decline 

of radial growth of the tree (Filip & Rosso 1999). The disease originates in the root 

system, where the death of the tissue precedes defoliation of the crown (Havrylenko 

and others 1989). Death of the roots is followed, many times, by the development of 

brown rots in the sapwood (Varsavsky and others 1975, Havrylenko and others 1989, 

Rajchenberg and Cwielong 1993, Barroetaveña & Rajchenberg 1996). Resin 

exudation has been pointed out either as related (Varsavsky and others 1975) or as 

unrelated (Havrylenko 1989) to the disease. The death of the tree may occur slowly, 

as a progressive defoliation, or rapidly, in which case foliage changes from chlorotic 

to red with little or no defoliation (Filip & Rosso 1999, Greslebin & Hansen 2006). 

Affected stands are usually associated with poorly drained sites (La Manna & 

Rajchenberg 2004a, b). The origin of the disease in the roots and its association with 

poorly drained sites suggested the action of a Pythiaceous organism (Pythiaceae, 

Oomycota). Several species of Phytophthora were isolated from streams and soils in 

affected stands (Greslebin and others 2005), but attempts to isolate from necrotic 

tissues were negative up to recently when a new Phytophthora species was detected 

on them (Greslebin and others 2007). 

The new species, Phytophthora austrocedrae, was the first organism consistently 

associated with cypress mortality suggesting it might be the primary cause of MDC. 

To confirm this hypothesis and fulfill Koch’s postulates, pathogenicity of P. 

austrocedrae on Austrocedrus chilensis was evaluated. P. syringae, the principal 

species recovered from soil near symptomatic trees, was also tested. This work 

presents the results of pathogenicity tests and their implications on the aetiology of 

the disease. 

Materials and Methods 

Isolation

Isolates of P. austrocedrae (Table 1) used for pathogenicity tests were obtained from 

the advancing necrotic zone of phloem lesions of symptomatic trees studied in 

previous work (Greslebin and others 2007). The isolate of P. syringae (Table 1) was 

recovered from soil in an Austrocedrus stand exhibiting symptoms of MDC. Isolates 

were identified by their morphological features and ITS sequences (Greslebin and 

others 2007). 
GENERAL TECHNICAL REPORT PSW-GTR-221

66 

Koch’s Postulates 

Pathogenicity tests were performed: 

a) In the field through stem and root inoculation of adult trees; and 

b) In the laboratory through stem inoculation of five-year-old seedlings; and 

c) Through soil infestation. 

Field root and stem inoculation 

Field inoculations were performed in an A. chilensis stand located at INTA (National 

Institute of Farming Research) Trevelin, where P. austrocedrae had been previously 

recorded. Ten trees showing no symptoms were selected for stem inoculation. 

Inoculations were made in 2 different seasons: summer (January 2005) and fall (May 

2005). Five trees were inoculated (stem and roots) in each season. Results were 

combined for analysis. 

Root inoculation 

Up to four superficial main roots of each tree were excavated and four treatments 

[T0: control; T1: P austrocedrae (strain CIEFAP Py 190); T2: P. austrocedrae (strain 

ATCC MYA-4074); T3: P. syringae (strain CIEFAP Py 5)] (Table 1) were randomly 

assigned. Two inoculations per root were made in those trees with less than 4 main 

roots. Cores (7 mm diam) of bark were aseptically removed using a borer. V8-agar 

discs from the edges of 15 day old cultures of P. austrocedrae and 7 day old cultures 

of P. syringae were placed in the holes and covered with the removed bark. A piece 

of sterilized, moist cheese-cloth was placed over each inoculation point, covered with 

aluminum foil, and sealed with adhesive tape. Controls received uninfested V8-agar 

discs. After inoculation roots were covered again with the removed soil. 

Stem inoculation 

Each tree received 4 inoculations corresponding to the following treatments: T0: 

control; T1: P austrocedrae (strain CIEFAP Py 190); T2: P. austrocedrae (strain 

ATCC MYA-4074); T3: P. syringae (strain CIEFAP Py 5) (Table 1). Inoculations 

were made on the sides of the tree facing each cardinal direction following the same 

procedure as in roots. Treatments were randomly assigned to each side of the tree. 

After 4 months bark was removed to expose the phloem, and the length and width of 

the lesion (necrotic phloem) was recorded. Re-isolation was attempted from the top 

and the bottom edges of the lesions and ELISA immunoassays to detect 

Phytophthora were performed on necrotic tissues associated with each treatment 

including controls. A Kruskal-Wallis Nonparametric Analysis of Variance was 

applied in order to detect significant differences between mean lesion size of 

treatments. Significant differences between mean lesion size of each treatment and 

control were tested through a Mann-Whitney test. Homogeneity of variances was 

tested using the Levene test. 

Stem inoculation of seedlings 

Thirty 5-year-old seedlings were selected for stem inoculation. Two strains of P. 

austrocedrae were tested (Table 1): CIEFAP Py 190 (T1) and CIEFAP Py 232 (T2). 

Treatments were randomly assigned to each seedling (ten seedlings for each 

treatment and ten controls). Cores (5 mm diam) of bark were aseptically removed 

using a borer. T-agar discs from the edges of 15-day-old cultures of P. austrocedrae

were placed in the holes and covered with the removed bark. A piece of sterilized, 

moist cheese-cloth was place over each inoculation, covered with aluminum foil and 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

67 

sealed with adhesive tape. Controls received uninfested T-agar discs. Lesion length 

and width in phloem were recorded after 2 months. Re-isolation was attempted from 

the edges of the lesions, and ELISA immunoassays to detect Phytophthora were 

performed on necrotic tissues associated with each treatment and controls. A 

Kruskal-Wallis Nonparametric Analysis of Variance was applied in order to detect 

significant differences between mean lesion sizes of treatments. Significant 

differences between mean lesion size of each treatment and control were tested 

through a Mann-Whitney test. Homogeneity of variances was tested using the Levene 

test. 

Soil infestation 

Inocula consisted of agar discs with sporangia. Agar discs were cut from the edges of 

15-day- old P. austrocedrae cultures (T1: strain CIEFAP Py 190, T2: strain CIEFAP 

Py 232) (Table 1) and placed in T-broth for 4 days. Then, agar blocks were rinsed 10 

times in distilled water to remove nutrients and placed in soil extract for 4 days. Pots 

with a 1:1 mix of tindalized soil and sterilized volcanic sand were used for planting 

the seedlings. Twenty pots were assigned to each treatment and the control. Two 

seedlings were planted in each pot and one agar block with sporangia was placed in 

the same hole where each seedling was planted. Pots were flooded for 48 hours every 

14 days. Controls received uninfested T-agar discs in the same hole where each 

seedling was planted. Mortality of seedlings was recorded weekly. 

Table 12Isolates used for pathogenicity tests 

Strain number Species Origin 

ATCC MYA-4074 

CIEFAP 203 

P. 

austrocedrae 

Argentina, Chubut, Los Alerces National 

Park, Braese stream, isolated from necrotic 

inner bark of A. chilensis, Oct. 2005 

CIEFAP 190 P. 

austrocedrae 

Argentina, Chubut, Futaleufú, Río Grande 

Valley, “La 106” Ranch, isolated from 

necrotic inner bark of A. chilensis, Set. 2005 

CIEFAP 232 P. 

austrocedrae 

Argentina, Chubut, Los Alerces National 

Park, near to Los Pumas stream, isolated 

from necrotic inner bark of A. chilensis, Oct. 

2005 

CIEFAP 5 P. syringae Argentina, Chubut, Futaleufú, Río Grande 

Valley, “Los cerezos” Ranch, isolated from 

soil of A. chilensis stand. June 2001 

Results 

Pathogenicity of Phytophthora austrocedrae on 

Austrocedrus chilensis

Root and stem inoculations of adult trees 

Since Levene test showed unequal variances (P-value < 0.001) for lesion size in both 

roots and stems, non parametric analyses were applied. All roots inoculated with 

Phytophthora austrocedrae developed brown, necrotic lesions that affected the 

phloem reaching and staining the sapwood (fig 1). Lesions developed after P. 

austrocedrae inoculations (both T1 and T2) were significantly longer and wider (one-
GENERAL TECHNICAL REPORT PSW-GTR-221

68 

Stem

Control

P. austrocedrae (T1)

P. austrocedrae (T2)

P. syringae

0

5

10

15

20

25

M ean lesion length (cm )

Roots

Control

P. austrocedrae (T1)

P. austrocedrae (T2)

P. syringae

0

5

10

15

20

25

M ean lesion length (cm )

Roots

Control

P. austrocedrae (T1)

P. austrocedrae (T2)

P. syringae

0

2

4

6

8

10

M ean lesion w idth (cm )

Stem

Control

P. austrocedrae (T1)

P. austrocedrae (T2)P. syringae

0

2

4

6

8

10

M ean lesion w idth (cm )

Figure 2—Mean length and width of necrosis (cm) produced by P. 

austrocedrae and P. syringae on roots and stems of A. chilensis adult 

trees. 

sided P-value < 0.0001) than those in the Controls (fig. 2). Mean lesion length and 

width of P. syringae lesions did not differ from those of the controls (fig. 1). 

Stem inoculations with P. austrocedrae developed lesions similar to those observed 

in root inoculations. Two exceptions were found: one tree developed small and 

superficial lesions and another did not develop lesions in the stem but it did in the 

roots. Lesions developed after P. austrocedrae inoculations (both T1 and T2) were 

significantly longer (one-sided P-value < 0.0001) and wider (T1: one-sided P-value < 

0.0001, T2: one-sided P-value = 0.003) than those in Control inoculations (fig. 2). 

Mean lesion length and width of P. syringae inoculations did not differ from Control 

lesions (fig. 2). 

Mean lesion length and width of T1 treatments were greater than mean lesion length 

and width of T2 treatments in both root and stem inoculation but these differences 

were not significant except for mean lesion width of stem inoculations (one-sided Pvalue = 0.02). 

Figure 12Lesion caused by Phytophthora austocedrae on a root on an 

adult tree of Austrocedrus chilensis. Left: bark removed showing necrotic 

 phloem. Right: phloem removed showing sapwood superficially affected.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

69 

Trees showed no external foliar symptoms at the end of the study. A few trees did 

exhibitresin exudation from the inoculation point. 

Phytophthora austrocedrae was re-isolated from 78 percent of the lesions and ELISA 

tests were positive from 100 percent of them. Phytophthora syringae was not 

reisolated from inoculation points, and ELISA tests were negative for both P 

syringae and control treatments. 

Stem inoculation on five year old seedlings 

After two months all seedlings inoculated with Phytophthora austrocedrae developed 

brown, necrotic lesions that affected the phloem, reaching and staining the sapwood. 

A few seedlings showed external symptoms (i.e. dark red foliage) (fig. 3) but most 

showed no symptoms even though they developed lesions similar to those of 

symptomatic seedlings. Since Levene test showed unequal variances (P-value < 

0.002) non parametric analyses were applied. Lesions of both treatments were 

significantly longer (one-sided P-value < 0.0001) than those in Controls (Fig. 4). 

Phytophthora austrocedrae was successfully re-isolated twice (from one plant of 

each treatment) the other re-isolation attempts failed, being over-grown by 

Deuteromycetes. ELISA tests were positive for 100 percent of the lesions and 

negative for the Controls. Mean lesion length of T2 was greater than T1 one (fig. 4) 

but the difference was not significant (one-side P-value = 0.11) 

Figure 32Five year old seedlings inoculated with Phytophthora austrocedrae after 

two month of inoculation. A) Symptomatic seedling and it respective lesion. B) 

Asymptomatic seedling and it respective lesion. 

A B
GENERAL TECHNICAL REPORT PSW-GTR-221

70 

Five year old seedlings

Control

P. austrocedrae (T1)

P. austrocedrae (T2)

0

5

10

15

20

M ean lesion length (cm )

Soil Infestation 

In P. austrocedrae treatments (T1 and T2) 95 percent of the seedlings were dead after 

the second flooding (20 days of treatment). Mortality was mostly observed after each 

flooding (about 40 percent of the seedlings after the first flooding and about 55 

percent after the second one). Roots of dead seedlings were brown or were severely 

rotted. Only two control seedlings were dead at the end of the study (one month) and 

they died the first week of treatment. 

Isolations from rotted roots of dead seedlings were overgrown by Mortierella spp. 

and P. austrocedrae could not be recovered, but ELISA tests were positive. ELISA 

tests from the two dead seedlings found in the Controls were negative. 

Discussion 

The experimental evidence confirms that Phytophthora austrocedrae is the causal 

agent of MDC. The pathogenicity tests indicate that P. austrocedrae is an aggressive 

pathogen of Austrocedrus chilensis. Adult trees and seedlings were susceptible to 

infection and lesions developed quickly (up to 4.7 cm month-1 in adult trees and up to

11.5 cm month-1 in seedlings). A root pathogen has been frequently suggested as 

cause of MDC (Havrylenko and others 1989, Rosso and others 1994, Rajchenberg 

and others 1998, Filip & Rosso 1999), but a specific organism had not been 

implicated. P. syringae has been isolated from soil and streams in diseased stands, 

but never from trees (Greslebin and others 2005), and was not pathogenic in stem or 

root inoculations in these tests. 

The apparent aggressiveness of P. austrocedrae was unexpected. MDC has 

traditionally been described as a slow decline, with progressive defoliation 

(Rajchenberg and others 1998), where death of individual trees may take several 

Figure 42Mean length of necrosis (cm) 

produced by P. austrocedrae on stems of A. 

chilensis seedlings.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 71 

decades (Calí 1996, Filip & Rosso 1999). It is not unusual to find apparently healthy 

trees very close to dead or dying ones. This also disagrees with an aggressive 

pathogen. The questions are: why do some trees die quickly while others die decades 

after they begin to decline, and why do some trees remain unaffected even when they 

are next to dying trees? We have observed that some lesions in the stem become 

inactive and trees begin to wall off old lesions with callus tissues (fig. 5). Whether 

the lesions become inactive because of tree defenses or because of ambient 

conditions unfavorable for the pathogen is an issue that remains unclear and should 

be considered in future research. P. austrocedrae growth in vitro is affected by high 

temperatures (Greslebin and others 2007), showing no growth at 25 C. Maximum air 

temperature in summer in areas where Austrocedrus grows can reach 35 C 

(exceptionally more). High summer temperatures could be responsible for the 

inactivation of the lesions on some trees. The effect of ambient temperatures on 

lesion development as well as the variation of A. chilensis’ resistance/susceptibility to 

P. austrocedrae should be evaluated in future work. 

MCD symptomatology is consistent with a Phytophthora disease but similar crown 

symptoms may result from other causes. We propose that the disease name Mal del 

Ciprés be reserved for the disease of Austrocedrus caused by Phytophthora 

austrocedrae characterized by root infection and necrotic lesions extending up the 

bole. This pathogen has now been isolated from dying A. chilensis trees in Isla 

Victoria (Nahuel Huapi National Park) the place where MDC was first reported, and 

in most of the other areas throughout the tree’s range that have been affected by 

MDC (Greslebin and others 2007). 

Another very important issue that should be resolved is the geographic origin of P. 

austrocedrae. Is it a native species or an introduced pathogen? The area where the 

disease was first observed (Isla Victoria) was planted early in the 20th century with 

exotic tree species from many places in the world and a nursery was built to provide 

seedlings to many other places in Patagonia. P. austrocedrae could be an exotic 

pathogen that was introduced in Isla Victoria and spread from this point. On the other 

hand, today it is a widely distributed species present throughout the cypress 

Old, inactive lesion

Figure 5—Callus tissues 

walling off an old, inactive 

lesion. 
GENERAL TECHNICAL REPORT PSW-GTR-221

72 

distributional range, and its closest genetic relative is Phytophthora syringae, another 

species that is also present in A. chilensis forests. Perhaps it is a native species that 

acts as a pathogen under favorable site conditions including poor soil drainage (La 

Manna & Rajchenberg 2004a,b) or has increased it activity because of the climate 

change. To elucidate the origin of P. austrocedrae as well as P. syringae will be the 

objective of our future work. 

Acknowledgments 

We want to thank Dr. Charles G. Shaw III and Dr. Paul Hennon, USDA Forest 

Service, Alaska Region, Forest Health Protection and ICSU-TWAS-UNESCOUNU/IAS Visiting Scientist Programme for making possible this collaborative work 

by supporting AGG and EMH visits to OSU and to CIEFAP respectively. Dr. Carlos 

Buduba, Instituto Nacional de Tecnología Agropecuaria (National Institute of 

Farming Technology) INTA of Argentina, is warmly acknowledged for kindly 

permits the realization of pathogenicity tests in Cypress stands of INTA Trevelin. 

This work was supported by Agencia Nacional de Promoción Científica y Técnica 

(ANPCyT, FONCyT) of Argentina (PICT 1-13305) and National Research Council 

(CONICET) of Argentina (PIP 5660). AGG is researcher for CONICET. 

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Barroetaveña, C.; Rajchenberg, M. 1996. Hongos Aphyllophorales (Basidiomycetes) que 

causan pudriciones en Austrocedrus chilensis en pie. Bol Soc Argen Bot 31: 201-216. 

Bran, D.; Pérez, A.; Barrios, D.; Pastorino, M. & Ayesa, J. 2002. Eco-región valdiviana: 

distribución actual de los bosques de “ciprés de la cordillera” (Austrocedrus chilensis) - 

Escala 1:250.000. INTA, APN, FVSA. Informe preliminar, Bariloche, Argentina. 12 p. 

Calí, S.G. 1996. Austrocedrus chilensis: estudio de los anillos de crecimiento y su relación 

con la dinámica del “Mal del Ciprés”en el P. N. Nahuel Huapi, Argentina. Tesis de 

Licenciatura en Cs. Biológicas, Universidad Nacional del Comahue, Bariloche. 143 p. 

Filip, G.M.; Rosso, P.H. 1999. Cypress mortality (mal del ciprés) in the Patagonian Andes: 

comparison with similar forest diseases and declines in North America. European Journal of 

Forest Pathology 29: 89-96. 

Greslebin, A.G.; Hansen, E.M. 2006. Novedades sobre el “mal del ciprés”. Patagonia 

Forestal 12(1): 11-14. 

Greslebin, A.G.; Hansen, E.M.; Winton, L. Rajchenberg, M. 2005. Phytophthora species 

from declining Austrocedrus chilensis forests in Patagonia, Argentina. Mycologia 97: 218-

228. 

Greslebin, A.G.; Hansen, E.M.; Sutton, W. 2007. Phytophthora austrocedrae sp. nov., a 

new species associated with Austrocedrus chilensis mortality in Patagonia (Argentina). 

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Havrylenko, M.; Rosso, P.H.; Fontela, S.B. 1989. Austrocedrus chilensis. Contribución al 

estudio de su mortalidad en Argentina. Bosque 10: 29-36. 

La Manna, L.; Rajchenberg, M. 2004a. Soil properties and Austrocedrus chilensis forest 

decline in Central Patagonia, Argentina. Plant Soil 263: 29-41. 
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La Manna, L.; Rajchenberg, M. 2004b. The decline of Austrocedrus chilensis forests in 

Patagonia, Argentina: soil features as predisposing factors. Forest Ecol Manag 190: 345-357. 

Rajchenberg, M.; Cwielong, P.P. 1993. El Mal del Ciprés (A. chilensis): su relación con las 

pudriciones radiculares y el sitio. Actas del Congreso Forestal Argentino y Latinoamericano, 

Paraná, Entre Ríos. 

Rajchenberg, M.; Barroetaveña, C.; Cwielong, P.P.; Rossini, M.; Cabral, D.; Sívori, A. 

1998. Preliminary Survey of Fungi Associated with the decline of Ciprés in Patagonia. In 

Delatour C, Guillaumin JJ, Lung-Escarmant B, Marçais B (eds.). Proceedings of the 9th 

International Conference on Root and Butt-Rots (Carcans, France), 1-7 Sept., 1997. INRA, 

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GENERAL TECHNICAL REPORT PSW-GTR-221 

74 

Kauri (Agathis australis) Under 

Threat From Phytophthora?1

Ross E. Beever,2 Nick W. Waipara,2 Tod D. Ramsfield,3 

Margaret A. Dick,3

 and Ian J. Horner4

Abstract 

Five species of Phytophthora have been recorded from Agathis australis (kauri) or soil 

in kauri forests: P. cinnamomi, P. cryptogea, P. kernoviae, P. nicotianae and 

Phytophthora taxon Agathis (PTA) initially recorded as P. heveae. P. cinnamomi has 

been found widely in natural stands and has been linked with ill-thrift and occasional 

tree death, especially in regenerating stands on poorly drained sites. PTA, which is 

known from fewer natural stands, is associated with a collar rot, causing large 

bleeding lesions near the ground, yellowing foliage, and tree death. The other three 

species have only been reported once. ITS sequence studies of PTA show it belongs 

with, but is distinct from, P. heveae in ITS clade 5. Cultural and molecular studies 

indicate a close relationship with P. katsurae. It is proposed that PTA may be 

introduced to New Zealand, but too few isolates are available to determine whether 

genetic variability of this species provides support for this hypothesis. Recent surveys 

have found collar rot is widely distributed across the natural range of kauri. Typically, 

affected stands are relatively small in size. Size class distributions indicate trees of 

many ages are affected and a disease front can sometimes be detected. Pathogenicity 

tests show PTA is highly pathogenic to kauri. We propose that collar rot caused by 

PTA is an emerging disease caused by an introduced pathogen that is spreading slowly 

from widespread disease foci. It poses a threat to kauri, both at the individual icon 

level and at the population level, with flow-on effects to kauri ecosystems. 

Introduction 

Kauri (Agathis australis), a conifer in the Araucariaceae, is a dominant tree of 

lowland stands in northern New Zealand, frequently occurring in a mosaic of mixed 

forests of diverse composition (Ecroyd 1982, Ogden and Stewart 1995, Wardle 

1991). Following excessive exploitation for timber during the 19th and early 20th

centuries, it is today virtually restricted to relatively small reserves. Giant individual 

trees, which can reach over 4.5m in trunk diameter and exceed 1000 years age, are 

accorded special status by Maori, Pakeha and tourists. Tane Mahuta (‘God of the 

Forest’) and Te Matua Ngahere (‘Father of the Forest’) are major tourist attractions in 

Waipoua Forest, the largest remaining kauri stand in the country. Kauri seeds are 

winged allowing wind dispersal (Ecroyd 1982). Vegetation patterns indicate kauri 

regeneration occurs following disturbance, which can range from large-scale wind 

and fire events to small-scale canopy gaps resulting from fall of giant trees (Ogden 

and Stewart 1995). Many present day stands result from human-induced fires 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Landcare Research, Private Bag 92170, Auckland 1044, New Zealand. 

3

 Scion, Private Bag 3020, Rotorua, New Zealand. 

4

 HortResearch, Private Bag 1401, Havelock North, New Zealand. 

Corresponding author: BeeverR@landcareresearch.co.nz. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

75 

associated with European settlement from the mid-1800s to the mid-1900s. These 

stands are often seral in character, with kauri establishing in a shrubland ‘nursery’ 

dominated by the myrtacean tea trees, Leptospermum scoparium and Kunzea 

ericoides (Ogden and others 1987). 

Five species of Phytophthora have been recorded from kauri and/ or kauri 

ecosystems (Gadgil 2005, McKenzie and others 2002, Ramsfield and others 2008): 

P. cinnamomi (var. cinnamomi), P. cryptogea, P. kernoviae, P. nicotianae, and a 

species we refer to here by the tag name Phytophthora taxon Agathis (PTA). Erwin 

and Ribeiro (1996) in error cite Gadgil (1974) as reporting P. hibernalis from A. 

australis. He does not, nor does Gadgil (2005) list P. hibernalis from this host. 

P. cinnamomi is found widely in natural stands and has been linked with ill-thrift and 

occasional tree death, especially in regenerating stands on poorly drained sites 

(Podger and Newhook 1971). Symptoms included severe chlorosis, canopy thinning 

and dieback, and sometimes tree death. Phytophthora taxon Agathis (PTA) was first 

reported from a natural stand of unhealthy kauri on Great Barrier Island (fig. 1), a 

small island off the northern New Zealand coast (Gadgil 1974). Symptoms included 

yellowing of foliage, canopy thinning and occasional tree death. Additonally, 

affected trees frequently had lesions on the lower trunk and main roots. These lesions 

sometimes encircled the stem and were bleeding copious amounts of resin (kauri 

gum). The affected trees occupied c. 1.5 ha in a block of forest regenerating after 

disturbance associated with forestry operations in 1931–32 and trees up to 30 cm 

diameter at breast height (DBH) were affected. A homothallic Phytophthora was 

isolated from the lesions and from soil samples collected in the vicinity of affected 

trees and determined, albeit with some reluctance, as P. heveae on the advice of IMI 

staff. Pathogenicity tests on kauri seedlings using both wound and soil inoculation 

methods showed it was highly pathogenic. 

Of the species associated with kauri, P. cinnamomi, P. cryptogea, and P. nicotianae

were likely introduced to New Zealand following human contact about 600 years 

ago. Whether or not PTA, and P. kernoviae (Ramsfield and others, these 

proceedings), are indigenous or have been introduced is problematic. Recent 

observations indicate PTA is more widespread than previously realised and may pose 

a significant risk both to kauri ecosystems and to individual iconic trees. This paper 

summarises these studies and reviews them in relation to existing knowledge of the 

associations of Phytophthora species with kauri. 

Methods 

Culturing and Morphological Studies 

Isolations from lesion margins were made to common lab media including P5ARP 

and from soil using lupin baits and apple baiting (Erwin and Ribeiro 1994). The 

strains used are listed in Table 1. ICMP isolates are maintained long term in liquid 

nitrogen storage. Cultures were grown (18–200

C in the dark) on V-8 juice agar for 

morphological studies, and PDA for DNA sequencing studies. Gametangial images 

were taken by interference light microscopy using a 40x objective. 
GENERAL TECHNICAL REPORT PSW-GTR-221

76 

Tree Health Studies 

Health status in relation to tree diameter was examined in a highly affected stand of 

about 1.4 ha at Huia, Waitakere Ranges. All trees were examined in a 20m x 20m 

plot in the middle of the stand, as well as along two transects extending at right 

angles from the plot to the disease margin of the affected stand. DBH was measured 

with a diameter tape and the health status assessed as: healthy, living but with 

bleeding lesions, or dead (often also with evidence of old oozing gum indicating past 

lesion presence). 

Phylogenetic Analysis 

DNA was extracted from mycelium using a QIAGEN DNeasy Plant Mini kit 

(Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The internal 

transcribed spacer (ITS) region of the ribosomal DNA repeat was PCR amplified 

using the primers ITS-4 and ITS-6 following the PCR protocol of Cooke and others 

(2000). 

Table 1—List of clade 5 isolates used in this study, including those for which 

GenBank sequences were downloaded 

Species* ICMP** GenBank Other 

collection 

numbers*** 

Country 

of 

isolation 

Host Substrate 

heveae

(isotype 

strain)

- AF266770 IMI180616 

= CBS296.29 

Malaysia Hevea 

brasiliensis 

Fruit 

heveae 16914 - SCRP361 

= IMI131093 

Malaysia Theobroma 

cacao 

- 

heveae 16691 - DAR27023 Australia - soil under 

Eucalyptus

katsurae - AF266771 IMI360596 Ivory 

Coast 

Cocos 

nucifera 

- 

katsurae 16948 - Uchida 

H1024 

Hawaii Cocos 

nucifera 

Fruit 

katsurae 16950 - Uchida 

H1027 

Hawaii Cocos 

nucifera 

Fruit 

katsurae 16951 - Uchida 

H1031-1 

Hawaii Cocos 

nucifera 

Fruit 

katsurae 16915 - SCRP388 

= Panabieres 

F441 = 

CBS587.85 

= 

ATCC36818 

= IMI325914 

Taiwan - soil 

taxon 

Agathis 

(PTA) 

16471 EF067922 ATCC32256 

= FRI135 

New 

Zealand 

Agathis 

australis 

taxon 

Agathis 

(PTA) 

17021 - Beever 

REB316-1 

New 

Zealand 

Agathis 

australis 

Bleeding 

lesion 

* names as used in this study, but see text for further discussion. 

** International Collection of Microorganisms from Plants, maintained by Landcare 

Research. 

*** the first number quoted is the immediate source of the ICMP isolate. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

77 

Following PCR, the PCR products were column purified and sequenced in both 

directions with ITS-4 and ITS-6. Phytophthora katsurae, P. heveae and P. citricola

ITS sequences were retrieved from GenBank to represent authentic isolates of the 

species that were being analysed. The DNA sequence data were trimmed to (5’-

CCACACC....TCTCAA-3’) to anchor the alignment and the sequences were aligned 

with Clustal X (Thompson and others 1997). Gap opening and gap extension 

parameters were adjusted to achieve the best visual alignment. The aligned sequences 

were then imported into Mega 4 (Tamura and others 2007) and a Neighbour-Joining 

analysis, using the Kimura 2-parameter model was used to construct a phylogeny, 

which was rooted with P. citricola. 

Results 

Field Observations 

PTA was found in 2006 in a stand of regenerating kauri on the Maungaroa Ridge in 

the Waitakere Range near Auckland city, following an enquiry from the public about 

unusual kauri deaths. As at the Great Barrier site, affected trees showed foliage 

yellowing, canopy thinning and bleeding lower trunk lesions sometimes girdling the 

trunk and extending to the major roots. PTA was isolated from the margin of 

bleeding lesions. A distinct disease front could be identified along some parts of the 

stand. These observations are very similar to those of Gadgil (1974), who in addition 

demonstrated rapid death of saplings following wound inoculation on the lower stem. 

We conclude that PTA causes the trunk and root lesions and we designate the disease 

caused by PTA as kauri collar rot. As such, this designation does not exclude the 

possibility that more field work may link kauri collar rot with other species of 

Phytophthora in the future. Kauri collar rot has been observed in two other stands of 

regenerating trees in the Waitakere Range and in addition, large relatively isolated 

trees in mature forest have been observed with collar rot symptoms. For example, in 

the Cascades region of the Waitakere Range, which has not been modified by past 

logging, large mature trees with partly dead stag-like canopies and a sector of their 

trunks dead occur sporadically. Examination of some such trees shows extensive 

gummosis and lesion development at the margins of the dead tissue at base of the 

trunk, which we interpret as collar rot infection established many years earlier and 

gradually ring-barking the tree. On the other hand, other trees showing putative collar 

rot symptoms show no obvious leaf or canopy symptoms, and in some instances, it 

appears as if the putative lesion has been walled off as only old gummosis is 

apparent. No attempts have yet been made to isolate Phytophthora from such mature 

trees. 

Detailed examination of trees at a regenerating site near Huia in the Waitakere Range 

where symptoms are abundant indicates that trees of all size classes are affected (fig 

1). PTA was obtained from both trunk lesions and soil apple baiting at this site. The 

vegetation at this site includes senescent trees of Kunzea ericoides and is interpreted 

as a seral successional site following timber extraction and fire perhaps >100 years 

ago (Ogden 1983). The death of kauri at this site is likely to lead to a shift towards 

dominance by podocarp tree species such as Dacrydium cupressinum (rimu), which 

is present at the site but appears unaffected. 

 
78

GENERAL TECHNICAL REPORT PSW-GTR-221

Figure 1—Size class distribution of Agathis australis (kauri) in a collar rot-affected forest 

stand at Huia, Waitakere Range. In total, 156 trees were scored. Health status was 

assessed as: healthy, showing gummosis (lesions oozing gum) or dead. 

Collar rot symptoms have also been observed in Waipoua Forest and nearby Trounson 

Kauri Park, and PTA was recovered by soil baiting at Trounson (M. Braithwaite pers. 

comm. 2007). However, no symptoms were observed in two brief surveys in forests in 

the Coromandel Peninsula (fig 2). 

The Great Barrier site described by Gadgil (1974) was revisited in 2006 by one of us 

(REB). Affected trees covered an area of c.10ha, representing a 5~10-fold increase since 

1972. This corresponds to a rate of spread of c.3m per year on the assumption of circular 

areas of infection, a rate comparable to that of P. cinnamomi spread in southwestern 

Australia (Strelein and others 2006). An ill-defined disease front could be identified 

from a distance. PTA was recovered from a bleeding lesion (ICMP 17027).

Based on symptomatology, the occurrence of PTA in soil samples, the estimated rate 

of spread at the Great Barrier site, and the presence of distinct disease fronts at some 

localities, we propose that PTA is a soil-borne species that spreads predominantly 

through soil and soil water movement. 
GENERAL TECHNICAL REPORT PSW-GTR-221 

79 

Figure 2—Map showing location of New Zealand in the Pacific and northern 

New Zealand showing main locations mentioned in text. Trounson Park is 

close to Waipoua Forest. The approximate southern limit of kauri is shown by 

the dotted line. 

Taxonomic Studies 

The recent revolution in Phytopthora taxonomy warranted a re-examination of the 

nature of PTA. Recent isolates from the Waitakere Ranges, from Gt Barrier Island 

and from Trounson were compared with an isolate from the 1970s study (Gadgil 

1975) that had been stored by ATCC (Table 1). All isolates were culturally and 

morphologically similar, being homothallic with large relatively smooth to slightly 

rugose oogonia resembling those of P. heveae (fig. 3). All isolates had identical ITS 

sequences and phylogenetic analysis places them close to, but distinct from, P. 

heveae in the ITS clade 5 of Cooke and others (2000) (fig 4). PTA is closer in ITS 

sequence to isolates identified as P. katsurae, being identical to one isolated from soil 

in Taiwan (ICMP16915) and differing by 3 base pairs (substitutions) from isolates 

from Cocos (AF266771, ICMP 16948, ICMP 16950, ICMP 16951). 

Figure3—Gametangia of selected Phytophthora isolates from clade 5 of Cooke 

and others (2000). Cultures were grown for 19 d in the dark on V8 agar. (A) 

PTA (ICMP 17021), (B) P. heveae (ICMP 16914), (C) P. katsurae (ICMP 

16950), (D) P. katsurae (ICMP 16915). 
GENERAL TECHNICAL REPORT PSW-GTR-221

80 

Figure 4—Neighbour joining phylogenetic tree based on ITS sequences of 

selected Phytophthora strains obtained in this study. Representative 

GenBank sequences used by Cooke and others (2000) for clade 5 (AF26671 

for P. katsurae, AF26670 for P. heveae) are included along with AF266788 

(P. citricola) as an outgroup. 

Discussion 

Despite PTA’s high pathogenicity relative to P. cinnamomi, Gadgil (1974) did not 

conclude it posed a particular threat to kauri, at least in part because it was known 

only from one relatively isolated island site. This assessment must be reviewed in the 

light of our findings of its more widespread occurrence on the mainland including the 

Waipoua region. We consider it unlikely that these new occurrences represent recent 

translocations from Gt Barrier Island. Rather we suspect it has been present, but 

undetected, at these sites for many years. Our rationale is that while the bleeding 

lesion symptoms are relatively obvious, kauri responds to any injury by producing 

resin and indeed this feature has been used in the past to promote commercial gum 

production by purposely injuring the trees. Thus examples of collar rot-affected trees 

are likely to be overlooked by the casual observer. More work is needed to better 

clarify the relationships between PTA presence, lesion production and rates of 

spread. In this regard, we note that Gadgil (1974) reported PTA from soil under 

apparently healthy trees on Gt Barrier some distance from the collar rot-affected site, 

and concluded that the fungus was widespread and that some environmental factor 

was involved in triggering disease expression. This site has not been revisited to 

observe whether or not disease was expressed subsequently. Nevertheless, we 

conclude there is sufficient a priori evidence to propose that PTA poses a threat to 

stands of regenerating kauri, where its effect may be to change the composition of the 

forest to one dominated by podocarps. Additionally, we propose that PTA poses a 

threat to large iconic kauri, where infection may lead to premature death, albeit over 

a period of many years. 

Three of the species associated with kauri have only been recorded once. 

Phytophthora cryptogea was reported from dying kauri seedlings in a nursery in the 

1950s (Newhook 1959) and there is no reason to suspect this was more than an 

opportunistic infection associated with poor nursery hygiene. Similarly, P. kernoviae
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

81 

was recovered only once, from soil beneath a large recently dead kauri at Trounson 

Kauri Park during a survey of northern kauri forests (see below). 

However, little significance can be attached to this finding in relation to kauri health, 

especially as P. kernoviae has now been found, also in soil, at a number of other 

locations encompassing the natural range of kauri (Ramsfield and others 2008). 

Phytophthora nicotianae has been reported once from a bleeding lesion on a young 

tree (Brien and Dingley 1959). No collections or isolates are available to confirm this 

identification, and we conclude this species plays no more than a minor role in kauri 

health.

In contrast, P. cinnamomi, is widespread both in mature and regenerating kauri 

forests (Newhook and Podger 1972, Podger and Newhook 1971). Furthermore, P. 

cinnamomi has also been associated with seedling mortality in nursery beds 

(Newhook 1959), and pathogenicity tests indicate kauri seedlings and sapling are 

indeed susceptible to P. cinnamomi (Horner 1984, Johnston and others 2003, Podger 

and Newhook 1971). Death of trees associated with unnatural disturbance such as 

track and road building has sometimes been attributed to P. cinnamomi infection. 

Nevertheless, in natural communities, P. cinnamomi has seldom been associated with 

tree ill-health. For example, in May 2003 Beever and Horner (unpublished) surveyed 

Phytophthora associated with adult kauri in Waipoua Forest and the nearby Trounson 

Park using lupin-baited soil sampling. P. cinnamomi was recovered frequently at both 

Waipoua (47 percent of 36 samples) and Trounson (25 percent of 25 samples). 

However, no association was detected between recovery of P. cinnamomi and tree 

health, with P. cinnamomi recovered in similar numbers under both healthy and nonthrifty trees. PTA was not recovered during this survey. 

However, Podger and Newhook (1971) do implicate P. cinnamomi in a disease 

outbreak in natural stands in the Waitakere Range where kauri was showing 

symptoms similar to those of littleleaf disease of Pinus echinata in the southern US. 

These symptoms included severe chlorosis, canopy thinning and dieback, and 

sometimes death. Affected trees were observed in two small areas of dense stands of 

regenerating forest. P. cinnamomi was isolated from kauri roots as well as the roots 

of many associates in the stands. The authors attributed the disease to severe fine root 

damage caused by P. cinnamomi associated with abnormally heavy summer rainfall 

and unusual waterlogging of the soil at the affected sites. While Podger and 

Newhook (1971) point out that stands of naturally regenerating kauri are common 

and raise the possibility that P. cinnamomi-associated mortality might increase in 

such stands in the future, casual observation suggests this has not occurred to date. 

We (Horner and Beever) revisited the primary site studied by Podger and Newhook 

(1971) in August 2006 and, while rotting fallen kauri trunks were observed, the 

remaining trees look in good health. We conclude that P. cinnamomi probably plays a 

minor role in health of adult kauri, perhaps as part of a root disease complex that 

under abnormal conditions at some sites leads to disease. Nevertheless, P. 

cinnamomi may be playing a more subtle role in forest dynamics through pre- and 

post-emergence damping off of kauri seedlings (Johnston and others 2003). 

Experimental treatment with metalaxyl in regenerating forest plots suppressed P. 

cinnamomi root rot in kauri seedlings, and resulted in a substantial increase in 

seedling growth (Horner 1984). 
GENERAL TECHNICAL REPORT PSW-GTR-221

82 

Phytophthora katsurae was described from Japan (as P. castanea), where it was 

associated with lower trunk lesions of chestnut (Katsura 1976). Subsequently it was 

recorded from natural forest soil in Taiwan and renamed (Ko and Chang 1979). 

Based on cultural and morphological characters, including strongly bullate oogonia, 

Ko and Chang (1979) concluded the Taiwan soil fungus (ATCC 36818 = 

ICMP16915) was identical with a ‘type’ culture of P. katsurae obtained from K. 

Katsura, which they also examined. ICMP16915 shows these strongly bullate 

oogonia (fig. 3). P. katsurae coconut isolates have moderately bullate oospores (fig. 

3, Ko and others 2006). Recently, Oh and others (2007) report a chestnut trunk lesion 

pathogen from Korea resembling the type of P. katsurae in its strongly bullate spores 

and pathogenicity to chestnut and with ITS sequence identical to both PTA as well a 

chestnut isolate (C. Hong 22H6) from Japan (E. Oh pers. comm. 2008, these 

proceedings). Isolate C. Hong 22H6 is a derivative of P. Tsao P990, which was 

received from Japan in 1970 as #031 (M. Gallegly, C Hong pers. comm. 2008) and is 

linked by the latter number to the holotype (Kyoto Herbarium no. 1971-031), and is 

thus very likely an ex type culture. The Korean chestnut pathogen is thus reasonably 

determined as P. katsurae. However, despite PTA having an identical ITS sequence, 

we suspect PTA is not this species based on its smooth to slightly rugose oogonial 

ornamentation (fig. 3) and different cardinal temperatures (Horner unpublished). We 

propose that PTA may be introduced to New Zealand, in view of its relatively recent 

first report (Gadgil 1974) coupled with its distinctive symptoms on kauri, and the 

cultural similarity of isolates recovered to date along with their identical ITS 

sequences. However, we have not located reports of isolates matching PTA in both 

spore ornamentation and ITS sequence from other parts of the world. Whether or not 

it is introduced, we suggest it should be treated as introduced for management 

purposes until the situation is clarified. Such management could include restricting 

access to affected sites and ensuring high health plants are used in restoration 

projects. 

In considering the origin of PTA, it is useful to consider the records of P. heveae and 

P. katsurae, especially those from apparently natural plant communities. Arentz 

(1986) reports both species are present in soils in Papua New Guinea, including 

rainforests containing Agathis robusta and Araucaria cunninghamii and Araucaria 

hunsteinii, although without obvious linkage to disease. Brown (1999), in a study 

focused on the association between P. cinnamomi and patch death in tropical 

rainforests of northern Queensland (containing both Agathis and Araucaria), also 

reports both P. heveae and P. katsurae, with P. heveae more frequent at higher 

elevations under dead patches of rainforest. Ko and others (2006) report both species 

from natural forests in Taiwan, neither in obvious association with disease. However, 

none of these authors provide DNA sequence data for their isolates and so their 

precise relationships are not clear, although the oogonial ornamentation of the 

Taiwan P. katsurae isolates matches ICMP16915, also from Taiwan. Ko and others 

(2006) point out that their Hawaiian Cocos isolates of P. katsurae, which include 

isolates H1024 and H1027 for which we obtained sequence data, are much less 

ornamented than the Taiwan isolates and indeed some approach P. heveae and PTA 

in their lack of ornamentation, although differing from both in ITS sequence. While it 

is reasonable to conclude both P. heveae and P. katsurae are indigenous to eastern 

Asia and Australasia, whether records from further afield (Erwin and Ribeiro 1996) 

represent indigenous populations or recent introductions must await further study. A 

recent multi-locus study (Blair and others 2007) confirms that P. heveae and P.

katsurae, the only named members of clade 5 of Cooke and others (2000), are closely 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

83 

related but phylogenetically distinct species. Morphologically, they are distinguished 

largely by the ornamentation on the oogonium (Erwin and Ribeiro 1996, Ko and 

others 2006, Oudemanns and Coffey 1991). It is apparent that a more comprehensive 

study of cultural, morphological and molecular variation of isolates from around the 

world, including in particular areas of possible natural occurrence, is warranted to 

better clarify the number and relationships of the clade 5 species, including PTA. 

Acknowledgments 

Maureen Fletcher, Duckchul Park, Sarah Tsai, Nic Faville (Landcare Research), 

Mark Braithwaite, Mark Bullians and Heather Pearson (MAF Biosecurity), Jenni 

Stanley and Asher Jones (Auckland University) assisted with laboratory and field 

work and figure preparation. Alex Nathan (Te Iwi O Te Roroa), Stephen King 

(Waipoua Forest Trust), and Dept of Conservation (Northland) facilitated our 

sampling at Waipoua and Trounson. David Cooke (Scottish Crop Research Institute, 

Scotland), Janice Uchida (University of Hawaii, US) and Michael Priest (Orange 

Agricultural Institute, Australia) supplied cultures. Auckland Regional Council (Jack 

Craw) supported our work in the Waitakere Range. ATCC cared for the PTA isolate 

deposited in 1970s, and facilitating our obtaining it for the present study. Don 

Woodcocks (Dept of Conservation, Gt Barrier), who assisted in the 1972 field work 

on Gt Barrier, guided us to the original site in 2006. Peter Gadgil encouraged the 

continuation of his pioneering study on PTA. This work was funded by New 

Zealand's Foundation for Research, Science and Technology and Ministry of 

Research, Science and Technology, and MAF Biosecurity New Zealand. 

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86 

Pythiaceous Fine Feeder Root Pathogens 

Associated With Eucalyptus 

gomphocephala (Tuart) Decline in 

Southwest Western Australia1

Peter M. Scott,2,3 Bryan L. Shearer,4 Paul A. Barber,2,3 and 

Giles E.St.J. Hardy2,3 

Abstract 

Eucalyptus gomphocephala is a keystone canopy species endemic to a narrow (5-10 km wide) 

coastal strip approximately 300 km in length in south-west Western Australia. Eucalyptus 

gomphocephala is undergoing a significant decline that was first identified as a spot decline in 

1994 and now occurs throughout large sections of its remnant distribution within Yalgorup 

National Park, in some areas resulting in 100 percent mortality. Multiple factors, including 

soil-borne pathogens, have been identified as possibly contributing to the decline. Fewer fine 

roots are associated with trees on declining sites compared to those on healthy sites. Foliar 

analysis indicates that declining trees have lower concentrations of some micronutrients, 

including zinc, which uptake is typically impaired by fine feeder root loss. A range of 

Pythiaceous microorganisms have been isolated from declining roots and these may be 

contributing to the loss of fine roots. Glasshouse trials are currently underway to determine 

whether these isolates are indeed pathogenic. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Centre of Phytophthora Science and Management, School of Biological Science and Biotechnology, Murdoch 

University, Murdoch, Western Australia 6150, Australia. 

3

 Tuart Health Research Group, School of Biological Science and Biotechnology, Murdoch University, Murdoch, 

Western Australia 6150, Australia. 

4 Science Division, Department of Environment and Conservation, Locked Bag 104, Bentley Delivery Centre, WA 

6983, Australia. 

Corresponding author: P.Scott@murdoch.edu.au. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

87 

Phytophthora Species Associated 

With Tanoak Stem Cankers in 

Southwestern Oregon1

Paul Reeser,2 Wendy Sutton2, and Everett Hansen2

Abstract 

From 2001 through 2006 stem cankers on tanoak (Lithocarpus densiflorus) were 

sampled during surveys to detect and eradicate Phytophthora ramorum from forests 

in southwestern Oregon. Pieces of bark from stem canker margins were plated on 

cornmeal agar amended with 10 ppm natamycin, 200 ppm Na-ampicillin, and 10 

ppm rifampicin. P. ramorum was usually identified on the isolation plates by the 

presence of characteristic hyphae, chlamydospores, and sporangia. Other colonies 

resembling Phytophthora were isolated into pure culture and identified by 

examination of morphological features and by DNA sequence analysis. 

Phytophthora species other than P. ramorum isolated from tanoak stem cankers 

include P. cambivora, P. cinnamomi, P. species "Pgchlamydo," P. gonapodyides, P. 

nemorosa, P. pseudosyringae, and P. siskiyouensis. Additional isolates may 

represent new species of Phytophthora and have not yet been identified. Selected 

isolates were tested for pathogenicity by inoculating healthy tanoak stems. Lesion 

margins were plated for re-isolation of the pathogen. 

Introduction 

Since 2001 the Oregon Department of Forestry (ODF) has conducted an intensive 

quarantine and eradication effort in southwestern Oregon to limit the spread of 

Phytophthora ramorum, cause of sudden oak death (SOD). Diseased tanoak 

(Lithocarpus densiflorus) trees were sampled to verify infection by P. ramorum. In 

some cases sampled infections yielded a species of Phytophthora other than P. 

ramorum. We identified these species and tested some of them for their pathogenicity 

on tanoak. 

Materials and Methods 

Aerial surveys were conducted by the Oregon Department of Forestry twice yearly 

(2001 through 2006) to locate dead or dying tanoak trees. Cause of death or disease 

was determined by follow-up ground surveys. Cankers or lesions in tanoak stems 

were sampled for the presence of P. ramorum by plating bark from the leading edge 

in corn meal agar amended with 10 ppm Na-natamycin, 200 ppm Na-ampicillin, and 

10 ppm rifampicin (CARP). Phytophthora colonies were purified by re-plating on 

fresh CARP and then sub-cultured on corn meal agar amended with 20 ppm β-

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Department of Botany and Plant Pathology, Oregon State University, Corvallis OR 97331, USA. 

Corresponding author: reeserp@science.oregonstate.edu. 
GENERAL TECHNICAL REPORT PSW-GTR-221

88 

sitosterol (CMAβ) for DNA extraction and long-term storage. Isolates were stored at 

room temperature as agar plugs in sterile de-ionized water. 

For morphological identification isolates from 2006 were grown on 15 percent 

clarified V8 agar amended with 20 ppm β-sitosterol (V8S). Plugs from colony 

margins were transferred into stream water to generate sporangia. Isolates which did 

not form oogonia in single culture on V8S were paired with standard A1 and A2 

tester strains on V8S to generate oospores and determine mating type. Structures for 

microscopic observation were fixed and preserved in 3.7 percent formaldehyde. 

Isolates were identified by comparison of features with published descriptions 

(Brasier and others 2003, Hansen and others 2003, Jung and others 2003, Reeser and 

others 2006, Waterhouse 1963). Representative isolates from each species identified 

were selected for DNA sequencing. DNA extracted from mycelium grown on CMAβ

was used as template to amplify nuclear rDNA internal transcribed spacer (ITS) for 

sequencing. ITS sequences were compared with Genbank database using BLAST 

Search to aid identification. 

Isolates from 2001 through 2005 were identified by sequencing the mDNA COX 

spacer and comparing with sequences of known reference isolates (Frank Martin, 

personal communication). Representatives from each group identified were 

confirmed by morphological examination as described above. 

Five isolates of P. cambivora, four isolates of P. gonapodyides, one isolate of P. 

nemorosa, and one isolate of P. siskiyouensis were inoculated into green twigs of 

tanoak seedlings by placing a CMAβ culture plug over a pinprick wound and sealing 

with parafilm. Each inoculated twig also received an agar control. Lesion size was 

measured after 10 to 15 days, and lesion margins were plated on CARP for reisolation of the pathogen. 

One isolate each from P. cambivora, P. cinnamomi, P. gonapodyides, P. nemorosa, 

P. siskiyouensis, and P. species 'Pgchlamydo' was tested for pathogenicity on tanoak 

stems in the field. A 5 mm diameter plug from the margin of a V8S culture was 

placed in a hole in the bark, covered with wet cheesecloth and sealed with aluminum 

foil and duct tape. Each isolate was inoculated into five different stems. Each stem 

received three different isolates and an agar control. After four weeks bark was 

removed to reveal lesion development. Lesions were measured (length by width), and 

pieces from four points on the lesion margin were plated in CARP to re-isolate. 

Results 

Results of tanoak stem canker isolations are listed in table 1. More than one half of 

the tanoak cankers sampled were culture negative for Phytophthora. P. ramorum and 

P. nemorosa were the species most frequently isolated. Eight additional species were 

identified, three of them being un-named ('Pgchlamydo') or undefined ('ilicis clade' 

sp.1 and sp.2). Each of these eight species occurred only rarely. 

Infection in green twigs of tanoak seedlings was variable. All four isolates of P. 

gonapodyides tested produced relatively large lesions in green tanoak twigs. Only 

one of the five isolates of P. cambivora tested produced lesions in green tanoak 

twigs. The one isolate of P. nemorosa tested produced moderately sized lesions in 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

89 

green tanoak twigs, and the one isolate of P. siskiyouensis tested did not form any 

lesion. 

Table 1—Phytophthora species isolated from tanoak stem cankers sampled 

during sudden oak death surveys in southwest Oregon from 2001 through 

2006. 

Species isolated Number of stem cankers 

Negative culture for Phytophthora 575 

P. cambivora A1 4 

P. cinnamomi A2 1 

P. species 'Pgchlamydo' 1 

P. gonapodyides 6 

P. nemorosa 102 

P. pseudosyringae 3 

P. 'ilicis clade' sp.1 1 

P. 'ilicis clade' sp.2 1 

P. ramorum 359 

P. siskiyouensis 4 

Total stem cankers sampled 1057 

In contrast, when inoculated in stems of field-grown tanoak, P. cambivora, P. 

cinnamomi, P. gonapodyides, P. nemorosa, P. siskiyouensis, and P. species 

'Pgchlamydo' were all pathogenic after 4 weeks, causing substantial bark lesions 

relative to agar control (table 2). In all cases, the species used for inoculation was 

recovered on re-isolation from lesion margins. 

Table 2—Pathogenicity of selected isolates of Phytophthora from tanoak 

stems. Isolates were inoculated into tanoak stems in the field. 

Isolate Species n mean lesion area (cm2)

 a s.d. 

control 12 0.2 0.10 

9641.1 P. cinnamomi 5 11.5 10.48 

9569 P. siskiyouensis 5 13.4 84.63 

9675-BK P. cambivora 5 16.5 5.55 

9364 P. sp. 'Pgchlamydo' 5 18.1 6.13 

9684.2 P. nemorosa 5 18.4 14.75 

9616 P. gonapodyides 5 18.6 7.59 

a lesion area computed as approximate rhomboid (l x w)/2 - inoculum area

Discussion 

Prior to the recent SOD epidemic, no species of Phytophthora were known to cause 

cankers in forest grown tanoak. The discovery of P. ramorum as a pathogen of 

tanoak in California was quickly followed by the discovery of P. nemorosa and P. 
GENERAL TECHNICAL REPORT PSW-GTR-221

90 

pseudosyringae associated with tanoak cankers. Six years of diagnostic support of 

survey and detection for P. ramorum in tanoak forests of southwest Oregon has 

revealed the occurrence, at very low frequency, of at least five additional species of 

Phytophthora causing stem cankers in tanoak. 

Literature Cited 

Brasier, C.M.; Cooke, D.E.L.; Duncan, & Hansen, E.M. 2003. Multiple new phenotypic 

taxa from trees and riparian ecosystems in Phytophthora gonapodyides-P. megasperma ITS 

Clade 6, which tend to be high-temperature tolerant and either inbreeding or sterile. Mycol. 

Res. 107: 277-290. 

Hansen, E.M.; Reeser, P.W.; Davidson, J.M.; Garbelotto, M.; Ivors, K.; Douhan, ; 

Rizzo, D.M. 2003. Phytophthora nemorosa, a new species causing cankers and leaf blight of 

forest trees in California and Oregon, U.S.A. Mycotaxon 88: 129-138. 

Jung, T.; Nechwatal, J.; Cooke, D.E.L.; Hartmann, G.; Blaschke, M.; Oßwald, W.F.; 

Duncan, J.M.; Delatour, C. 2003. Phytophthora pseudosyringae sp. nov., a new species 

causing root and collar rot of deciduous tree species in Europe. Mycol. Res. 107: 772-789. 

Reeser, P. W.; Hansen, E.M.; Sutton, W. 2007. Phytophthora siskiyouensis, a new species 

from soil, water, myrtlewood (Umbellularia californica) and tanoak (Lithocarpus densiflorus) 

in southwestern Oregon. Mycologia 99:639-643. 

Waterhouse, G.W. 1963. Key to the species of Phytophthora de Bary. Mycological Papers 

92. Commonwealth Mycological Institute, Kew, U.K. 22 p. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

91 

Monitoring Occurrence and Distribution 

of Phytophthora Species in Forest 

Streams in North Carolina Using 

Baiting and Filtration Methods1

Jaesoon Hwang,2 Steven W. Oak,3 and Steven N. Jeffers2 

Abstract 

The recent epidemics of sudden oak death, caused by Phytophthora ramorum, in the coastal 

forests of California and southwest Oregon have drawn attention to other species of 

Phytophthora present in natural ecosystems that may threaten forest tree health. Since 

Phytophthora species are well adapted to aquatic environments, the species present in a 

strategically-selected stream may represent the occurrence and distribution of Phytophthora 

species over the relatively large land area drained by this stream. Based on this assumption, 

five streams in three watersheds in the Pisgah National Forest in western North Carolina were 

monitored monthly for Phytophthora species. from April 2005 to March 2006. Filtration and 

two baiting methods were used to detect propagules of Phytophthora species in stream water. 

A 1-liter water sample was collected from each stream and vacuum-filtered with Nuclepore 

and Durapore membrane filters. In addition, wounded or non-wounded leaves of 

Rhododendron maximum were used as baits and were exposed in a stream for 3 days or 2 to 3 

weeks, respectively. 

Phytophthora species were detected from all five streams throughout the 12-month 

monitoring period. P. cambivora, P. cinnamomi, P. citricola, P. citrophthora, P. 

gonapodyides, P. heveae, P. pseudosyringae, and seven morphologically and genetically 

distinct groups of isolates were identified from 1560 isolates collected. P. gonapodyides was 

the most prevalent (1353 isolates) and was detected consistently throughout the monitoring 

period. P. citricola, P. gonapodyides, and P. pseudosyringae were widely distributed and 

occurred in all five streams. Diversity of Phytophthora species was greatest in July, when 11 

species/groups were detected and least in February when only one species was detected. P. 

gonapodyides and P. pseudosyringae were the only two species recovered over winter months 

(from November to February). Thirteen of the 14 species/groups were detected by filtration 

while only eight species/groups were isolated with each baiting method. Overall, filtration 

was superior to baiting for detecting Phytophthora species in forest streams: it provided 

quantitative data on propagule density; it was more efficient than either baiting method; and it 

was more effective at detecting greater species diversity. 

Introduction 

The recent epidemics of sudden oak death, caused by Phytophthora ramorum, in the 

coastal forests of California and southwest Oregon have drawn attention to other 

species of Phytophthora present in natural ecosystems that may threaten forest tree 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Department of Entomology, Soils, and Plant Sciences; Clemson University; Clemson, SC 29634-0315, USA. 3

 USDA Forest Service, Southern Region FHP; Asheville, NC 28802, USA. 

Corresponding author: jaesooh@clemson.edu. 
GENERAL TECHNICAL REPORT PSW-GTR-221

92 

health. Since species of Phytophthora are well adapted to aquatic environments, the 

species present in streams may represent the occurrence of Phytophthora species over 

most or all of the relatively large land area drained by them. Consequently, five 

streams were sampled monthly for 12 months to determine if streams could be used 

to monitor occurrence and diversity of Phytophthora spp. in a forest watershed. 

Materials and Methods 

Five streams in four distinct watersheds in the Pisgah National Forest in western 

North Carolina were sampled monthly from April 2005 to March 2006: Bent Creek, 

Big Creek, Fletcher Creek, Davidson River, and South Mills River. Filtration and two 

baiting methods were used to detect propagules of Phytophthora spp. in stream 

water. For filtering, a 1-liter sample of water was collected from each stream, and the 

samples were processed within 10 h of collection. Nine 100-ml sub-samples of water 

were vacuum-filtered through two types of membrane filters (47 mm in diameter) 

with three pore sizes: Nuclepore filters with 1- and 3-μm pores and Durapore filters 

with 5-μm pores. The filters were inverted onto PARPH-V8 selective medium in 

100-mm-diameter disposable petri plates, and the plates were placed in the dark at 

20°C for two to three days. Filters then were removed and colonies of Phytophthora

spp. were counted. Representative colonies were subcultured and stored for later 

identification. 

For baiting, either four wounded leaves or four non-wounded leaves of 

Rhododendron maximum were placed in a mesh bag rigged to float just below the 

water surface. Leaves were wounded by making cuts (5 to 10 mm in length) 

perpendicular to the leaf edge at approximately 5- to 7-mm intervals along the leaf 

edge (fig 1). Wounded leaves were exposed in a stream for three days while nonwounded leaves were exposed in the same stream for two to three weeks. To isolate 

Phytophthora spp. from wounded leaves, five rectangular pieces (approximately 5 to 

7 mm on a side) with water-soaked lesions (fig 1) were cut from the edge of each leaf 

and embedded in PARPH-V8 medium. To isolate from non-wounded leaves, five 

pieces (approximately 5 mm × 5 mm) were removed from the margin area of watersoaked or necrotic lesions on each leaf and transferred to PARPH-V8 medium. 

Isolation plates were held at 20°C in the dark for 14 days and examined regularly for 

mycelia characteristic of Phytophthora spp. Representative colonies were subcultured 

and stored for later identification. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

93 

Phytophthora species were identified based on morphological and molecular 

characters. Isolates grown on PARPH-V8 medium were examined for the presence of 

sporangia, chlamydospores, and oospores. In addition, agar plugs with actively 

growing hyphae were removed, flooded with 1.5 percent non-sterile soil extract 

solution, and placed under fluorescent lights at room temperature (22 to 24°C) for 24 

h to induce sporangium production. For molecular characterization, each isolate was 

placed in pea broth medium at room temperature for five to seven days to produce a 

mycelium mat for DNA extraction. The ITS region of ribosome DNA and cox region 

of mitochondrion DNA were amplified using ITS4 and ITS6 or FM75 and FM77/83 

primers sets. RFLP analysis was done with AluI and MspI restriction enzymes for 

ITS region DNA and with AluI, RsaI, and TaqI restriction enzymes for DNA from 

the cox region (fig 2). 

Over the course of monitoring these streams, native plants of Rhododendron 

maximum and Kalmia latifolia with symptoms of tip dieback and leaf blight were 

observed in the riparian zone along all five streams. Shoots on some of these plants 

appeared to have been submerged in the streams during periods of high water flow. 

Foliage samples from symptomatic plants were collected, and tissue pieces from 

lesions were embedded in PARPH-V8 medium to isolate Phytophthora spp. as 

described above. In addition, composite soil samples were collected from the riparian 

zone of all five streams and assayed for Phytophthora spp. using a baiting bioassay. 

Approximately 1-liter of soil was collected along each stream. Samples were mixed 

thoroughly, and three replicate 100-ml aliquots of each soil sample were flooded with 

distilled water (200 ml of water per 100 ml of soil) and baited with leaf pieces of 

camellia and rhododendron. After three days at room temperature, leaf-piece baits 

were transferred to PARPH-V8 medium and plates were placed in the dark at 20°C 

for seven to ten days. Colonies of Phytophthora spp. were isolated and identified as 

described above. 

Figure 1—Water-soaked 

lesions on a wounded 

rhododendron leaf after 

3 days of exposure in a 

forest stream. 
GENERAL TECHNICAL REPORT PSW-GTR-221

94 

Figure 2—Identification of isolates of Phytophthora spp. by RFLP analyses: A, 

DNA from the ITS region of 34 isolates was amplified and digested with AluI; 

B, DNA from the cox region of 15 isolates was amplified and digested with 

AluI (top) and RsaI (bottom). Lanes (both top and bottom) 1, 10, and 20 in A

and lanes 1, 10, and 18 in B contain a molecular marker. 

Results and Discussion 

Species of Phytophthora were detected in all five streams throughout the 12-month 

monitoring period. From 1,560 isolates collected, seven recognized species—P. 

cambivora, P. cinnamomi, P. citricola, P. citrophthora, P. gonapodyides, P. heveae, 

and P. pseudosyringae—and seven other morphologically and genetically distinct 

groups were identified. P. gonapodyides was most prevalent (1,353 isolates) and was 

detected consistently throughout the monitoring period. P. citricola, P. 

gonapodyides, and P. pseudosyringae were widely distributed and occurred in all five 

streams. The number and diversity of species of Phytophthora recovered varied 

depending on the month, location, and detection method. Diversity of Phytophthora

spp. was greatest in July, when 11 species and groups were detected, and least in 

February—when only one species was detected. P. gonapodyides and P. 

pseudosyringae were the only two species recovered in winter months (November to 

February). The greatest diversity in a single stream occurred in the South Mills River 

where ten species and groups were detected over the 12-month sample period. 

Diversity was least in Big Creek where only four species and groups were found. 

Thirteen of the 14 species and groups were recovered by filtration while only eight 

species and groups were recovered with each baiting method. Overall, filtration was 

superior to either baiting method for detecting Phytophthora spp. in forest streams; it 

provided quantitative data on propagule density, recovered a greater diversity of 

species, and was more efficient because only one trip was needed to sample each 

stream. P. citricola and P. heveae were the only two species isolated from the 

samples of symptomatic plants collected along the five streams, and only P. 

cinnamomi and P. heveae were recovered from the riparian soil samples. In 

conclusion, Phytophthora spp. were abundant throughout the year in the five North 

Carolina forest streams we monitored, and there appeared to be a greater diversity of 

A B
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

95 

species of Phytophthora in the streams than in the soils and plants along these 

streams—based on the methods employed in this study. 

Acknowledgments 

We thank Lynn Luszcz, Frank Martin, Patricia de Sa, Pedro Uribe, and Drew Zwart 

for technical assistance in isolation and species identification; Jill Baker for 

assistance with stream sampling; North Carolina Arboretum and Pisgah Ranger 

District in Pisgah National Forest for providing access to study locations. This project 

was funded by USDA ARS Foreign Disease-Weed Science Research Unit, USDA 

Forest Service Pacific Southwest Research Station, and Armstrong Wood Products 

Inc. 
GENERAL TECHNICAL REPORT PSW-GTR-221

96 

Phytophthora Species in Forest Trees 

in Hungary: A Genetic Approach1

Ferenc Lakatos2 and Ilona Szabó1 

Abstract 

Disease symptoms caused by different Phytophthora species were studied in Hungary since 

1999. A collection of more than 300 cultures from 6 main localities and from four different 

hosts (Alnus glutinosa, Juglans nigra, Quercus petraea and Q. cerris) was established. The 

identification of the isolates presented sometimes difficulties because of the absence of 

adequate morphological characters. In 2006 we have started to use molecular markers for 

species identification based on the methods and results published on the identification of 

different Phytophthora species and the advances of the available molecular methods at our 

institute. 

Altogether 80 isolates were analyzed. DNA extraction and PCR assay were performed using 

the REDExtract-N-Amp kit from Sigma. The amplification of the DNA was carried out 

followed the manufacturer’s instruction with the primers “ITS4” and „ITS6”. Initial 

denaturation step of 3 min at 94° C, followed by 35 cycles of 94° C (30 sec), 55° C (30 sec) 

and 72° C (90 sec) and a final extension step at 72° C (10 min). PCR was performed in an 

Eppendorf Mastercycler and the sequence reactions on an AB 3730XL automatic sequencer 

respectively. 

The DNA sequence was alignment by ClustalX using default settings. Distance analysis was 

performed by the Neighbor Joining NJ algorithm. The distance matrix was calculated based 

on the Tamura-Nei substitution model, while the robustness of the topology was tested by 

bootstrapping with 1000 repetitions. For comparison also several GenBank 

(http://www.ncbi.nlm.nih.gov/) entries of different Phytophthora taxa were taken. 

74 percent of the amplified DNA resulted in sequences (59 isolates). Altogether seven already 

described and at least three undescribed (or at least in the GenBank as Phytophthora sp. 

documented) Phytophthora taxa were found. Eight of them were found in Alnus (P. alni, P. 

citricola, P. gonapodyides, P. inundata, P. megasperma and P. sp1, P. sp2, P. sp3), four in 

Juglans (P. cactorum, P. citricola, P. hedraiandra, P. sp1) and two in Quercus (P. citricola, 

P. gonapodyides). The sequences of the Hungarian isolates represents 4 major clades: 1: P. 

inflata/citricola, 2: P. cactorum/ideae/hedraiandra, 3: P. 

inundata/megasperma/gonapodyides and 4: P. alni. The mostly widespread is the P. 

citricola/inflata group, having four haplotypes (17 isolates) from all four investigated hosts. 

P. gonapodyides showed the highest polymorphism, having 7 haplotypes (11 isolates) on two 

hosts (Alnus glutinosa and Quercus petraea). 

Three undefined taxa (P. sp1, sp2, sp3) didn’t result in an exact species determination even 

after an intensive BLAST search in the GenBank. All haplotypes found were submitted to the 

GenBank and are under revision at the time of abstract submission. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 University of West Hungary, Institute of Sylviculture and Forest Protection, H-9400 Sopron, Hungary 

Corresponding author: flakatos@emk.nyme.hu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

97 

Introduction 

Disease symptoms caused by different Phytophthora species were studied intensively 

in Hungary since 1999 (Szabó and others, 2000). In the recent years a collection of 

more than 300 cultures from 8 localities (Barcs, Csorna, Hévíz, Jánossomorja, 

Homorúd, Kaszó, Sopron and Tornyosnémeti) and from four different hosts (Alnus 

glutinosa, Juglans nigra, Quercus petraea and Q. cerris) was established. The 

identification of the isolates presented sometimes difficulties because of the absence 

of adequate morphological characters. In 2006 we have started to use molecular 

markers for species identification based on the methods and results published on the 

identification of different Phytophthora species (Cooke and Duncan, 1997; Schena 

and Cooke, 2006; Garnica and others, 2006) and the advances of the available 

molecular methods at our institute. 

Materials and Methods 

The molecular identification was performed by sequencing the ITS1 and ITS2 

regions of rDNA and comparing with the known Phytophthora sequences accessible 

in GenBank. Altogether 80 isolates were analyzed so far. The DNA was extracted 

from pure culture using REDExtract-N-Amp kit from Sigma-Aldrich. PCR was 

performed using specific primers ITS6 and ITS4 (Cooke and Duncan 1997) in an 

Eppendorf Mastercycler and the sequence reactions on an AB 3730XL automatic 

sequencer respectively. 

The DNA sequence was aligned by ClustalX using default settings. Distance analysis 

was performed by the Neighbor Joining NJ algorithm with Kimura 2 parameter. The 

distance matrix was calculated based on the Tamura-Nei substitution model, while 

the robustness of the topology was tested by bootstrapping with 1000 repetitions. For 

comparison also several GenBank entries of different Phytophthora taxa were taken. 

Results 

Seventy-four percent of the amplified DNA resulted in sequences (59 isolates). 

Altogether seven already described and further 4 undescribed (in the GenBank as 

Phytophthora sp. documented) Phytophthora taxa were found. Ten of them were 

found in Alnus (P. alni, P. citricola, P. gonapodyides, P. hungarica, P. inundata, P. 

megasperma and P. sp1, P. sp2, P. sp3, P. sp4), three in Juglans (P. cactorum, P. 

citricola, P. sp1) and two in Quercus (P. citricola, P. gonapodyides) (Table 1.). 

Table 1—Phytophthora species found in different forest stands 

Phytophthora species Alnus 

glutinosa

Juglans 

nigra

Quercus 

petraea

Quercus 

cerris

Phytophthora alni X 

Phytophthora cactorum X 

Phytophthora citricola X X X X 
GENERAL TECHNICAL REPORT PSW-GTR-221

98 

Phytophthora gonapodyides X X 

Phytophthora hungarica X 

Phytophthora inundata X 

Phytophthora megasperma X 

Phytophthora sp1 X X 

Phytophthora sp2 X 

Phytophthora sp3 X 

Phytophthora sp4 X 

Total 10 3 2 1 

P. alni was found at two locations in Alnus stands solely. The three sequenced 

isolates from one location were genetically identical. P. cactorum was found on one 

location in a Juglans stand. The four DNA fragments showed minimal genetic 

variation (1bp, 0.16 percent). P. citricola was the most widely distributed species 

found in all four forest types. Also the investigated 17 isolates showed the highest 

genetic diversity (11bp, 1.44 percent). P. gonapodyides was found on two hosts on 

Alnus glutinosa and Quercus petraea. The investigated 11 isolates showed moderate 

genetic diversity (5 bp, 0.61 percent). P. hungarica, P. inundata and P. megasperma

was found only in Alnus glutinosa stands with no (P. hungarica and P. megasperma; 

two isolates each), or limited (P. inundata; 8 isolates) genetic variation. 

Four additional Phytophthora taxa were found in Alnus glutinosa stands. None of 

them (P. sp1, sp2, sp3 and sp4) turned out as a known species even after an intensive 

BLAST search in the GenBank. These 15 investigated isolates have a fragment 

length between 817-822bp. Some of them showed some similarities to closely related 

species in the clade 6 (regarding Cooke and others, 2000), which is one of the most 

complicated clade within the genus (Brasier and others 2003). 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

99 

 P. citricola/inflata

 P citrophthora

Clade 2

 P idaei

 P hedraiandra

 P pseudotsugae

 P. cactorum

Clade 1

 P cinnamomi

 P cambivora

 P fragariae rubi

 P fragariae fragariae

 P. alni

Clade 7

 P. inundata

 P sp3

 P sp1

 P hungarica

 P sp4

 P megasperma

 P sp2

 P gonapodyides

Clade 6

67

61

51

100

60

61

97

100

100

70

85

96

96

99

100

79

60

85 96

59

31

49

87

0,01

Figure 1—The Neighbour-Joining tree of the investigated Phytophthora

isolates (Kimura-2 parameter). In Italics: species found in Hungary, others 

were taken from GenBank for comparison. 

The sequences of the Hungarian isolates represent 4 caldes regarding the 

classification of Cooke and others (2000) (fig 1.): Clade 1: P. cactorum ; Clade 2: P. 

citricola/inflata, Clade 7: P. alni and Clade 6: P. 

inundata/hungarica/megasperma/gonapodyides including all undescribed species (P. 

sp1, sp2, sp3 and sp4). The mostly widespread is the P. citricola/inflata group, 

having four haplotypes (17 isolates) from all four investigated hosts. P. gonapodyides

showed the highest polymorphism, having 7 haplotypes (11 isolates) on two hosts 

(Alnus glutinosa and Quercus petraea). 

All haplotypes found were submitted to the GenBank and are under revision at the 

time of submission. 

Conclusions 

Seven described and further four until now undescribed species of Phytophthora

were found in Hungary: P. alni, cactorum, citricola, gonapodyides, hungarica, 

inundata and megasperma. Large genetic diversity was found in the investigated 

Phytophthora isolates regarding the host tree species and location. Large intraspecific 

variabiliy was found in P. citricola and P. gonapodyides. The analysis of the ITS 
GENERAL TECHNICAL REPORT PSW-GTR-221

100 

fragment doesn’t allow the species determination in some cases. Here the analysis of 

further DNA parts is desired. 

Acknowledgment

Our research was supported by the Hungarian Research Fund (OTKA T049077). 

Literature Cited 

Brasier C. M.; Cooke, D. E. L.; Duncan, J. M.; Hansen E. M. 2003. Multiple new 

phenotypic taxa from trees and riparian ecosystems in Phytophthora gonapodyides – P. 

megasperma ITS clade 6, which tend to be high-temperature tolerant and either inbreeding or 

sterile. Mycol. Res. 107(3): 277-290. 

Cooke, D. E. L.; Duncan, J. M. 1997. Phylogenetic analysis of Phytophthora species based 

on the ITS1 and ITS2 sequences of ribosomal DNA. Mycol. Res. 101, 667-677. 

Cooke, D. E. L.; Drenth, A.; Duncan, J. M.; Wagels, G.; Brasier C. M. 2000. A molecular 

phylogeny of Phytophthora and related Oomycetes. Fungal Genetics and Biology 30, 17–32. 

Garnica D. P.; Pinzon A. M.; Quesada-Ocampo L. M.; Bernal A.; Barreto E.; Grünwald 

N.; Restrepo S. 2006. Survey and analysis of microsatellites from transcript sequences in 

Phytophthora species: frequency, distribution, and potential as markers for the phylum 

Oomycota. BMC Genomics 7, 245. 

Schena, L.; Cooke, D. E. L. 2006. Assessing the potential of regions of the nuclear and 

mitochondrial genome to develop a “molecular tool box” for the detection and 

characterization of Phytophthora species. J. of Microbiol. Methods 67, 70-85. 

Szabó, I.; Nagy, Z.; Bakonyi, J.; T. Érsek, T. 2000: First Report of Phytophthora Root and 

Collar Rot of Alder in Hungary. Plant Disease 84 (11): 1251. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 101 

Phytophthora Biodiversity: How Many 

Phytophthora Species Are There?1

Clive Brasier2

Introduction 

Many new Phytophthoras species are currently being identified associated with the 

nursery trade or natural ecosystems. Many of these may be recent invasives: a result 

of the increasing international movement of plants leading to export and spread of 

exotic Phytophthora species. Examples include P. ramorum and P. kernoviae. The 

frequency of the new discoveries, the obvious taxonomic and phylogenetic interest 

involved, and the possible plant health risk to forests prompted me to take a look at 

the theoretical question: how many Phytophthoras species are there? To explore this 

question, it seems best to begin with the history of Phytophthora species over time. 

Phytophthora species 1876–1999 

The numbers of known Phytophthora species represented in various taxonomic 

treatise’s between de Bary’s (1876) description of P. infestans and Erwin and 

Ribeiro’s (1995) encyclopaedic compilation of Phytophthora knowledge 

‘Phytophthora Diseases Worldwide’ is shown in table 1. 

Table 1—Phytophthora species over time 1876–1996 

Date Author(s) Number of known or 

described species 

1876 Anton de Bary P. infestans 

(1876 ~1920s) (uncertain ~ some generic 

confusion) 

1917 J. Rosenbaum ca 11 species 

1931 C.M. Tucker ca 21 species 

1963 G.M. Waterhouse ca 41 species 

1976 F.J. Newhook, G.M. Waterhouse 

and D.J. Stamps ca 50 species 

1996 D.C. Erwin and O.K. Ribeiro ca 54 species 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Forest Research, Alice Holt Lodge, Farnham, Surrey GU10 4LH, U.K. 

Corresponding author:clive.brasier@forestry.gsi.gov.uk. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 102

The “early period” ca 1880–1920 involved a considerable degree of taxonomic 

uncertainty as criteria to separate Oomycete genera were consolidated. Among the 

historic land marks, Rosenbaum (1917) made one of the first scholarly compilations 

of Phytophthora species using morphological criteria such as antheridial type, 

sporangial papillum type, chlamydospore presence and biometric measurements such 

as sporangial length/breadth ratio. Tucker (1930), in a sizeable monograph and key, 

investigated both the morphological and physiological aspects of described 

Phytophthoras species including host specificity and temperature–growth 

relationships. Waterhouse (1963) organised the described species into her six well 

known morphological groups, based mainly on the antheridial and sporangial criteria 

used by Rosenbaum and Tucker. She also produced a workable key based on the 

same criteria. Newhook, Waterhouse and Stamps (1976) updated the Waterhouse 

approach with a tabular key. Erwin and Ribeiro’s (1995) volume bought all the 

available information on Phytophthora species together. 

Erwin and Ribeiro (1995) was the ultimate word on biology and pathology of 

Phytophthora species prior to the era of Phytophthora population biology and 

molecular genetics. It marked the end of the period in which Phytophthoras were 

treated primarily as morphological entities for taxonomic purposes. A curve fitted to 

the number of species known and described over the period 1876–1995 is shown in 

figure 1. It shows a steady but unremarkable rate of increase, rising to about 54 

described species at the time Erwin and Ribeiro’s volume went to press. This figure 

had increased only to 55 species by 1999 (table 2), with the description of P. 

quercina (Jung and others 1999). 

Figure 1—Number of described Phytophthora species over time, 1876–1995. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 103 

How Many Phytophthoras? 

Hawksworth (2001) estimated that only 10 percent of fungi were known to science. 

Crous and Groenwald (2005) proposed that only 7 percent of fungi were known. One 

could use these authors’ estimates as a simple, though crude, rule of thumb from 

which to estimate the possible total number of extant Phytophthora species, albeit 

that they should be treated with caution, since they are hedged around with various 

assumptions. 

A necessary assumption for the present purpose is that species diversity in Oomycete 

fungi follows similar principles to that in “true fungi.” The estimation process also 

involves some rather fanciful play with dates and numbers. In the world of estimates 

it depends which figures you use and how you use the figures. As a starting point in 

the process, I am also going to assume that there were ~60 Phytophthora species 

known at the end of 1999. This is slightly higher than the figure of 55 species 

deriving from the 54 taxa listed by Erwin and Ribeiro (1995) plus P. quercina (table 

2). Even the figure of 54 species for Erwin and Ribeiro is not an absolute, since 

various equivocal taxa are listed in their treatise, such as possibly synonymous 

species (e.g. P. arecae and P. palmivora), varieties (e.g. P. cinnamomi var 

parvispora), and unconfirmed or improperly described spcies such as P. eriugena. 

Table 2—Phytophthora species described Pre 2000 

P. arecae P. erythroseptica P. japonica P. phaseoli 

P. boehmeriae P. fragariae frag. P. katsurae P. porri 

P. botryosa P. fragariae rubi P. lateralis P. primulae 

P. cactorum P. gonapodyides P. macrochlamy. P. pseudotsugae

P. cajani P. heveae P. meadii P. quercina

P. cambivora  P. hibernalis P. medicaginis P. quininea 

P. capsici P. humicola P. megakarya P. richardiae 

P. cinnamom P. idaei P. megasperm P. sinensis 

P. citricola P. ilicis P. melonis P. sojae 

P. citrophthora P. infestans P. mexicana P. syringae 

P. clandestina P. inflata P. mirabilis P. tentaculata 

P. colocasiae P. insolita P. multivesiculata P. trifolii 

P. cryptogea P. iranica P. nicotianae P. vignae 

P. drechsleri P. italica P. palmivora ~11/55 = 20 

percent 

 Damaging to nursery trees and/or forests and natural ecosystems. 
GENERAL TECHNICAL REPORT PSW-GTR-221

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Taking Hawksworth’s 10 percent as the proportion of known fungal species, then, 

using the figure of ~60 described species at the end of 1999, one arrives at a possible 

total of 600 extant Phytophthora species i.e. currently living species including both 

the knowns and unknowns. That implies a further, rather daunting possibility that 

there may have been as many as ~540 Phytophthora species still unknown (meaning 

not yet discovered and not yet formerly described) at the beginning of the current 

millennium! 

However, instead of applying the raw figure of about 600 extant species, I propose 

using a more conservative, bracketed figure of between 200–600 extant species. On 

this basis, there may have been anything between 140 and 540 unknowns at the end 

of the last millennium. Another way to view this 140–540 range is to consider it as 

340 ± 200 unknowns. This figure seems to me more comfortable, although not 

necessarily more comforting. If it is a reasonable approximation to the truth, then 

whatever the precise figure is, the large numbers of still undiscovered Phytophthora

species are going to be a significant challenge to our diagnostic and taxonomic 

expertise, our phylogenetic expertise, and perhaps even to the patience of those who 

will have to write their descriptions. Such a large number of unknowns may also 

represent a threat to our plant health regimes, as I shall mention later. 

Developments Since 2000 

While the number of described Phytophthora at the end of 1999 was about 55 (table 

2), already, since 2000, a remarkable 50+ new species (species that have been 

morphologically and molecularly identified as unique though not yet necessarily 

formally described) have been discovered. These are listed in table 3. Again, the 

figure of 50+ species is an approximation. The precise number is unknown, and I 

have heard of more potential additions since this paper was originally prepared in 

August 2007. Also, some taxa, such as P. alni and its subspecies, could either be 

counted as one species or as multiple taxa. Nonetheless, the fact that the number of 

species described or under description in the seven years since the year 2000 is about 

the same as the number described in the previous 120 years is, surely, extraordinary. 

In figure 2, a new data point has been added to the species versus time diagram of 

figure 1. I have used the IUFRO Phytophthora meeting of 2007 as the cut-off point. 

The amended diagram clearly shows the dramatic upswing since 1995. What is the 

explanation for this upswing? I suggest that four main factors are responsible: 

• The emergence of more holistic, population-based species concepts since the 

1980s, leading to greater awareness of the subtlety and complexity of 

Phytophthora species, including the existence of uniquely evolutionarily adapted 

species within the old taxonomic morphospecies, and so to a greater willingness 

to identify and describe novel taxa. 

• The rapid application of molecular tools for defining evolutionary units in fungi. 

• Increasing international trade in rooted plants, which is having the effect of 

distributing more pathogens beyond their natural endemic ranges. Consequently, 

some of them become noticed as a problem within the nursery trade, or as threats 

to forests and natural ecosystems. 

• Increased environmental screening for Phytophthora species based on PCRbased detection of molecular signals. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 105 

Table 3—Phytophthora species / taxa identified Post 2000 (morphologically 

and molecularly unique species, some not yet formally described) 

P. alni (x 3) P. kernoviae P. frigida P. taxon salixsoil

P. andina P. nemorosa P. austrocedrae P. taxon pgchlamydo 

P. asparagi P. niederhauserii P. lagoariana P. taxon riversoil

P. bisheria P. pistaciae P. cuyabensis P. taxon oaksoil

P. brassicae P. polonica P. cact x hedr P. parvasperma

P. captios  P. pseudosyringae P. foliorum P. hungarica

P. europae  P. psychrophila P. sulawesiensis P. sylvatica

P. fallax P. ramorum P. siskiyouensis P. parsiana 

P. gallica P. sansomea P. uliginosa P. quercetorum 

P. glovera P. taxon Banksia P. rosacearum P. taxon meadii-like

P. hedraiandra P. taxon. chicory P. sansomeana P. taxon Acer

P. inundat  P. cactorum x

nicotianae P. taxon Agathis

P. ipomoeae P. tropicalis P. taxon orphan

P. kelmania P. alticola ~ 30 / 52 = 58 

percent 

 Associated with, or with research on, nursery trees, forests and natural ecosystems. 
GENERAL TECHNICAL REPORT PSW-GTR-221

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Figure 2—Number of described Phytophthora species over time, extended 

beyond 1995 (Erwin and Ribeiro) to IUFRO 2007, showing the remarkable 

increase over this period. 

If the surge in identification of new Phytophthora species continues for another 

decade or so, we will soon be on the way towards the minimum of 200 extant 

Phytophthora species estimated above. 

Demonstration of Phylotaxa 

A valuable practical application of modern PCR technology is the ability to sample 

environmental substrates such as soil, water and plant material for microbial 

diversity. Such studies are beginning to reveal unknown phylotaxa: taxa which are 

apparently unique on the basis of their molecular profile, but for which no living 

culture exists. During the 1990s, unique bacterial phylotaxa were shown to be 

abundant during investigations of the ecology of bacterial populations in soil. 

Phylotaxa are now beginning to be identified in comparable sampling for 

Phytophthora. Arcate, Kemp and Nelson (2006) recently applied PCR-based methods 

to search for Oomycetes in the rhizosphere of tomatoes and other crops in New York 

State. They found many Pythiums, a couple of Phytophthoras, and greater 

biodiversity than they obtained by direct plating methods. They were also able to 

sample the dormant oospore flora as well as active mycelium and propagules. 

Two research groups associated with IUFRO have recently began to apply these 

methods to screen for Phytophthoras in natural ecosystems, so far mostly in stream 

water. Hansen, Reeser and Sutton (these proceedings) have examined streams in 

south west Oregon using combined SSCP data of ITS and Cox loci. They have 

identified 30 phylospecies, of which ten are believed to be new or unknown taxa 

based on BLAST searches. In a similar study of a central Oregon stream near 

Corvallis, Remigi, Sutton, Reeser and Hansen (these proceedings) identified eleven 

phylospecies, of which five are probably unknown taxa. 

Scibetta, Cooke and Cacciola (also unpublished) have used nested PCR of ITS1, 

followed by cloning and sequencing, to examine water samples from streams in 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 107 

western Scotland. They found 17 phylospecies in the streams, of which three are 

believed to be new or unknown taxa. Scibetta and Cooke (unpublished) used the 

same methods to survey streams in Ecuador at 2–3000 m above sea level. They found 

ten phylospecies, including P. infestans, three Peronospora ‘species’, and three new 

or unknown taxa. 

These pioneering studies show that significant numbers of currently unknown 

Phytophthora species may be present in streams, even in relatively well documented 

locations such as western Oregon. Since, as yet, we have no cultures of these 

unknowns, these results are tantalising, to say the least! Particularly intriguing is the 

fact that DNA of typically aerially dispersed taxa with caducous sporangia, such as P. 

infestans and Peronospora species—not expected to be stream inhabitants—were 

detected in the streams in Ecuador. This raises interesting questions about how the 

DNA of these taxa came to be in the water. Does it perhaps reflect the ‘raining’ of 

aerially dispersed sporangia from the atmosphere into streams; or the percolation of 

plant debris containing inoculum into the stream from the surrounding environment? 

Or does it reflect a genuine inhabitation of the stream by the Phytophthora (or 

Peronospora) species concerned? 

New Species Revealed by Re-examination of 

Culture Collections 

Additional information is coming from the application of molecular diagnostics to 

Phytophthora culture collections. This is especially true where a collection has not 

previously been subjected to a rigorous taxonomic screening. A good example is the 

collection of the Vegetation Health Service (VHS) at CALM, western Australian. 

This has recently been subject to ITS sequencing by Stukely, Burgess and Hardy 

(unpublished). In this still ongoing analysis, 30 taxa have been identified of which 

seven (23 percent) are considered new or unknown taxa. Since (in contrast to 

phylotaxa) cultures are available, the unknowns can now be examined for their 

phenotypic and other molecular characteristics. In a similar study of the isolates in 

the ICMP Phytophthora culture collection of Landcare Research in New Zealand, 

Beever and others (2000) identified around 30 species on the basis of ITS 

sequencing. Among these were two new or unknown taxa. These have now been 

partly characterised and informally named P. taxon Agathis and P. taxon orphan 

(see also Beever, this volume). 

What Proportion of New Phytophthora Species are 

Associated With Forests and Natural Ecosystems? 

Of the ~55 Phytophthora species known in 1999, eleven could be considered as 

damaging to nursery trees, forests and natural ecosystems (my assessment). These are 

shown in bold in table 2. This represents about 20 percent of the total. About the 

same proportion might therefore have been expected among the 50 or so species 

characterised since 2000. But this is not the case. As can be seen in table 3, the 

proportion for these post-2000 species is ~30/50, or 60 percent. 

This difference between the pre- and post-2000 proportions (20 percent versus 60 

percent) might be explained by three factors: (1) before 2000, more species of 
GENERAL TECHNICAL REPORT PSW-GTR-221

 108

significance to agriculture may have been identified because of a research emphasis 

on agricultural problems, (2) since 1990, there has increasing interest and concern 

about the impact of Phytophthoras in forests and natural ecosystems, and (3) since 

the 1990s, there may have been an increased spread of new species from exotic 

habitats into nurseries and into natural environments as a result of growing 

international trade in plants. 

Whatever the precise factors involved, the fact that a greater proportion of the ‘new’ 

species appear to be associated with nurseries, forests and other natural environments 

clearly has implications for international plant biosecurity and plant health practice. 

That current international biosecurity protocols are inadequate has been discussed 

elsewhere (Brasier 2005, 2008). 

Phylogenetic Implications of the Increasing 

Number of New Phytophthora Species 

Numbers again 

The first comprehensive molecular phylogeny of Phytophthora and other Oomycetes 

(Cooke and others 2000) was, based on ITS sequences. It covered some 47 of the ca

55 Phytophthora morphospecies known at the time (table 2). The taxa were assigned 

to ten major Clades numbered 1–10 (fig 3). This overall structure is still retained in 

recent multi-gene trees, such as four locus tree of Kroon and others (2004) and the 

seven locus tree of Blair and others (2008; fig 4). It is interesting to consider how the 

many new species described since Cooke and others (2000) (table 3), or indeed the 

possible 200–600 species that may eventually be found, will affect the structure and 

interpretation of the trees. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 109 

Figure 3—Phytophthora Clades 1–8 as originally identified by Cooke and 

others (2000) from ITS sequences. Their Clades 9 and 10 are not shown. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 110

Figure 4—Phytophthora Clades 1–10. A recent seven locus concensus tree 

constructed by Blaire and others (2008). 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 111 

A typical example of the continuing numerical increase in species is represented by 

the expansion of Clade 8. This has already gone from nine species in Cooke and 

others (2000) to about 16 species today (fig. 4). In the Cooke and others tree, P. 

lateralis was something of a lone outrider. Now this species is part of a significant 

subcluster (subclade 8c, fig. 4), having been joined by P. hibernalis (cultures of 

which were not available to Cooke and others) and two new species P. ramorum and 

P. foliorum. Presumably, more new species will follow. 

The Ever Growing Clade 6 

The considerable increase in the number of Clade 6 species is one of the most 

interesting developments. When first constructed by Cooke and others (2000), Clade 

6 contained only P. megasperma, P. humicola, P. inundata (as P. sp. ‘O-group’) and 

P. gonapodyides. Subsequently, a combined phenotypic and molecular study was 

carried out on putative Clade 6 isolates of uncertain status from culture collections in 

Europe and North America (Brasier and others 2004). This study identified a number 

of unique taxa, many of them high temperature and/or sexually sterile species. It also 

extended the number of known Clade 6 species to 13. Their phylogenetic 

relationships are shown in figure 5. Some of these new taxa have yet to be formally 

described e.g. P. taxon salixsoil, P. taxon rasberry and P. taxon walnut (table 2). 

Another, P. taxon forestsoil, has recently been described as P. sylvatica sp. nov 

(table 2 and J. Bakonyi, personal communication). 

Figure 5—Phylogenetic relationships of the thirteen taxa of Clade 6 

recognised by Brasier and others (2004). Based on ITS sequences. 

To these 13 species can now also be added P. hungarica sp. nov. (table 3 and J. 

Bakonyi, personal communication) and P. taxon orphan identified in the ICMP 

culture collection in New Zealand (table 3 and Beever and others 2006). And that is 

not all. Five of the seven ‘new’ taxa recently identified by Stukely, Burgess and 
GENERAL TECHNICAL REPORT PSW-GTR-221

 112

Hardy in the VHS culture collection also fall within Clade 6, bringing the current 

number of species in Clade 6 to about 20. Overall, these additions represent an 

increase of 16 taxa since Cooke and others (2000). 

Of course, not all the additions are new discoveries. Some have languished in the 

culture collections owing to the considerable difficulty in identifying species units 

within this group before population-based species concepts and molecular phylogenetic tools were applied see Brasier and others 2004. And the Clade 6 saga does 

not end there. Of the ten unknown phylotaxa recently identified by Hansen and 

colleagues in south west Oregon streams, the majority probably also belong in 

Clade 6. 

So, how many extant species are there in Clade 6, including the current unknowns? 

Let’s play again! If we start with a figure of ~15 known or described Phytophthora

taxa in Clade 6 at the time of this meeting (based on data up to and including Cooke 

and others 2000, Brasier and others 2004, Beever and others 2006 plus P. sylvatica 

and P. hungarica) and use the figure of approximately 105 currently known or 

described Phytophthora species overall (Tables 2 and 3 combined), then the number 

of Phytophthora species presently assigned to Clade 6 is ca 14 percent of the total. If 

we allow my previous estimate of 200–600 extant species in the genus, then 14 

percent would indicate there may be between 28 and 84 extant species in Clade 6. 

This means that the number of species in Clade 6 might even be greater than the 

number of Phytophthora species known to science in 1999 ... surely a figure to 

conjure with! 

Many of the Clade 6 taxa in the 

ITS tree shown in Brasier and 

others (2004) are very closely 

related i.e. separated by only a 

few base substitutions, 

indicating recent divergence, 

especially those in Subclade II 

(fig. 5). It is therefore likely that 

many of the so far unidentified 

species in Clade 6 will be 

closely related to existing 

species and to each other. In 

which case Clade 6 could 

become very crowded indeed, 

as indicated in figure 6! 

Subclade 6 is already becoming 

too crowded to show all the taxa 

in a rooted tree without double 

or triple parking the labels. 

Furthermore, if the ‘missing’ 

taxa also prove to be 

phenotypically somewhat 

similar (aquatic, sterile or 

inbreeding, high temperature) 

species, such as P. 

gonapodyides, P. taxon 

Figure 6—How Clade 6 might look, 

theoretically, if the minimal estimate of 28 

extant species were represented (compare 

with figure 5). Note the now very crowded 

taxa of Subclade II. Consider what the tree 

might look like with the maximum estimate of 

84 extant species. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 113 

Pgchlamydo, P. taxon salixsoil etc (Brasier and others 2004), then we may have to 

rethink our Phytophthora species concept, at least for Subclade II. We will also face 

the challenge of identifying the evolutionary processes which have bought about such 

a high level of phylogenetic diversity. 

Phylogenetic Pitfalls? 

Since the ITS tree of Cooke and others (2000), we have been faced somewhat 

unexpectedly with a rapidly rather than a gradually expanding Phytophthora

phylogenetic tree. For the foreseeable future, trees produced every few years are 

likely to acquire more ‘twigs and branchlets’. They will be more sophisticated in 

terms of genetic analysis and will look increasingly impressive in size. But what do 

these trees provide in terms of our fundamental understanding of Phytophthora? 

Obviously, trees constructed from DNA should be an order of magnitude better than 

the morphological groups of Waterhouse (1963), or an earlier morphology-based 

phylogeny of my own (Brasier 1982). While Waterhouse’s groups and key provided 

a valuable taxonomic framework for forty years, and were presented by her as 

necessarily artificial constructs, we can be reasonably confident that our molecular 

phylogenetic trees are much more representative of ‘natural relationships’. However, 

can we also be confident about what they tell us of the evolutionary history of the 

known taxa? 

I suggest we should treat our fast growing phylogenetic trees with a degree of 

caution. First, many taxa, and therefore much data, are missing because of past 

extinctions. Some of these missing taxa could be key ancestral taxa that would have 

lain at or closer to the nodes and branches of the main clades, but disappeared during 

major evolutionary jumps and radiations. Second, much evidence of past reticulation 

(inter-specific hybridisation) events may be missing. Where new taxa have arisen via 

interspecific hybridisation or introgression in the ancient past, the genetic footprints 

of the process will often have been lost over time through genetic rearrangement and 

concerted evolution. Third, much information is missing because we may still have to 

add to the trees the estimated 100–500 unknown Phytophthora species. 

Figure 7—The real issues? Why did ancestral species ‘A’ give rise to Clades 

1 and 2? Why did ancestral species ‘W’ give rise to species X, Y and Z (P. 

cactorum, P. idaei, P. pseudotsugae) ? Diagram adapted from Cooke and 

others 2000. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 114

The addition of some of these new taxa may well alter the substructure within some 

of the Clades, as already seen for Clades 6 and 8. There might even be missing 

Clades. 

What is surely important in terms of interpreting the trees is not so much that species 

X and Y are closely related e.g. that P. ramorum and P. lateralis are related within 

Clade 8. So what? What does that really tell us? In Darwinian terms, what is more 

important is to understand are the processes and events that gave rise to their 

divergence: the processes that were responsible for the nodes or branches. Why did 

ancestral species A give rise to Clades 1 and 2? (fig. 7). Or why did ancestral species 

W give rise to three W species X, Y and Z? (fig. 7). Traditional fungal taxonomy lost 

its way in the middle of the last century by concentrating too much on the what 

(morphological and descriptive) and not enough on the how and why issues (the 

parameters which defined species boundaries in evolutionary terms; cf. Brasier 

1997). Maybe Phytophthora molecular phylogenetics is in danger of doing the same? 

Concluding Comments 

To me, this dip into Phytophthora numerical biodiversity and phylogenetic 

uncertainties has been another reminder of how little we know about the origins of 

the genus. Indeed, it seems reasonable to suggest that the construction of a 

phylogenetic tree is simply the point at which the real evolutionary challenges begin. 

Given that there are likely to be between 200 and 600 Phytophthora species out there, 

I see two key challenges for the future. 

One is to understand why there are so many Phytophthoras. What environmental 

processes and events (geographic, climatic, biotic) drove their evolution? Why did 

e.g. P. ramorum and P. lateralis diverge from a common ancestor, or from each 

other? Addressing this set of questions is the job of the evolutionary biologist. 

Another is to understand, at a basic genetic level, how the species are different e.g. 

how P. ramorum differs from P. lateralis. Addressing this set of questions is the job 

of the classical geneticist and the genomicist. In the context of the present paper, it 

was also the job of the next speaker (Howard Judelson, Chapter X). 

Fortunately, these two challenges are, of course, not mutually exclusive, and answers 

obtained for one could enormously benefit understanding in the other. 

Literature Cited 

Arcate, J.M.; Kemp, M.A.; Nelsen, E.B. 2006. Diversity of Perenosporomycete (Oomycete) 

communities associated with the rhizophere of different plant species. Microbial Ecology. 

51: 36–50. 

Blair, J.E.; Coffey, M.D.; Park, S-Y; Geiser, D.M.; King, S. [in press]. A multi-locus 

phylogeny for Phytophthora utilizing markers derived from complete genome sequences. 

Fungal genetics and Biology. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 115 

Beever, R.E.; Ramsfield, T.D.; Dick, M.A.; Park, D.; Fletcher, J.; Horner, I.J. 2006.

Molecular characterisation of New Zealand isolates of the fungus Phytophthora: Landcare 

Research Contract Report LCOJ06/155, MAF, Wellington, New Zealand. 43 p. 

Brasier, C.M. 1982. Problems and prospects in Phytophthora research. In: Erwin, D.C.; 

Bartnicki-Garcia, S.; Tsao, P.H., eds. Phytophthora, its Biology, Taxonomy, Ecology and 

Pathology. St Paul Minnesota: American Phytopathological Society: 351–364. 

Brasier, C.M. 1997. Fungal species in practice. Identifying species in fungi. In: Claridge, 

M.F.; Dahwah, H.A.; Wilson, M.R., eds. Species: the units of biodiversity. U.K.: Chapman 

and Hall: 135–170. 

Brasier, C.M.; Cooke, D.E.L.; Duncan, J.M.; Hansen, E. 2003. Multiple new phenotypic 

taxa from trees and riparian ecosystems in Phytophthora gonapodyides—P. megasperma ITS 

Clade 6, which tend to be high-temperature tolerant and either unbreeding or sterile. 

Mycological Research. 107: 277–290. 

Brasier, C.M. 2005. Preventing invasive pathogens: deficiencies in the system. The 

Plantsman. 4: 54–57. 

Brasier, C.M. 2008. The biosecurity threat to the U.K. and global environment from 

international trade in plants. Plant Pathology. (in press due out shortly)

Cooke, D.E.Ll.; Drenthe, A.; Wagels, G.; Duncan, J.M.; Brasier, C.M. 2000. A molecular 

phylogeny of Phytophthora and related Oomycetes. Fungal Genetics and Biology. 30: 17–32. 

Crous, P.W.; Groenewald, J.Z. 2005. Hosts, species and genotypes: opinions versus data. 

Australian Plant Pathology. 34: 463-470. 

De Bary, A. 1876. Researches into the nature of the potato fungus Phytophthora infestans. 

Journal of the Royal Agricultural Society of England, Series 2. 12:239–269. 

Erwin, D.C.; Ribeiro, O.K. 1996. Phytophthora diseases worldwide. St. Paul, Minnesota: 

American Phytopathological Society: 562 p. 

Jung, T.; Cooke, D.E.L.; Blashke, H.; Duncan, J.M.; Osswald, W. 1999. Phytophthora 

quercina sp.nov., causing root rot of European oaks. Mycological Research. 103: 785-798. 

Kroon, L.P.M.N.; Bakker, F.T.; van den Bosch, G.B.M.; Bonants, P.J.M.; Flier, N.G. 

2004. Phytogenetic analysis of Phytophthora species based on mitochondrial and nuclear 

DNA sequences. Fungal Genetics and Biology. 41: 766–782. 

Newhook, F.J.; Waterhouse, G.M.; Stamps, D.J. 1978. Tabular key to the species of 

Phytophthora de Bary. Mycological Paper 143. Commonwealth Mycological Institute, Kew, 

U.K. 20 p. 

Rosenbaum, J. 1917. Studies in the genus Phytophthora. Journal of Agricultural Research. 

8: 233–276. 

Tucker, C.M. 1931. Taxonomy of the genus Phytophthora de Bary. University of Missouri 

Agricultural Experiment Station Research Bulletin 153. 207 p. 

Waterhouse, G.M. 1963. Key to the species of Phytophthora de Bary. Mycological Paper 92. 

Commonwealth Mycological Institute, Kew, U.K. 22 p. 
GENERAL TECHNICAL REPORT PSW-GTR-221

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Interactions

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Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

119 

First Report of Ink Disease on Chestnut 

Caused by Phytophthora katsurae in Korea1 

Eunsung Oh,2 Keum Chul Shin,3 Sang Hyun Lee,2 Kyung Hee 

Kim,2

 and Jong Kyu Lee3 

Abstract 

Phytophthora is well known as a destructive pathogen on crops and nursery stocks. In the late 

1990s Phytophthora species, newly described or suddenly lethal to mature forest trees, have 

appeared in the United States and Europe. Some cultivars of chestnut (Castanea crenata X C. 

mollissima) in Korea have also died without knowing the causal agent in last two years. The 

typical symptom was black-oozed, reddish, sunken tissue on trunk. When bark was peeled off, 

a distinct necrotic region on basal trunk and the vertical spread of the discoloration were 

observed. In November 2006, we were able to isolate the agent from the necrotic areas, and 

thirteen isolates were obtained from three different areas. The isolates produced numerous 

homothallic oogonia (34.0~46.2 x 21.9~26.7 um) with warty protuberance on the surface and 

a long, funnel-shaped stalk enclosed by antheridium (amphigynous) at the base. No 

chlamydospores were observed, but the formation of papillate, ovoid to obpyriform sporangia 

(17.0~38.9 x 14.6~29.2 um) were induced by cold treatment in filtered creek water or deionized water. Based on morphological characteristics and rDNA ITS sequencing, the isolate 

showed 99.6 percent similarity with P. katsurae (AF266771), indicating three base pair 

differences. All isolates from 3 different areas were completely identical in the comparison of 

rDNA ITS sequence. Resistance or susceptibility of different cultivars of chestnut against the 

isolate showed variations among cultivars, resulting in potential impact on chestnut 

plantations with many cultivars in Korea. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Division of Forest Diseases and Insect Pests, Department of Forest Environment, Korea Forest Research Institute, 

Seoul, 130-712, South Korea. 

3

 Forest Pathology and Mycology Lab., Kangwon National University, Chunchon, 200-701, South Korea. 

Corresponding author: eunsungoh@foa.go.kr. 
GENERAL TECHNICAL REPORT PSW-GTR-221

120 

Development of Molecular Diagnostic Tools 

for the Detection of Phytophthora 

cinnamomi From Cryptic Soil Samples in 

Southern Australia1

Nari M. Williams,2 Emer O’Gara,2 Giles E.St.J. Hardy,2 and 

Phillip A. O’Brien2

Abstract 

Phytophthora cinnamomi is responsible for the widespread destruction of native forest and 

heathlands across southern Australia. In these ecosystems, P. cinnamomi is responsible for the 

death of a broad range of susceptible species including members of the Proteaceae, 

Epacridaceae, Papilionaceae and Myrtaceae, and results in significant changes in species 

composition, community structure and ecosystem function. Management of the pathogen in 

native ecosystems is centred on pathogen containment and relies on the identification of 

dieback boundaries and detection of the pathogen directly from soil. Once an infestation is 

defined, preventative measures may be taken to ensure infested soil and plant material are not 

spread during land management activities. Ongoing monitoring of the pathogen then forms an 

integral component of sustainable ecosystem management. 

Efficient management of P. cinnamomi is impeded by the inability to consistently detect the 

pathogen from infested soil samples. This is especially the case for cryptic sites in which there 

is no apparent expression of plant symptoms. Such situations commonly occur where sites are 

excavated during mining activities or are disturbed by fire. In the absence of a better 

alternative, land managers still use baiting analysis of soil samples in formulating 

management plans for P. cinnamomi containment. This is despite the fact that recoveries are 

often low and there is a high risk of false negatives. This in turn limits the confidence placed 

on the results of baiting analysis. DNA based detection offers improved sensitivity and higher 

sample throughput for the detection of P. cinnamomi than baiting assays. Through our 

research, comparative analysis using PCR based methods in parallel to baiting assays has 

shown a significant increase in the detection of P. cinnamomi by nested PCR. However, 

although the benefits of DNA based diagnostic tools have good promise for future disease 

management, low and variable target populations mean that sampling strategy and confidence 

levels remain the key issues in delivering a reliable and consistent diagnostic service. 

This presentation will examine the challenges encountered during the development and 

validation of nested and real time PCR protocols for the detection of P. cinnamomi from soil 

collected from native ecosystems throughout southern Australia. The implications for using 

molecular diagnostic assays as management and research tools for P. cinnamomi will be 

discussed. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2

 Centre for Phytophthora Science and Management, Murdoch University 

School of Biological Science and Biotechnology, South Street, Murdoch, Western Australia, Australia. 

Corresponding author:Nari.Anderson@murdoch.edu.au. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

121 

Host-Pathogen Interaction of Phytophthora 

citricola and P. pseudosyringae With 

European Beech (Fagus sylvatica)

1

Julia Koehl,2 Roberto Portz,2 Vera Martin,2 Angela Simonsmeier,2

and Wolfgang Oßwald2

Abstract 

Fagus sylvatica is an important tree species and due to its immense tolerance towards 

different climate conditions and habitats it is one of the major tree species in European 

forests. Over the past years the number of trees infected with Phytophthora increased 

dramatically. Particularly P. citricola was described as an important and an aggressive species 

in this context, that besides P. cactorum and P. cambivora lead to destruction of trees. 

Noticeable differences could be observed regarding virulence and symptom development: 

Infection with P. citricola that was identified as most aggressive species resulted in massive 

reduction of fine root mass. Before trees showed any wilting symptoms, rates of 

photosynthesis and transpiration decreased. Intriguingly also the nonaggressive Phytophthora 

species P. pseudosyringae reduced fine root system likewise, but neither wilting nor die back 

of plants occurred (Jung and others 2003; Fleischmann and others 2004). The root system of 

beech seedlings infected with P. pseudosyringae was reduced in length but far less destructed 

than P. citricola infected roots which points out some kind of development of resistance in 

beech seedlings after infection with P. pseudosyringae. This project aims to investigate these 

mechanisms by employing an aggressive and a non-aggressive Phytophthora species on 

beech, since until now any resistance mechanisms involved in Fagus sylvatica -Phytophthora 

interaction are completely unknown. Besides the above ground symptoms we investigated the 

extent and mode of action of the infection process of both Phytophthora species in beech 

roots. We applied histological, physiological (photosynthesis, water potential, transpiration) 

and molecular (qPCR and gene expression) methods to determine nature and progression of 

the infection in beech seedlings with P. citricola and P. pseudosyringae as well as a temporal 

dependence of symptom development on quantity of infestation. A biphasic course of 

infection of the root tissue could be observed after infection with Phytophthora, at which the 

first increase was due to zoospore encystment within the first 36 hours. The second peak is 

correlated with penetration and growth in beech root tissue. Most remarkably was the fast and 

immense colonization of xylem vessels by hyphae of P. citricola, from where rapid 

propagation of this aggressive Phytophthora species into other parts of the plant evolves. The 

activity of hydrolytic enzymes (as glucanases and invertases) in both Phytophthora species 

was investigated and content of soluble sugars, as well as starch were determined to elucidate 

their role for colonization by Phytophthora species with different pathogenic impact. At the 

present time our group is about to transform P. citricola and P. pseudosyringae with GFP 

(green fluorescent protein) to acquire three-dimensional data sets from intact, individual, 

pathogen encounter sites in planta and to open possibilities for analysis of functional genes 

involved in infection processes. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2

 Section Phytopathology of Woody Plants, Technische Universität München, 

Am Hochanger 13, 85350 Freising, Germany. 

Corresponding author:j.koehl@lrz.tu-muenchen.de. 
GENERAL TECHNICAL REPORT PSW-GTR-221

122 

Literature Cited 

Jung, T.; Nechwatal, J.; Cooke, D.E.L.; Hartmann, G.; Blaschke, M.; Oßwald, W.F.; 

Duncan, J.M.; Delatour, C. 2003. Phytophthora pseudosyringae sp. nov., a new species 

causing root and collar rot of deciduous tree species in Europe. Mycological Research 107: 

772-789. 

Fleischmann, F.; Göttlein, A.; Rodenkirchen, H.; Lütz, C.; Oßwald, W. 2004. Biomass, 

nutrient and pigment content of beech (Fagus sylvatica) saplings infected with Phytophthora 

citricola, P. cambivora, P. pseudosyringae and P. undulata. Forest Pathology 34, 79–92. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

123 

Investigation of the Effects of Phytophthora 

alni subsp. alni Collar Inoculation on CO2

Uptake, Transpiration, and Carbohydrate 

Content of Alnus glutinosa Saplings1

Julia Koehl,2 Frank Fleischmann2, Rainer Matyssek3 and 

Wolfgang Oßwald2

Abstract 

Three-year-old Alnus glutinosa trees were single or double-inoculated at the stem basis with 

Phytophthora alni subsp. alni under natural climate conditions to clarify local and systemic 

pathogenic effects on young alder saplings. Lesion formation on the bark showed a biphasic 

pattern with moderate extension rate in spring and strong development during late-summer. 

However, large variability was encountered in pathogen development within the collective of 

the infected plants, ranging between high susceptibility and almost resistant. Infection resulted 

in strong growth retardation, and finally 75 percent of all inoculated trees had died after two 

years of infection. During disease development, rates of CO2 uptake and transpiration were 

significantly reduced. Consequently, minimum levels of leaf water potential were less 

negative in infected than control trees, reflecting stomatal narrowing under infection. A linear 

regression was found, when all girdling values of the single and double inoculated saplings 

were plotted versus the corresponding mean values of photosynthesis measured in 2004, 

proving the inhibitory systemic effect of stem girdling on leaf photosynthesis. In the second 

year of infection the amount of girdling of all surviving saplings decreased to values below 50 

percent. All these saplings did no longer differ from controls in terms of photosynthesis, 

transpiration and water use efficiency (WUE). Starch levels of leaves of infected trees were 

significantly increased relative to control trees, possibly indicating destruction of the bark 

tissue by the pathogen to cause blockage of phloem transport from leaves to roots. Given such 

a syndrome scenario, stomatal closure appears to result from product-inhibited photosynthesis 

upon phloem disruption, preventing WUE to decline. Hence, the found gas exchange 

responses are systemic responses of the local pathogen infection at the stem basis. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2

 Section Phytopathology of Woody Plants, Technische Universität MünchenAm Hochanger 13, 85354 Freising, 

Germany. 

3

 Chair for Ecophysiology of Plants, Technische Universität MünchenAm Hochanger 13, 85354 Freising, Germany. 

Corresponding author:osswald@wzw.tum.de. 
GENERAL TECHNICAL REPORT PSW-GTR-221

124 

Phytophthora ramorum Tissue Colonization 

Studied With Fluorescence Microscopy1

Marko Riedel,2 Stefan Wagner,2 Monika Götz,2 Lassaad Belbahri,3

Francois Lefort,3

 and Sabine Werres2

Abstract 

The continual worldwide spread and the expanding host spectrum of P. ramorum has 

become a serious threat to natural plant communities. To counter this threat, detailed 

knowledge about infection pathways and tissue colonization is essential. To analyze 

these issues, histological studies of infected tissue with epifluorescence microscopy 

have been started. For first infection tests the Rhododendron has been taken as a model 

host. Roots of potted Rhododendron cuttings were infected with the P. ramorum 

isolate BBA 9/95 (ex-type strain). Inoculation of the non-injured plants was done by 

application of a zoospore suspension onto the surface of the pot soil. The plants were 

incubated with 16 hours of light at 20°C in a quarantine chamber. Samples of healthy 

looking plants and plants with typical symptoms were taken and fixed. Unstained 

hand-cuttings were analyzed with fluorescence microscopy. The development of 

zoosporangia on leaf surfaces was analyzed using the vital stain FUN®1 (Molecular 

Probes). Natural autofluorescence of P. ramorum and plant tissue is enhanced using 

the fixing fluid FAA (formaline-aceto-alcohol). 

Epifluorescence images showing P. ramorum structures in different tissues and in 

different stages of disease development are presented. An overview of the 

development of P. ramorum in Rhododendron root, twig and leaf tissue is presented 

for discussion. 

Introduction 

In Europe Rhododendron plants present the main risk for the spread of P. ramorum in 

natural ecosystems. Using Rhododendron as a model system for the study of woody 

plants was a logic consequence. In preceding infection trials with irrigation water in 

containered Rhododendron stands (Werres and others 2007) P. ramorum was 

detectable in healthy looking root balls of asymptomatic plants. At this time only 

limited knowledge of root infection in rhododendron plants was available. 

Furthermore little is known about the whole issue of P. ramorum latency. To 

approach these problems infection trials to investigate P. ramorum colonization in 

root-infected Rhododendron plants were started. 

Traditionally in fungal phytopathology transmission light microscopy is used to 

analyze tissue colonization and host-pathogen interaction. This technique usually 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2

 Institute for Plant Protection in Horticulture, Federal Biological Research Centre for Agriculture and Forestry 

(BBA), Messeweg 11/12, D-38104 Braunschweig, Germany. 

3

 Laboratory of Applied Genetics and Plant Health, Institute Earth Nature and Landscape, University of Applied 

Sciences of Western Switzerland, 150 Route de Presinge 1254 Jussy, Switzerland. 

Corresponding author: m.riedel@bba.de. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

125 

requires laborious pre-treatments of the samples like clearing, staining, embedding 

and thin sectioning (Pogoda and Werres 2004). To highlight fungal or oomycetic 

structures a wide range of stains including fluorescence dyes have been used (Zhang 

and Dickinson, 2001). Most of these dyes fail to specifically stain only the pathogen 

structures. Most often host tissue is stained too and not all of the pathogen structures 

showed the same staining intensities. 

In this study we found that P. ramorum tissue expresses a very strong and specific 

natural autofluorescence which is distinguishable from surrounding host tissue. Here 

we report a simple method for analyzing host-pathogen interaction at a histological 

level using the natural autofluorescence of Rhododendron tissue and P. ramorum

structures. 

Material and Methods 

Plant material and inoculations 

As host plants, rooted Rhododendron cuttings of the cultivar `Cunninghams White ́ 

were maintained in a quarantined chamber at 20 °C with 16 hours of light. 

Inoculations were done using a 5ml aqueous suspension containing 30.000 zoospores 

of the European P. ramorum isolate BBA 9/95. The inoculum was applied directly 

onto the freshly irrigated potting medium. 

Sample preparation and microscopic analysis 

Samples (segments of root stem and foliar tissue) from asymptomatic and 

symptomatic plants were taken at regular intervals starting seven days after 

inoculation. The samples were fixed using the fixing fluid formaline-aceto-alcohol 

(FAA). Prior to hand sectioning, the samples were washed in de-ionized water for 15 

minutes. Whole fine roots or hand sections were mounted in water on microscopic 

slides and then used for microscopic observation. 

For microscopic analysis a Zeiss Photomicroscope II with epifluorescence equipment 

was used. Observations were made using the × 10, × 16 or × 40 lenses and the Zeiss 

fluorescence filter set No. 05 (excitation filter: 395–440 nm, 460 nm beam splitter 

and 470 nm long pass filter). Emission spectra profiles (lambda scan) were generated 

using the Leica TCS SP2 confocal laser scanning microscope (CLSM) and the Leica 

LCS software. Excitation was done with a 488 nm Argon Krypton laser at power 

levels of 10 percent to 50 percent. Settings for the gain and offset were 700 to 750 

and 0 to -20 respectively. 

Results and Discussion 

P. ramorum emits a strong natural autofluorescence when excited with blue light 

(430 to 500 nm). Using confocal laser scanning microscopy (CLSM) the emission 

maximum could be located in the green-yellow area at approx. 550 nm (fig. 1). 

Excited with the same light the different types of host tissue show a specific emission 
GENERAL TECHNICAL REPORT PSW-GTR-221

126 

spectra too. Using this emission, pathogenic structures were clearly distinguishable 

from host tissue. 

Figure 1—Natural P. ramorum autofluorescence, emission spectra profile 

(CLSM lambda scan), A: CLSM image of a hypha, B: typical emission curves 

with maximum at approx. 550 nm 

Furthermore, dependent on the state of infection (asymptomatic, discolouration, 

necrosis, etc.) the spectra of host tissue exhibit specific variations. The red 

autofluorescence of chlorophyll indicates the asymptomatic state of green tissue in 

leaf palisade mesophyll or the cortex of stems. In pith and cortex, asymptomatic 

stages are marked by light green and light yellow autofluorescence respectively. With 

the beginning of discolouration the fluorescence shifts to yellow and brownish 

colours. Necrotic and collapsed tissue exhibits a typical brown colouration. Thus the 

determination of tissue type and state can be evaluated by shape and colour. 

In asymptomatic plants the presence of chlamydospores in roots was observable 7 to 

approx. 60 days after inoculation. The spores were predominantly located in the root 

exodermis on lateral sites of fine roots (fig. 2). The majority of chlamydospores 

exhibited a bright yellowish autofluorescence which enabled very easy detection in 

the root tissue. Occasionally chlamydospores with an unusual red autofluorescence 

were found. The germination of chlamydospores could be observed in some cases. 

First phaenotypic visible disease symptoms appeared 15 days after inoculation. In 

early stages of infection hyphae are the predominant P. ramorum propagules found in 

pith, xylem, phloem and the cortex of roots and stems (fig. 2). Especially the xylem is 

most extensively colonized by hyphae. In vessels, hyphae grow mainly intracellularly 

but penetration of cell walls (intercellular growth) occurs regularly. In later stages of 

infection massive colonization by hyphae was found in nearly all types of host tissue. 

Furthermore in xylem of infected plants an increased production of tyloses can be 

observed. The production of tyloses is often referred to as a possible reaction of 

plants to progressing infections. 

In progressed stages of infection the production of chlamydospores can be observed 

in the tissue of pith and cortex. While in pith tissue chlamydospores can be observed 

only occasionally, in cortex chlamydospores they can often accumulate in high 

numbers. The majority of these chlamydospores show, similarly to the spores found 

A B
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

127 

in roots, a typical bright yellow-greenish colouration. But also chlamydospores 

exhibiting red autofluorescence were found. Most abundantly these red spores were 

found in discoloured or necrotic tissue. 

Colonization of foliar tissue follows pathways similar to the situation observed in 

stems. Early stages of infection are characterized by the spread of hyphae via veins. 

Later a massive colonization of mesophyll tissue can be seen and hyphae start to 

grow out of stomata (fig. 2). At this stage foliar tissue still appears to be intact which 

is indicated by the red autofluorescence of chlorophyll. Under optimal conditions 

(humid and cool) zoosporangia start to develop on hyphae branching out of stomata 

(fig. 2). When leaves start to show signs of discolouration, chlamydospores can be 

found inside foliar tissue. In necrotic tissue chlamydospores can be found 

accumulated in huge numbers (fig. 2). 

The occurrence of chlamydospores with unusual red autofluorescence is, except for 

the roots, linked to the presence of discoloured or necrotic tissue. In vitro, cultured 

chlamydospores never exhibited such red autofluorescence. In progressed states of 

infection when host tissue starts to collapse most of the chlamydospores show red 

fluorescence (fig. 2I). Additionally at this stage of infection in the tissue of roots, 

stems and leaves, areas were found where a similar red autofluorescence occurs (figs.

2C and 3B). With CLSM lambda scans it could be shown that both cases of red 

autofluorescence have similar emission spectra profiles (fig. 3). It can be assumed that 

substances like phenolics which are emitted by the plant during the process of plant 

defence or in the progress of tissue-collapse, represent the origin of the red 

autofluorescence. 
GENERAL TECHNICAL REPORT PSW-GTR-221

128 

A B

C D

E F

G H I
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

129 

Figure 2 (preceeding page)2Microscopic images of P. ramorum propagules in 

rhododendron tissue. A, C–I: epifluorescence images, B: transmission light 

image 

A, B: Chlamydospores in exodermis of healthy looking roots, C: Growth of 

hyphae (arrows) inside xylem of discoloured roots and accumulation of 

substances with red autofluorescence. D: hyphae (arrows) growing alongside 

of vessels, E: production of chlamydospores in pith tissue of discoloured

stems, F: chlamydospores with yellow and red autofluorescence in collapsing 

cortex of discoloured stems, G: hyphae growing out of stomata on 

asymptomatic leaves, H: zoosporangia produced on hyphae growing out of 

stomata of discoloured leaves, I: accumulation of chlamydospores in necrotic 

mesophyll cells of foliar tissue, white bars indicate 50 μm 

Figure 32Red autofluorescence in P. ramorum chlamydospores and infected 

rhododendron tissue. A: chlamydospore in cortex of discoloured stem 

showing unusual red autofluorescence, B: CLSM emission spectra profile of 

red chlamydospore, C: red autofluorescence in cortex of discoloured 

rhododendron stem, D: CLSM emission spectra profile of stem tissue 

exhibiting red autofluorescence, white bars indicate 50 μm 

A B

A 

C D

A 
GENERAL TECHNICAL REPORT PSW-GTR-221

130 

Acknowledgments 

We would like to thank USDA-Forest Service, Pacific Southwest Research Station 

for funding the project. 

Literature Cited 

Pogoda, F.; Werres, S. 2004. Histological studies of Phytophthora ramorum in 

Rhododendron twigs. Canadian Journal of Botany-Revue Canadienne de Botanique. 

82: 1481-1489. 

Werres, S.; Wagner, S.; Brand, T.; Kaminski, K.; Seipp, D. 2007. Survival of 

Phytophthora ramorum in recirculating irrigation water and subsequent infection of 

Rhododendron and Viburnum. Plant Disease. 91: 1034–1044. 

Zhang, L.; Dickinson, M. 2001. Fluorescence from rust fungi: a simple and effective method 

to monitor the dynamics of fungal growth in planta. Physiological and Molecular Plant 

Pathology. 59: 137–141. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

131 

Spread of Infection Within Tanoak 

(Lithocarpus densiflorus) Trees Inoculated 

With Phytophthora ramorum1

Jennifer Parke,2,3 Bradley Collins,3 Gerri Buckles,2 Everett 

Hansen,3 and Barb Lachenbruch4

Abstract 

In an experiment with mature naturally infected tanoak trees, Phytophthora ramorum was 

observed in sapwood and appeared to interfere with stem water transport (Parke and others 

2007). We postulated that colonization of the xylem could contribute to rapid spread within 

the tree and lead to development of crown mortality symptomatic of sudden oak death. In this 

field experiment, we compared the initial spread of P. ramorum within xylem and phloem of 

tanoak trees inoculated at specific locations in the bole, basal sprouts, and belowground 

tissues. We determined hydraulic conductivity of sapwood from inoculated vs. wounded boles 

and investigated the spatial and temporal development of tyloses in response to infection. 

The experiment was conducted in a redwood-tanoak stand at El Corte de Madera Open Space 

Preserve in the Midpeninsula Regional Open Space District in San Mateo Co., California. 

Boles of understory tanoak trees (10-15 cm DBH) were inoculated in May 2006 with mycelial 

plugs of P. ramorum placed at the cambium. Bole controls received agar plugs without the 

pathogen. Twigs of basal sprouts and belowground tissue were inoculated similarly. There 

were twelve replicate trees for each of the four treatments (bole wounded control, boleinoculated, sprout-inoculated and root-inoculated). Half of the trees were harvested in 

September 2006 and the remaining trees were harvested in 2007. Sapwood samples were 

excised for hydraulic conductivity assays, and adjacent sapwood and phloem tissues were 

used for pathogen isolation, diagnostic PCR, and microscopy to determine the extent and 

location of P. ramorum infection. 

For both sampling times, inoculated boles with xylem infections had significantly more 

tyloses compared to wounded, non-inoculated boles (Collins, 2008). In addition, the increase 

in tyloses was associated with a decrease in specific hydraulic conductivity, suggesting that 

tyloses induced by infection with P. ramorum could interfere with stem sap flow. Over time, 

tylosis development increased in tissues further away from the inoculation site, preceding the 

vertical spread of infection. Results suggest that infected sapwood contains numerous tyloses 

which could significantly impede stem water transport. This is consistent with the hypothesis 

that tanoak mortality associated with P. ramorum infection results, at least in part, from 

impaired hydraulic conductivity (Collins and others, submitted). In this study, infections did 

not spread from inoculated basal sprouts to the boles, nor did they spread from inoculated 

belowground tissues to aboveground tissues. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2 Department of Crop and Soil Science, 3017 Agriculture and Life Sciences Building, Oregon State University, 

Corvallis, OR 97331, USA. 

3

 Department of Botany and Plant Pathology, 2082 Cordley Hall, Oregon State University, Corvallis, OR 97331, 

USA. 

4

 Department of Wood Science and Engineering, 118 Richardson Hall, Oregon State University, Corvallis 97331, 

USA. 

Corresponding author: Jennifer.Parke@oregonstate.edu. 
GENERAL TECHNICAL REPORT PSW-GTR-221

132 

Literature Cited 

Collins, B.R. 2008. The effects of Phytophthora ramorum infection on hydraulic conductivity 

and tylosis formation in tanoak sapwood. M.S. Thesis, Oregon State University, Corvallis. 61 

pp. 

Collins, B.R.; Lachenbruch, B.; Hansen, E.; Parke, J.L. The effects of Phytophthora 

ramorum infection on hydraulic conductivity and tylosis formation in tanoak sapwood.

(manuscript submitted)

Parke, J.L.; Oh, E.; Voelker, S.; Hansen, E.M.; Buckles, G.; Lachenbruch, B. 2007. 

Phytophthora ramorum colonizes tanoak xylem and is associated with reduced stem water 

transport. Phytopathology. 972: 1558-1567. 

 
 Epidemiology

133
134
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

135 

Studying Ink Disease Distribution Patterns 

on Heterogeneous Landscape by Means of 

Remote Sensing Activities and 

GIS Technology1

Andrea Vannini,2 Giuseppina Natili,2 and Anna Maria Vettraino2

Abstract 

Forest disease monitoring by means of remote sensing, using satellite or digital cameras 

carried by aircrafts, is getting more and more popular. The main advantages of this technique 

are the high image resolution (less than one meter), the possibility to acquire information on 

large surfaces in short time, the utilization of pre-defined spectral bands chosen to best 

discriminate the reflectance signature of healthy and diseased individual trees. Combination 

of remote sensing with GIS technology and geostatistic modules represents a powerful tool in 

order to develop risk maps based on disease variables, landscape features and climate, and to 

obtain important epidemiological information. 

Monitoring of Ink disease in Italy was carried by mean of spectral images acquisition with 

ASPIS technology. Following image interpretation and definition of foci, a disease map was 

developed and resolved in a GIS. The association of disease presence with landscape features 

including slope, presence of roads and drainages was investigated by analysing the different 

informative layers. A significant effect of road density (expressed as meters/hectare) and 

slope on disease presence was evidenced. Furthermore disease gradient from roads and 

drainages followed the Kiyosawa and Shiyomi model. Patterns of disease spread was studied 

at small scale (two hectares) suggesting that more factors, in addition to drainage and roads 

architecture, are associated to the foci expansion. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2 Department of Plant Pathology, University of Tuscia, via S. Camillo de Lellis, 01100-Viterbo, Italy. 

Corresponding author: vannini@unitus.it.
GENERAL TECHNICAL REPORT PSW-GTR-221

136 

Modelling the Potential Distribution of 

Phytophthora alni Root and Collar Rot of 

Alders in Bavaria and Preliminary 

Application of the Model to Estimate the 

U.S. Susceptibility to P. alni1

Thomas Jung,2 Marla Downing,3 Vernon Thomas,4 Markus 

Blaschke,5 Michael F. Tuffly,6 and Robin Reich7

Abstract 

Phytophthora alni formerly known as the “alder Phytophthora” is a recently evolved soil- and 

waterborne hybrid pathogen causing root and collar rot of Alnus species. P. alni has quickly 

spread across Europe via planting of infested nursery stock and has yet not been recorded 

from other continents. A model was developed to predict the potential distribution of P. alni

in Bavaria, Southern Germany in order to have a tool for assessing the potential hazard posed 

by P. alni to forests in other continents. Bavaria was chosen because of (i) being a hot spot of 

the disease, and (ii) the existence of both a large disease data base and expert knowledge. 

Logistic regression was used to identify environmental and site conditions important in 

describing alder infestations. Three models were evaluated. The models were based on 

categorical variables describing the establishment (planted/natural) and the flooding 

conditions (flooded/non-flooded) of 434 sample points of which 307 points were infested by 

P. alni; on GIS datasets on soil texture, aspect, slope and landform; and a Normalized 

Difference Vegetation Index (NDVI) calculated from satellite imagery. The non-spatial model 

included only the flooding condition of the sites and the establishment type while the spatial 

model included only GIS datasets and the vegetation index. The third model was a 

combination of the spatial and non-spatial model and included the flooding condition and 

establishment type of the stands and selected GIS datasets. Receiver Operating Curves (ROC) 

were used to assess the predictive accuracy of models. Both the spatial and the non-spatial 

model were highly accurate at predicting infected sites (95.8 and 82.1 percent accuracy) but 

their accuracy in predicting healthy sites was insufficient (66.1 and 61.1 percent). In contrast, 

the combined model predicted both healthy and diseased sites with high accuracy (85 percent 

and 86.6 percent, respectively). Preliminary results of an application of the Bavarian rules 

along with additional datasets on drainage, streams, climate, distribution of alder species, 

distribution of wholesale and retail nurseries, and urban settlements in a Multi-Criteria model 

to the United States indicate that there are regions with a considerable susceptibilty of alder 

forests to P. alni. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2 Independent scientist and consultant for tree diseases, Thomastrasse 75, D-83098 Brannenburg, Germany. 3 Forest Health Technology Enterprise Team, Forest Health Protection, USDA Forest Service, Suite 331, 2150 Centre 

Avenue, Building A, Fort Collins, CO 80526-1891, USA. 4

 Information Technology Experts, Inc. (ITX), 2120 S. College Ave., Fort Collins, CO, 80525, USA. 5

 Bavarian State Institute of Forestry (LWF), Section Forest Ecology and Forest Protection, Am Hochanger 11, D85354 Freising, Germany. 

6

 ERIA Consultants, LLC, 165 South 32nd Street, Boulder, CO, 80305, USA. 7 Department of Forest, Rangeland and Watershed Stewardship, Colorado State University, Fort Collins, CO, 80525, 

USA. 

Corresponding author: competence@tree-diseases.com. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

137 

Susceptibility of Some Italian Ornamental 

and Forestry Species to 

Phytophthora ramorum1

Anna Maria Vettraino,2 Barbara Ceccarelli2 and Andrea Vannini2

Abstract 

Phytophthora ramorum is the fungal-like pathogen which is the causal agent of a condition 

known in the USA as sudden oak death. Initially observed in a container-grown 

Rhododendron and Viburnum plants in nurseries, P. ramorum is now known to infect a wide 

range of hosts both in nursery and forest in Europe (Werres and others 2001; 

www.rapra.csl.gov.uk). Symptoms varied on different hosts and they are often restricted to 

leaf spots, stem and twig blight or occasionally seedling blight or tip dieback and bleeding 

cankers (Davidson and others 2003). The aim of this work was to assess the susceptibility of 

Mediterranean potential hosts. We assessed the reaction of 69 coniferous and broad-leaved 

tree species to stem and leaf inoculation with this quarantine pathogen. Three different P. 

ramorum isolates have been used: isolates P1376 (from Viburnum tinus) and P1577 (from 

Rhododendron catawbiensis), both A1 mating type, European isolates and isolate P1403 

(from Vaccinium ovatum), A2 mating type, American isolate. Leaves were inoculated with 

4x105

 zoospores mL-1. Stems were inoculated by placing a plug of mycelium in a wound cut, 

diameter 7mm. Foliar and stems lesions were examined after 10 days. Moreover the asexual 

sporulation ‘in planta’ was examined on each host. 

Leaf lesions caused by P. ramorum appeared variable in colour and size depending on the 

host. Symptoms ranged from scattered spots to large necrotic areas; sometimes lesions 

appeared surrounded by diffuse margins and a halo of yellow (fig. 1). 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2

 Department of Plant Pathology, University of Tuscia, via S. Camillo de Lellis, 01100-Viterbo Italy. 

Corresponding author: vannini@unitus.it. 
GENERAL TECHNICAL REPORT PSW-GTR-221

138 

Figure 12Symptoms of P. ramorum infection on inoculated hosts:1) Alnus 

incana; 2) Acer campestre; 3) Arbutus unedo; 4) Castanea sativa; 5) Olea 

europea; 6) Celtis australis; 7) Juniperus communis L.; 8) Fagus sylvatica; 9) 

Fraxinus ornus; 10) Rhododendron sp.; 11) Arbutus unedo; 12) Quercus 

robur; 13) Ulmus minor; 14) Quercus cerris; 15) Quercus ilex; 16) Quercus 

macrolepis; 17) Tilia cordata; 18) Sorbus aria; 19) Cornus sanguinea. 

The 65 hosts varied widely in susceptibility, ranging from 0 percent to 100 percent leaf area 

infected. No symptoms appeared on Phyllirea angustifolia, Phyllirea latifoglia, Photinia 

fraseri, Pistacia lentiscus, Rhamnus alaternus and Myrtus communis. Hosts varied widely in 

their reaction to inoculation regarding both disease incidence (2=109; P<0.0001) and lesions 

size (ANOVA; F= 15,978; P<0,0001). Stem inoculations resulted on all host species in 

necrotic lesions extending beyond the margin of the inoculation wound. Disease incidence 

was 100 percent for all the 65 hosts species analysed. The lesion length was highly variable 

among individual plants (ANOVA; F=16.2; P<0.001). Phytophthora ramorum was always reisolated form symptomatic tissues and never from controls. Sporangia were observed on all 

the 51 species tested including the 6 symptomless. Significant variability in sporangia 

production was observed among hosts (ANOVA; F=37.9; P<0.0001).

This study provides the first record of the reactions to P. ramorum of a broad range of host 

species growing from sea level to 1200 a.s.l. Moreover it contributes to update the list of the 

potential hosts of P. ramorum, with 35 species not hitherto tested and defines a list of SOD 

risk Mediterranean hosts. Our results indicate that most of the species tested can support both 

infection and sporulation following artificial inoculations with P. ramorum (Hansen and 

others 2005). Not always host species with high leaves susceptibility showed to be greatly 

sensible to stem inoculation, confirming results from other authors. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

139 

The presence of wide range of symptoms, even similar to those caused by other Phytophthora

spp., the broad host range associated with P. ramorum together with the presence of sporangia 

on symptomless hosts species could make difficult to detect the pathogen. For this reason 

potentially there is a very high risk of inadvertent introduction of P. ramorum plant through 

trade due to a hidden infection. 

Literature Cited 

Davidson, J.M.; Werres, S.; Garbelotto, M.; Hansen, E.M.; Rizzo, D.M. 2003. Sudden 

oak death and associated diseases caused by Phytophthora ramorum. Online. Plant Health 

Progress doi:10.1094/PHP-2003-0707-01-DG. 

Hansen, E.M.; Parke, J.L.; Sutton, W. 2005. Susceptibility of Oregon forest trees and 

shrubs to Phytophthora ramorum: A comparison of artificial inoculation and natural infection. 

Plant Disease 89, 63–70. 

Werres, S.; Marwitz, R.; Man in`t Veld, W.A.; DeCock, A.W.A.M.; Bonants, P.J.M.; 

DeWeerdt, M.; Themann, K.; Ilieva, E.; Baayen, R.P. 2001. Phytophthora ramorum sp. 

nov., a new pathogen on Rhododendron and Viburnum. Mycological Research 105, 1155–

1165. 
GENERAL TECHNICAL REPORT PSW-GTR-221

140 

Widespread Phytophthora Infestations of 

Nurseries in Germany and Austria and 

Their Role as Primary Pathway of 

Phytophthora Diseases of Trees1

Thomas Jung,2 Jörg Schumacher,3 Sindy Leonhard,3 

Günter Hartmann,4 and Thomas Cech5

Abstract 

Between 2001 and 2007 extensive nursery surveys were carried out across Germany and 

Austria. In Northern Germany beech, oak and maple fields of 14 nurseries were investigated 

and 54 percentwere found infested by P. cactorum (23 percent), P. syringae (23 percent) and 

P. cambivora (15 percent). Other Phytophthora spp. were isolated infrequently. In Lower 

Saxony, Northwestern Germany beech fields were surveyed in six nurseries and five were 

found infested by P. cactorum, P. cambivora, P. citricola, P. gonapodyides (each two 

nurseries) and P. pseudosyringae (one nursery). In Bavaria, Southern Germany the beech 

fields of all nine nurseries tested were infested by a range of eight Phytophthora species with 

P. citricola, P. cactorum (each seven nurseries) and P. cambivora (five nurseries) being most 

widespread. In Southern and Western Germany all oak fields (Q. robur, Q. rubra, Q. petraea) 

of the eight tested nurseries were infested by Phytophthora spp. P. quercina, P. citricola

(each five nurseries) and P. cactorum (four nurseries) were most common. 

Alder fields were investigated in Bavaria, Southern Germany, in Brandenburg, Eastern 

Germany and in Austria with the specific purpose of detecting P. alni which is responsible for 

the epidemic alder mortality across Europe. In Bavaria P. alni was recovered from rootstocks 

of alders from three out of four nurseries which regularly bought in alder plants for resale, but 

not in rootstocks from four nurseries that grew their own alders from seed. In addition, P. 

cambivora, P. cactorum, P. gonapodyides and P. taxon ‘Pgchlamydo’(each 37.5 percent), P. 

megasperma (50 percent) and P. citricola (62.5 percent) were isolated. In Brandenburg P. alni

was found in five out of ten nurseries. In addition, P. cambivora, P. cactorum and P. syringae

were recovered with annually changing isolation frequencies. In both countries the infested 

nurseries used water from infested water courses for irrigation. In Austria P. alni was found in 

one of the four nurseries studied. As a result alders in Bavaria and Brandenburg were 

produced according to a code of good practice. Control isolations showed that P. alni but not 

the other Phytophthora spp. could be eliminated. 

Extensive field studies in young forest and amenity plantations in Southern and Northwestern 

Germany, and in more than 3000 alder and more than 200 mature beech and oak stands across 

Germany demonstrate the ubiquitous involvement of Phytophthora species in the devastating 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Independent scientist and consultant for tree diseases, Thomastrasse 75, 

D-83098 Brannenburg, Germany. 

3

 Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection in Forests, 

Messeweg 11/12, D-38104 Braunschweig, Germany. 

4

 Lower Saxony Forest Reseach Station, Grätzelstrasse 2, D-37079 Göttingen, Germany. 5

 Federal Research and Training Centre for Forests, Natural Hazards and Landscape (BFW), Seckendorff-GudentWeg 8, A-1131 Vienna, Austria. 

Corresponding author: competence@tree-diseases.com. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

141 

broadleaf tree declines, and the role of infested nursery stock as a primary pathway of 

Phytophthora diseases of trees. The implications of our results for the nursery and the forest 

industries are discussed. 
GENERAL TECHNICAL REPORT PSW-GTR-221

142 

Survival and Chlamydospore Production of 

Phytophthora ramorum in 

California Bay Laurel Leaves1

Elizabeth J. Fichtner2, David M. Rizzo,2 Shannon C. Lynch,2

Jennifer Davidson3, Gerri Buckles,4 and Jennifer Parke4

Abstract 

Sudden oak death manifests as non-lethal foliar lesions on bay laurel (Umbellularia 

californica), which support sporulation and survival of Phytophthora ramorum in forest 

ecosystems. The pathogen survives the dry summers in a proportion of attached bay leaves, 

but the propagules responsible for survival are unknown. This study focuses on summer 

pathogen survival associated with bay laurel in redwood-tanoak and mixed-evergreen forests 

with specific objectives including:i) detection of P. ramorum in leaf litter and soils throu 

ghout the annual disease cycle, ii) quantification of chlamydospores on and within attached 

symptomatic leaves, and in fresh and aged litter, iii) determination of chlamydospore 

germination, and, iv) assessment of pathogen survival within litter and canopy leaves, 

addressing the location of viable inoculum within foliar tissues. Ten trees were tagged for 

repetitive sampling in four redwood-tanoak and four mixed-evergreen forests. Sampling was 

conducted at four times between May 2006 and September 2007 to target different points 

during the disease cycle. To determine pathogen presence in leaf litter and soil, three soil 

samples and 20 symptomatic litter leaves were collected and independently bulked from each 

tree. Samples were then baited for P. ramorum with rhododendron leaves. Chlamydospore 

populations on surfaces of attached leaves, and fresh and aged litter were determined by 

scrubbing individual leaves with a moistened toothbrush and filtering the resulting suspension 

through 35μM nylon mesh. Chlamydospores were then counted under a dissecting 

microscope and a subsample of chlamydospores was placed on selective medium to observe 

germination potential. To evaluate chlamydospore production within tissue, leaves were 

cleared with KOH and then observed with light microscopy. Pathogen survival and 

colonization was determined by subdividing symptomatic tissue from each leaf for detection 

by PCR, culture, and microscopy. Chlamydospore populations on attached leaf surfaces were 

higher in redwood-tanoak than in mixed-evergreen forests, but chlamydospore germination 

was never observed. Pathogen recovery was highest in the late spring, but declined by the end 

of the summer survival period. Isolation recovery was higher in attached leaves than in freshly 

cast leaves, but was rare from aged leaf litter tissues. High pathogen detection frequency was 

achieved by PCR, even when recovery in culture was not possible. Pathogen detection by 

PCR and isolation did not correspond with the heightened chlamydospore production 

observed at some sites. Though bay laurel supports chlamydospore production, the quantity of 

chlamydospores produced per leaf varies between sites and the lack of germination of these 

survival propagules contributes to the mystery of their potential role in the epidemiology of 

sudden oak death. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26±31, 2007, Monterey, California. 2 Department of Plants Pathology, University of California, One Sheilds Ave., Davis, CA 95616 USA. 3

 Department of Ecology, Evolution, and Conservation Biology, University of Hawaii, Honolulu, HI, 96822 USA. 4

 Department of Crop and Soil Science, Oregon State University, Corvalis, OR, 97331 USA. 

Corresponding author: ejfichtner@ucdavis.edu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

143 

Introduction 

Phytophthora ramorum, the cause of sudden oak death (SOD) in California (CA), is 

an exotic plant pathogen that has caused extensive mortality of susceptible oak 

(Quercus spp.) and tanoak (Lithocarpus densiflorus) in coastal California forests 

since the mid-1990s (Rizzo and others 2002, Rizzo and others 2005). The pathogen 

causes non-lethal infections on numerous hardwood and coniferous forest tree 

species, understory shrubs, and herbaceous plants (Davidson and others 2003, 

Garbelotto and others 2003). The non-lethal foliar lesions formed on California bay 

laurel (Umbellularia californica) by P. ramorum support prolific sporulation during 

rain events and summer survival of the pathogen in forest ecosystems (Davidson and 

others 2005b). 

Though symptoms of P. ramorum on bay laurel are limited to foliar lesions, the 

sporulation and survival potential of the pathogen on this host elevate its 

epidemiological significance in the forest ecosystem. At an ecological level, presence 

of bay laurel correlates with occurrence of P. ramorum (Rizzo and others 2005), and 

on individual leaves the pathogen may produce as many as 5x103

 zoospores during a 

single rain event (Davidson and others 2005a). Foliar infections of bay laurel 

generally precede the infection of adjacent oaks (Rizzo and Garbelotto, 2003) and 

isolate aggressiveness on bay laurel correlates directly with aggressiveness on oak 

(Hϋberli and others 2005). The pathogen survives the hot, dry summer months in a 

proportion of symptomatic bay leaves (Davidson and others 2005a); but summer 

survival potential in bay leaves varies between sites. The phenomenon of siteinfluenced survival of P. ramorum within bay laurel has been documented in both a 

redwood-tanoak forest and a mixed-evergreen forest (dominated by coast live oak) at 

Jack London State Park (Glen Ellen, CA) and Fairfield Osborne Preserve (Pengrove, 

CA), respectively. Summer foliar survival in attached symptomatic leaves is higher in 

the redwood-tanoak forest (70 percent recovery) than in the mixed-evergreen forest 

(20 percent recovery), yet these two sites are located only 5km apart on opposite 

sides of Sonoma Mountain (J. Davidson, unpublished data). Furthermore, sporangia 

production is detected earlier and both disease spread and severity are higher in the 

redwood-tanoak forest than in the mixed-evergreen forest. The difference in the 

disease progress curve between the two sites suggests a disparity in amount of 

primary inoculum at the onset of the rainy season; however, the propagules 

responsible for pathogen survival are yet unknown. 

Based on the hypothesis that primary inoculum is more abundant in redwood-tanoak 

forests than in mixed-evergreen forests at the onset of the fall rainy season, the 

overall focus of this work is to assess summer pathogen survival associated with bay 

laurel in the two forest types. Because infected bay laurel leaves are more likely to 

abscise than uninfected leaves, our investigations target both symptomatic attached 

leaves, and symptomatic leaves in the litter layer. Specific objectives include: i) 

detection of P. ramorum in leaf litter and soils throughout the annual disease cycle, 

ii) quantification of chlamydospores on attached symptomatic leaves, and in fresh 

and aged litter, iii) determination of chlamydospore germination, and, iv) assessment 

of pathogen survival within litter and canopy leaves, addressing the location of viable 

inoculum within foliar tissues. 
GENERAL TECHNICAL REPORT PSW-GTR-221

144 

Materials and Methods 

Ten trees were tagged for repetitive sampling in four redwood-tanoak (Jack London 

State Park, Pfeiffer Big Sur State Park, Henry Cowell Redwoods State Park, and 

Samuel P. Taylor State Park) and four mixed-evergreen forests (Fairfield Osborne 

Preserve, Pacheco, Skyline Regional Park, and China Camp State Park). The 

redwood-tanoak and mixed-evergreen forest sites spanned latitudinal gradients of 

over 225 km, and 125 km, respectively. Sampling was conducted in May and August 

2006 and in March and September 2007. At each sampling event, all samples were 

collected from the eight sights within two days. Three types of bay foliage were 

collected: symptomatic attached leaves, freshly cast litter leaves, and aged litter 

leaves. The September 2007 collection diverged from the normal sampling strategy 

because freshly cast leaf specimens were not available at all sites. 

Baiting of Litter and Soil 

To determine pathogen presence in leaf litter and soil, 20 leaves were collected and 

bulked from the forest floor and three soil samples were collected and bulked from 

under the canopy of each tree. Litter and soil samples were then baited with 

rhododendron cv. Colonel Coen leaves for P. ramorum. Lesion margins from 

symptomatic baits were placed on PARPH selective medium containing 0.05 g 

hymexazol/L for determination of presence of P. ramorum. A subsample of soil from 

each tree was passed through a 2 mm sieve and a 30-40 g sample was weighed and 

oven-dried for determination of soil moisture content. 

Determination of Chlamydospore Populations 

At each sample time, one symptomatic attached leaf, one symptomatic freshly cast 

leaf and one symptomatic aged litter leaf were collected for determination of 

chlamydospore presence on leaf surfaces of each of the 80 tagged trees. Additionally, 

one asymptomatic leaf was sampled from each of two trees at each site to assess for 

asymptomatic sporulation. Individual leaves were scrubbed on both sides with a 

moistened toothbrush and rinsed with deionized water before filtering the resulting 

suspensionthrough a 35 μM nylon mesh. Chlamydospores were then counted under 

the dissecting microscope and the total number of chlamydospores per leaf was 

recorded. 

To look for chlamydospores within leaf tissue, another set of symptomatic attached-, 

freshly cast-, and aged litter leaves was gathered from each tagged tree and cleared in 

1N KOH to render tissue translucent. After approximately three weeks in the KOH 

solution, leaves were clear enough to observe under the compound microscope. 

Determination of Chlamydospore Germination Potential 

At each sample time chlamydospore germination potential was determined by 

individually selecting 10 mature chlamydospores from the symptomatic attached-, 

freshly cast-, and aged litter leaves at each of the eight sites. For some sets of leaves, 

particularly those collected from mixed-evergreen forests, fewer than ten 

chlamydospores were available for germination studies. Individual chlamydospores 

were placed on PARPH selective medium, incubated in the dark at room temperature, 

and observed periodically over two weeks for chlamydospore germination. 

Additionally, chlamydospores dislodged from symptomatic attached leaves in 

September 2007 were transferred to uninfected bay laurel and rhododendron leaves to 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

145 

determine infection potential. Immediately after scrubbing leaves from Samuel P. 

Taylor State Park and Henry Cowell Redwoods State Park, chlamydospores were 

counted and 10 chlamydospores were placed on three detached, moistened bay laurel 

and rhododendron leaves. Leaves were then stored in moist chambers for 

approximately 2 weeks for observation of symptom development and subsequent 

isolation of symptomatic and asymptomatic tissue onto PARP. 

Pathogen Survival and Colonization 

To assess pathogen survival and colonization in leaf tissue, one symptomatic 

attached-, one freshly-cast-, and one aged litter- leaf was collected from each tagged 

tree. For each leaf, the area along the lesion margin was subdivided for detection by 

PCR, culture, and scanning electron microscopy (SEM). For detection in culture, 

tissue from attached symptomatic leaves was placed in PARP, whereas tissue 

sampled from the forest floor was placed in PARPH. Isolation plates were then 

stored in the dark for up to three weeks at room temperature for assessing recovery of 

P. ramorum and other Phytophthora spp. present. Only samples lacking recovery of 

P. ramorum in culture were assessed for pathogen presence using PCR. For visual 

observation of colonization, samples were fixed and preserved in formalin acetic acid 

(FAA) for future observation using scanning electron microscopy. 

Results 

Baiting of Litter and Soil 

P. ramorum was baited from 60–90 percent of soil samples at all sites in May 2006 

(fig. 1A), but was undetectable by August 2006. In May 2007, P. ramorum was baited 

from 0-70 percent of the soil samples (fig. 1B), with samples from redwood-tanoak 

forests exhibiting generally higher recovery than those from mixed-evergreen forests. 

In September 2007, the pathogen was only baited from one soil sample at Henry 

Cowell Redwoods State Park, a redwood-tanoak site. Phytophthora ramorum was 

only baited from the bulk leaf litter in March 2007, and only at sites experiencing a 

rain event concurrent with collection. 

Figure 12Percent recovery from soil baits sampled at the end of the winter/spring rainy 

seasons. Graph A represents recovery in May 2006 and B from March 2007. 

March 2007: Bulk soil 

baiting

% recovery

Redwood-tanoak forests Mixed-evergreen forests

0

10

20

30

40

50

60

70

80

90

100

Jack

London

Big Sur Henry

Cowell

S.P. Taylor FOP Skyline Pacheco China

Camp

May 2006: Bulk soil baiting

% recovery 0

10

20

30

40

50

60

70

80

90

100

Jack

London

Big Sur Henry

Cowell

S.P. Taylor FOP Skyline Pacheco China

Camp

Redwood-tanoak 

forests Mixed-evergreen 

forests

A B March 2007: Bulk soil 

baiting

% recovery

Redwood-tanoak forests Mixed-evergreen forests

0

10

20

30

40

50

60

70

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London

Big Sur Henry

Cowell

S.P. Taylor FOP Skyline Pacheco China

Camp

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Big Sur Henry

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S.P. Taylor FOP Skyline Pacheco China

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% recovery 0

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Jack

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Big Sur Henry

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Camp

Redwood-tanoak 

forests Mixed-evergreen 

forests

A B
GENERAL TECHNICAL REPORT PSW-GTR-221

146 

Determination of Chlamydospore Populations 

Chlamydospore populations on attached leaf surfaces were higher in redwood-tanoak 

than in mixed-evergreen forests at all sample dates (fig 2). Chlamydospore 

populations were consistently higher at Henry Cowell Redwoods and Samuel P. 

Taylor State Park over all sample times. Chlamydospore levels were variable in fresh 

and aged litter. 

Chlamydospores were not observed in leaf tissues cleared in KOH, even in leaves 

from sites supporting high chlamydospore loads. To determine whether 

chlamydospores were sloughed off leaf surfaces while incubating in KOH, a drop of 

KOH was pipetted from the bottom of multiple test tubes and observed under the 

compound microscope. Chlamydospores were observed to accumulate at the bottom 

of test tubes containing leaves incubating in KOH. 

Chlamydospore Germination 

Chlamydospore germination was never observed on selective medium at any 

collection times. Similarly, chlamydospores placed on bay laurel and rhododendron 

leaves in September 2007 did not initiate infections of either leaf tissue. 

Pathogen Survival and Colonization 

Pathogen isolation from attached leaves ranged from 40-100 percent at each site in 

May 2006 and declined to a range of 0–40 percent in August 2006 (fig. 3). In August 

2006 (fig 3B) and September 2007 (fig 3D), P. ramorum was not recovered from 

0

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120

140

JLSP Big Sur Henry Cowell SP Taylor FOP Skyline Pacheco China Camp

May 2006

August 2006

March 2007

September 2007

# chlamydospores/leaf

Redwood-tanoak forests Mixed-evergreen forests

Figure 22Chlamydospore populations on attached symptomatic bay laurel 

leaves. Values represent average number of chlamydospores per leaf at 

each of 8 sites and over four sample dates. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

147 

attached leaves at four sites. The summer of 2006 was characterized by excessive 

heat, with numerous days having high temperatures over 38o C at most sites. Over the 

rainy season between August 2006 (fig 3B) and March 2007 (fig. 3D), recovery from 

attached leaves increased at five sites and remained static at three sites. 

PCR resulted in higher detection of P. ramorum than isolation in culture (figs. 3, A 

and B). 

Fresh leaf litter was only collected at all eight sites in August 2006 (fig. 4A) and 

March 2007 (fig 4B), whereas aged litter was collected from all eight sites in August 

2006 (fig. 5A), March 2007 (fig. 5B), and September 2007. Phytophthora ramorum

was recovered from freshly cast leaves with higher frequency in March 2007 than in 

August 2006 (fig 4). Difference in isolation recovery of P. ramorum between 

attached leaves and freshly cast leaves was not more than 30 percent, with the 

exception of samples collected from Henry Cowell Redwoods in March of 2007. At 

this time, isolation recovery from fresh litter was 50 percent higher than recovery 

from attached leaves. 

P. ramorum was isolated in culture from one aged leaf at each of two sites in March 

of 2007 (fig. 5B); however, the collection at these two sites was concurrent with a 

Figure 32Recovery of Phytophthora ramorum from attached 

symptomatic bay laurel leaves. Graph A and B represent recovery by 

PCR and isolation in culture for samples collected in May and August 

2006, respectively. C and D represent recovery in culture for 

samples collected in March and September 2007, respectively. 

Banksia attenuata 

May 2006: Attached Symptomatic Leaves August 2006: Attached Symptomatic Leaves

0

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JLSP Big Sur Henry Cowell SP Taylor FOP Skyline Pacheco China Camp

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September 2007: Attached Symptomatic Leaves

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A B

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GENERAL TECHNICAL REPORT PSW-GTR-221

148 

heavy rain event. PCR detection of P. ramorum was possible in both fresh litter (fig.

4A) and aged litter (fig. 5A), even when the pathogen was not recovered in culture. 

Discussion 

After four collection events spanning different times within the annual disease cycle 

of P. ramorum, our data suggest that bay laurel in redwood-tanoak forests support 

higher levels of foliar chlamydospore production than bay laurel in mixed-evergreen 

forests. Two sites in particular, Henry Cowell Redwoods and Samuel P. Taylor State 

Parks, exhibit higher chlamydospore loads on bay laurel than the other sites 

examined in this study. Presumably the heightened chlamydospore loads at these 

sites result in more primary inoculum at the onset of the disease cycle; however, 

0

10

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30

40

50

60

70

80

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JLSP Big Sur Henry Cowell SP Taylor FOP Skyline Pacheco China Camp

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% positive recovery

Redwood-tanoak forests Mixed-evergreen forests

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JLSP Big Sur Henry Cowell SP Taylor FOP Skyline Pacheco China Camp

culture

% positive recovery

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March 2007: Freshly cast litter

A B

Figure 42Recovery of Phytophthora ramorum in freshly cast leaf litter. 

A represents recovery by PCR and isolation in August 2006 and B 

represents isolation recovery in March 2007. 

0

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JLSP Big Sur Henry Cowell SP Taylor FOP Skyline Pacheco China Camp

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JLSP Big Sur Henry Cowell SP Taylor FOP Skyline Pacheco China Camp

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% positive recovery

Redwood-tanoak forests Mixed-evergreen forests

A B

Figure 52Recovery of Phytophthora ramorum in symptomatic, aged leaf litter. 

Pathogen detection by isolation in culture and PCR was completed for the 

sampling in August 2006 (A), and recovery by isolation was completed for 

samples collected in March 2007 (B). 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

149 

pathogen recovery at the end of the summer survival periods was not higher in sites 

exhibiting high chlamydospore loads. Furthermore, because chlamydospores 

produced on naturally-infected bay leaves did not germinate in vitro, one can only 

speculate on their viability, survival potential, and infectivity. 

Infected bay laurel leaves are more likely to abscise than uninfected leaves, resulting 

in deposition of inoculum at the forest floor (Davidson and others 2005a). Pathogen 

recovery by isolation was higher in freshly cast litter than in aged litter, suggesting 

that survival declines rapidly after abscission. Detection of P. ramorum by PCR in 

fresh and aged litter remained high, even when the pathogen could not be recovered 

by isolation. PCR positives, however, may result from detection of non-viable 

pathogen tissue. Though PCR positives may result from presence of chlamydospores, 

the two sites exhibiting high chlamydospore loads did not have a higher frequency of 

PCR positives in aged or fresh litter than sites with low chlamydospore loads. 

P. ramorum survives the hot, dry summers in a portion of infected bay laurel leaves, 

providing a source of primary inoculum at the onset of the fall rainy season. 

Chlamydospores are produced on infected leaves, however, their potential role in 

survival and infectivity is yet unknown. Chlamydospore production on bay laurel 

foliage varies between sites, but their potential role in shaping disease progress in 

epidemics in different forest types remains open for conjecture. 

Literature Cited 

Davidson, J.M.; Fichtner, E.J.; Patterson, H.A.; Falk, K.R.; Rizzo, D.M. 2005a.

Mechanisms underlying differences in inoculum production by Phytophthora ramorum in 

mixed-evergreen versus tanoak-redwood forest in California. Sudden oak death second 

science symposium, January 18–21, Monterey, CA. 

Davidson, J.M.; Werres, S.; Garbelotto, M.; Hansen, E.M.; Rizzo, D.M. 2003. Sudden 

oak death and associated diseases caused by Phytophthora ramorum. Online. Plant Health 

Progress doi:10.1094/PHP-2003-0707-01-DG. 

Davidson, J.M.; Wickland, A.C.; Patterson, H.A.; Falk, K.R.; Rizzo, D.M. 2005b.

Transmission of Phytophthora ramorum in mixed-evergreen forest in California. 

Phytopathology 95:587–696. 

Garbelotto, M.; Davidson, J.M.; Ivors, K.; Maloney, P.E.; Hüberli, D.; Rizzo, D.M. 

2003. Non-oak native plants are the main hosts for the sudden oak death pathogen in 

California. Cal. Agric. 57:18–23. 

Huberli, D.; Harnik, T.; Meshriy, M.; Miles, L.; Garbelotto, M. 2005. Phenotypic 

variation among Phytophthora ramorum isolates from California and Oregon. Page 131 in: 

Proceedings of the sudden oak death Second Science Symposium: The State of our 

Knowledge. Pacific Southwest Research Station Gen. Tech. Rep. PSW-GTR-196. 

Rizzo D.; Garbelotto M. 2003. Sudden oak death: endangering California and Oregon forest 

ecosystems.Front. Ecol. Environ. 2003; 1(5): 197–204. 

Rizzo, D.M.; Garbelotto, M.; Davidson, J.M.; Slaughter, G.W.; Koike, S.T. 2002.

Phytophthora ramorum as the cause of extensive mortality of Quercus spp. and Lithocarpus 

densiflorus in California. Plant Dis. 86:205–214. 
GENERAL TECHNICAL REPORT PSW-GTR-221

150 

Rizzo, D.M.; Garbelotto, M.; Hansen, E.M. 2005. Phytophthora ramorum: Integrative 

research and management of an emerging pathogen in California and Oregon forests. Annu. 

Rev. Phytopathol. 43:309–335. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

151 

Survival of Phytophthora cinnamomi in Soil 

After Prescribed Fire in a Southern 

Appalachian Mountain Forest1

Inga M. McLaughlin,2 Steven N. Jeffers,2 and Thomas A. Waldrop3

Abstract 

The use of prescribed fire has become a common management strategy in fire-adapted forests 

of the southern Appalachian Mountains. Little is known about the direct effects of fire on 

survival of species of Phytophthora naturally present in forest soils. Therefore, the effects of 

fire on survival of P. cinnamomi was assessed during two low-intensity prescribed fires that 

were set in a mixed oak-pine forest within the Jocassee Gorges natural area in northwestern 

South Carolina during February 2007. P. cinnamomi is known to occur throughout this forest 

site. Before the first fire, six plots were established in the area to be burned and two plots 

were established in adjacent areas that would not be burned. Before the second fire, four plots 

were established in the area to be burned and one plot was established in an area that would 

not be burned. In each plot, seven 100-ml aliquots of forest soil naturally infested with P. 

cinnamomi in aluminum-mesh packets were placed at 2 cm and 10 cm below the soil surface. 

Temperature sensors attached to dataloggers were placed with each packet and recorded 

temperature in the soil at 1.5-second intervals. Sensors also were used to measure aboveground temperature during the second fire. After the fires, the soil in each packet was assayed 

for P. cinnamomi using a baiting bioassay procedure. 

During the second fire, maximum temperatures 30 cm above the soil surface in each plot 

ranged from 47 to 111°C. Soil temperatures in the non-burned plots during both fires ranged 

from 7 to 13°C at both 2 cm and 10 cm below the soil surface. However, in the burned plots, 

soil temperatures ranged from 7 to 14°C at 10-cm depths and from 11 to 42°C at 2-cm depths. 

After the two fires, P. cinnamomi was recovered from all 42 soil aliquots from both depths in 

the three plots in non-burned areas. P. cinnamomi also was recovered from all 70 soil 

aliquots buried at 10 cm and from 69 of 70 aliquots buried at 2 cm in the burned areas; it was 

not recovered from one soil aliquot placed 2 cm beneath the soil surface during the first fire—

where the soil temperature reached 42°C for 40 min. In this study, the direct effect of lowintensity, prescribed fire had minimal impact on the survival of P. cinnamomi in soil. 

Introduction 

The use of prescribed fire has become a common management strategy in fireadapted forests of the southern Appalachian Mountains to reduce fuel accumulation 

and encourage regeneration of declining forest ecosystems (Barden and Woods 1976; 

Brose and Waldrop 2000; Waldrop and others 1987). Little is known about the 

effects of fire on survival of species of Phytophthora naturally present in forest soils. 

Therefore, two prescribed fires were used to assess the direct effect of fire on the 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26–31, 2007, Monterey, California. 2

 Department of Entomology, Soils, and Plant Sciences; Clemson University, 120 Long Hall; Clemson, SC 29634-

0315, USA. 

3

 USDA Forest Service; Southern Research Station, 239 Lehotsky Hall; Clemson, SC 29643-1003, USA. 

Corresponding author: sjffrs@clemson.edu. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 152

survival of P. cinnamomi in soil in a mixed oak-pine forest within the Jocassee 

Gorges Natural Area of northwestern South Carolina in February 2007. P. cinnamomi

is known to occur throughout this forest site (Wood and others 2001). 

Materials and Methods 

Before the first fire, six 

plots were established in 

the area to be burned and 

two plots were established 

in adjacent areas that 

would not be burned. 

Before the second fire, 

four plots were 

established in the area to 

be burned and one plot 

was established in an area 

that would not be burned. 

In each plot, seven 100-ml 

aliquots of forest soil 

naturally infested with P. 

cinnamomi in aluminummesh packets were placed 

at 2 cm and 10 cm below the soil surface (fig. 1) depths at which P. cinnamomi has 

been recovered consistently (McLaughlin and Jeffers, unpublished; Wood and others 

2001). Temperature sensors attached to dataloggers were placed within each packet 

and recorded temperature in the soil at 1.5-second intervals (fig. 1). Sensors also 

were used to measure temperature approximately 30 cm above the soil surface during 

the second fire (fig. 1). After the fires, the soil in each packet was assayed for P. 

cinnamomi using a baiting bioassay. Soil from each packet was placed into a plastic 

freezer box (450 ml) and flooded with approximately 200 ml of distilled water. Six 

camellia leaf disks (5 mm in diameter) were floated on the surface of the water in 

each box as baits for P. cinnamomi, and boxes were kept at room temperature (22 to 

25 °C) for 3 days. Baits then were removed, blotted dry, and embedded in PARPHV8, a medium selective for Phytophthora spp. (Ferguson and Jeffers 1999), to 

recover P. cinnamomi. Numbers of baits and soil packets that were positive for 

P. cinnamomi were counted and percentages were calculated. 

Results and Discussion 

During the second fire, maximum temperatures 30 cm above the soil surface in the 

four burned plots were 47, 62, 91, and 111 °C. Maximum soil temperatures were 

slightly elevated at 2 cm beneath the soil surface in the burned plots (table 1) and 

ranged from 13 to 42 °C during the first fire and 9 to 16 °C during the second fire. 

Maximum soil temperature at 10 cm beneath the soil surface in the burned plots 

ranged from 9 to 18 °C during the first fire and from 11 to 13 °C during the second 

fire. Maximum soil temperatures in the non-burned plots during both fires were 

similar and ranged from 7 to 15 °C at 2 cm and 10 cm below the soil surface. 

Figure 1—Diagram of the layout of each plot in the 

study site at the Jocassee Gorges Natural Area in 

northwestern South Carolina. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 153 

After the two fires, P. cinnamomi was recovered from 99 to 100 percent of leaf baits 

and soil packets at 2 and 10 cm beneath the soil surface in the non-burned, control 

plots (table 1). P. cinnamomi also was detected from 100 percent of leaf baits and soil 

packets at 10 cm beneath the soil surface in the burned plots. However, presence of 

P. cinnamomi was slightly less in soil packets (99 percent) and leaf baits (97 percent) 

at the 2-cm depth in the burned plots (table 1) where soil temperatures in some plots 

reached 42 °C for 40 min, temperatures adequate to inactivate propagules of P. 

cinnamomi (Benson 1978; Jaurez-Palacios and others 1991). In this study, soil 

temperatures were not elevated consistently for a long enough period of time to 

inactivate propagules of P. cinnamomi. Therefore, typical prescribed fires may not be 

adequate to eliminate or significantly reduce propagules of Phytophthora spp. from 

forest soils in the southern Appalachian Mountains. 

Table 1—Summary of survival of P. cinnamomi in soil after and soil 

temperatures during two prescribed burns in the Jocassee Gorges Natural 

Area in northwestern South Carolina in 2007 

Packetsy Baitsz Max. soil 

Treatment temp. (°C) Depth 

(cm) 

Plots 

(no.) Total No. Percent Total No. Percent Mean Range

Burn 2 10 70 69 99 420 408 97 17 9-42 

 10 10 70 70 100 420 418 100 10 9-18 

Control 2 3 21 21 100 126 126 100 11 9-13 

 10 3 21 21 100 126 125 99 10 7-15 

y

 Seven packets were used in each plot, and each packet contained 100 ml of 

naturally-infested soil. Values are the numbers (No.) and percentages of packets 

in which P. cinnamomi was detected after the fires. z

 The soil in each packet was baited with six camellia leaf disks; values are the 

numbers (No.) and percentages of baits from which P. cinnamomi grew. 

Acknowledgments 

This research was funded by the U.S. Department of Agriculture, Forest Service. 

Special thanks to Mark Hall and Richard Baker for technical assistance and to 

William Bridges, Jr., for statistical advice. 

Literature Cited 

Barden, L.S.; Woods, F.W. 1976. Effects of fire on pine and pine-hardwood forests in the 

southern Appalachian Mountains. Forest Science. 22: 399–403. 

Benson, M.D. 1978. Thermal inactivation of Phytophthora cinnamomi for control of Fraser 

fir root rot. Phytopathology. 68: 1373–1376. 

Brose, P.H.; Waldrop, T.A. 2000. Using prescribed fire to regenerate table mountain pine in 

the southern Appalachian Mountains. In: Moser, W.K.; Moser, C.E., eds. Fire and forest 

ecology: Innovative silviculture and vegetation management. Tall Timbers Fire Ecology 

Conference Proceedings, No. 21. Tallahassee, FL: Tall Timbers Research Station: 191–l96. 

Ferguson, A.J.; Jeffers, S.N. 1999. Detecting multiple species of Phytophthora in container 

mixes from ornamental crop nurseries. Plant Disease. 83: 1129–1136. 
GENERAL TECHNICAL REPORT PSW-GTR-221

154 

Jaurez-Palacios, C.; Felix-Gastellum, R.; Wakeman, R.J.; Paplomatas, E.J.; DeVay, J.E. 

1991. Thermal sensitivity of three species of Phytophthora and the effect of soil solarization 

on their survival. Plant Dis. 75:1160–1164. 

Waldrop, T.A.; Van Lear, D.H.; Lloyd, F.T.; Harms, W.R. 1987. Long-term studies of 

prescribed burning in loblolly pine forests of the southeastern coastal plain. Gen. Tech. Rep. 

SE-45. Asheville, NC: U.S. Department of Agriculture, Forest Service, Southern Research 

Station. 23 p. 

Wood, A.K.; Tainter, F.H.; Jeffers, S.N. 2001. Distribution of Phytophthora species in 

forest soils of the Jocassee Gorges Natural Area in South Carolina, USA. In: McComb, J.A.; 

Hardy G.E.St.J.; Tommerup, I.C., eds. Phytophthora in Forests and Natural Ecosystems, 2nd 

International IUFRO Working Party 7.02.09 Meeting. Murdoch, W. Australia: Murdoch 

University Print: 83. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

155 

Persistence of Phytophthora lateralis After 

Wildfire: Preliminary Monitoring Results 

From the 2002 Biscuit Fire1

Frank Betlejewski2

Abstract 

Port-Orford-cedar (Chamaecyparis lawsoniana) is an economically and ecologically valuable 

tree species that occurs on approximately 107,044 ha in southwest Oregon and northwest 

California. Roughly 35,400 ha are infested with Phytophthora lateralis, the pathogen that 

causes Port-Orford-cedar root disease. The pathogen has been shown to kill more than ninety 

percent of the Port-Orford-cedar stocking on an infested site. Biologically valuable overstory 

Port-Orford-cedar are especially prone to infection and mortality. Loss of large Port-Orfordcedar can reduce the ecological function of the species and limit economic availability. 

Phytophthora lateralis is sensitive to heat. Hansen and Hamm (1996) showed that survival of 

the pathogen is minimal in soil exposed to temperature of 40 °C (104 °F) or greater, especially 

if conditions are dry. The Biscuit fire burned approximately 196,800 acres in southwest 

Oregon and northwest California in 2002 including over 1,180 ha infested with P. lateralis. 

In the spring of 2004, twenty-five monitoring plots were established inside the Biscuit fire 

perimeter on sites known to be infested with P. lateralis. The nineteen Silver Creek plots were 

baited at 0.61 m (two foot) spacing with forty-eight susceptible seedlings. The six Game Lake 

plots were baited at the same spacing with forty-four susceptible seedlings. 

Baiting is a bio-assay that uses Port-Orford-cedar seedlings to determine the presence of P. 

lateralis. Non-resistant Port-Orford-cedar seedlings are planted in soil where P. lateralis is 

suspected to occur. After an exposure period of six to eight weeks, the seedlings are 

recollected and analyzed for the presence of P. lateralis. The Biscuit fire seedlings were 

removed from the bait plots after eight weeks and analyzed to determine the number of 

infected seedlings and establish a baseline for P. lateralis presence. 

The plots were baited again in the fall of 2004. Fifty percent of the bait seedlings were the 

same susceptible stock used spring, 2004 and fifty percent of the bait seedlings were resistant 

to P. lateralis. The same contractor was used and seedlings were planted in the same holes as 

the spring, 2004 baiting. Dead seedlings were collected during the summer in 2005, 2006, 

and 2007 and analyzed to determine the number infected with P. lateralis. 

Twenty-two of the original twenty-five plots remain in 2007. Twenty-one of twenty-two plots 

planted in spring 2004 had mortality caused byP. lateralis (table 1). Phytophthora lateralis

mortality in the fall 2004 planting has declined from that seen in spring, 2004. Fewer plots 

showed P. lateralis-caused seedling mortality and fewer seedlings overall were infected.

Phytophthora lateralis mortality declined to thirteen, nine, and six plots respectively in 2005, 

2006, and 2007. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26–31, 2007, Monterey, California. 2

 USDA Forest Service, Southwest Oregon Forest Insect and Disease Service Center, 2606 Old Stage Road, Central 

Point, OR 97502, USA. 

Corresponding author: fbetlejewski@fs.fed.us.
GENERAL TECHNICAL REPORT PSW-GTR-221

156 

Table 12Decline in number of plots showing P. lateralis mortality 

Table 22Decline in number of seedlings showing P. lateralis mortality: 

Phytophthora lateralis requires a live host to reproduce. Port-Orford-cedar mortality due to 

the Biscuit fire has diminished the live host pool. Other factors potentially affecting 

Phytophthora lateralis survival include the heat pulse of the Biscuit fire itself and subsequent 

warming and drying of infested sites due to loss of canopy and darkening of soil post fire. 

In laboratory studies, Ostrofsky and others (1977) were unable to recover P. lateralis from 

infested organic matter after sixteen weeks at 25 °C (77 F). Hansen and Hamm (1996) 

showed P. lateralis survival is minimal in dry soil at temperatures greater than 40 °C (104 F). 

And DeBano, Neary, and Folliott (1998) estimated a heat pulse between 200 and 300 °C (392 

and 572 °F) in high severity fire at one centimeter (0.4 inch) soil depth. 

Preliminary indications are that the soil heat pulse and soil surface darkening from the Biscuit 

fire, Port-Orford-cedar fire mortality, and overall loss of canopy resulting in higher 

temperatures and lower relative humidities may be combining to create an unfavorable 

environment for persistence of P. lateralis. 

There are additional factors that require further analysis: 

1) The resistance effect: How does survival vary when comparing fall 2004 susceptible 

seedling survival versus fall 2004 resistant seedling survival? 

2)The fire/time effect: How does survival vary when comparing spring 2004 susceptible 

seedling survival to fall 2004 susceptible seedling survival? 

Management Implications: Results from the Biscuit fire monitoring may lead to the design 

and implementation of silvicultural and prescribed fire treatments that potentially could 

reduce or eliminate P. lateralis from currently infested sites. USDA Forest Service PortOrford-cedar management has focused on reducing P. lateralis spread and resistance 

breeding. Little work has been done to remove the pathogen from sites where it currently 

exists. If P. lateralis eradication treatments can be developed and utilized routinely, the need 

for other management practices could be reduced in the long term. 

Literature Cited 

DeBano, L.F.; Neary, D.G.; Ffolliot P.F. 1998. Fire’s Effects on Ecosystems. New York: 

John Wiley and Sons; 333 p.

 Plots with Infected Port-Orford-cedar 

2004 21 

2005 13 

2006 9 

2007 6 

 2004 2005±2007

Game Lake 63 21 

Silver Creek 123 71 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

157 

Hansen, E.M.; Hamm, P.B. 1996. Survival of Phytophthora lateralis in infected roots of 

Port-Orford-cedar. Plant Disease. 80: 1075-1078. 

Ostrofsky, W.D.; Pratt R.G.; Roth, L.F. 1977. Detection of Phytophthora lateralis in soil 

organic matter and factors that affect it’s survival. Phytopathology. 67:79- 
GENERAL TECHNICAL REPORT PSW-GTR-221

158 

Operational Program to Develop Resistance 

to Phytophthora lateralis in Port-Orfordcedar (Chamaecyparis lawsoniana): 

the First Ten Years1

Richard A. Sniezko,2 Leslie J. Elliott,3 Everett M. Hansen,4 Frank 

Betlejewski,5 Donald J. Goheen,5 Kirk Casavan,6 Scott E. Kolpak,2

Charles L. Frank,7 Peter A. Angwin,8 and Jim Hamlin9

Abstract 

The USDA Forest Service and USDI Bureau of Land Management (BLM) operational 

program to develop populations of Port-Orford-cedar (POC) with genetic resistance to the 

non-native root pathogen Phytophthora lateralis has been underway since 1996. Earlier, 

smaller scale efforts focused on making a few hundred field selections from a portion of the 

native range and confirming the presence of genetic resistance. The Forest Service, BLM, 

Oregon State University and others have all played an important role in the rapid development 

of this resistance program. The Forest Service provides genetic and pathology expertise, 

coordinates and designs the overall development of the program and manages the 

containerized seed orchards. The Forest Service and BLM have supplied program funding and 

have made many of the field selections, and established field trials to test resistant families 

and delineate and validate breeding zones. Oregon State University provides research 

pathology expertise and oversees the large annual artificial inoculation and assessment of 

seedlings and rooted cuttings. Many other landowners have permitted tree selections from 

their lands or have provided sites for field resistance trials. Program goals include developing 

populations of resistant trees, maintaining genetic variation and adaptability, and producing 

resistant seed for reforestation and restoration. 

Through 2006, over 12,500 field selections have been made in Oregon and California (fig 1). 

The first cycle of selection and testing (fig 2) is well underway. The field selections have been 

evaluated in the first phase of testing, using a stem dip inoculation test. Approximately ten 

percent of the parent trees in each stem dip trial are selected for a second phase of testing 

using a root dip inoculation test. Nearly a third of the stem dip selections have been root dip 

tested. In this second test phase, rooted cuttings or seedling progeny of a parent are 

inoculated and assessed for mortality to rate the resistance of the parent. In addition, root dip 

testing of controlled crosses (including selfs) is underway as part of the second cycle of 

selection and to examine the inheritance of resistance and to increase resistance. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 USDA Forest Service, Dorena Genetic Resource Center, 34963 Shoreview Road, Cottage Grove, OR 97424, USA. 3

 USDA Forest Service, Willamette National Forest, Sweet Home Ranger District, Sweet Home, OR 97386, USA. 

4 Department of Botany and Plant Pathology, Oregon State University, 2082 Cordley Hall, Corvallis, OR, 97331, 

USA. 

5 USDA Forest Service, Southwest Oregon Forest Insect and Disease Service Center, 2606 Old Stage Road, Central 

Point, OR, 97502, USA. 

6

 USDI Bureau of Land Management, Roseburg District Office, Roseburg, OR 97471, USA. 7 USDA Forest Service, Klamath National Forest 1312 Fairlane Road, Yreka, CA 96097, USA. 8

 USDA Forest Service, Shasta-Trinity National Forest, 3644 Avtech Parkway, Redding, CA, 96002, USA. 9

 USDA Forest Service, Umpqua National Forest, Roseburg, OR 97471, USA. 

Corresponding author: rsniezko@fs.fed.us. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

159 

^

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^

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Galice

Bandon

Ashland

Coos Bay

Roseburg

Brookings

Gold Beach Grants Pass

Port Orford

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Cottage Grove

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Arcata

Redding

Map Produced by Heather May

Dorena Genetic Resource Center

5/9/2005 Umpqua National Forest

p y ( ) ( ),

0 10 20 30 40 5 Miles

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Camas Valley

Legend

Breeding

Zone

110 0 - 1000'

125 1001 - 2500'

210 0 - 1000'

225 1001 - 2500'

315 0 - 1500'

325 1501 - 2500'

340 2501 - 4000'

350 > 4000'

425 0 - 2500'

545 1200 - 4500'

550 > 4500'

450 > 4000'

440 2501 - 4000'

Elevation

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POC Stem Dip Tested

POC Stem Dip Winner

Figure 1—Geographic range of Port-Orford-cedar and location of field 

selections for testing for Phytophthora lateralis resistance. 

Figure 2—Overview of the steps in breeding cycles to develop Phytophthora 

lateralis resistance for Port-orford-cedar.

Root Dip

Testing

Root branch 

cuttings or sow

seed from 

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CYCLE 1

CYCLE 2

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Seed

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Seed 

Orchard

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Repeat as needed to Cycle 3 and beyond

Stem Dip

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select top

10%

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trees in

forest

CYCLE 3

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Root branch 

cuttings or sow

seed from 

selected parents

Select

Best parents

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Seed

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Seed

CYCLE 1

CYCLE 2

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Seed

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Seed 

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Production

Seed

Repeat as needed to Cycle 3 and beyond

Stem Dip

Test –

select top

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trees in

forest

CYCLE 3

Root Dip

Testing

Root branch 

cuttings or sow

seed from 

selected parents

Select

Best parents

Within family

Selection

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Seed

Orchard

Seed

Orchard

Production

Seed

CYCLE 1

CYCLE 2

Breeding Sow

Seed

Resistance

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Within 

Family

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Seed 

Orchard

Production

Seed

Repeat as needed to Cycle 3 and beyond

Stem Dip

Test –

select top

10%

Select 

trees in

forest

CYCLE 3
GENERAL TECHNICAL REPORT PSW-GTR-221

160 

The native range of Port-Orford-cedar has been divided into 13 breeding zones (USDA Forest 

Service 2006), and screening for resistance is underway in all zones. For eight breeding zones, 

first-generation containerized seed orchards have been established using rooted cuttings from 

selected parents. Additional parent trees are needed to increase genetic diversity in most 

zones. Parent trees will be added or removed from orchards as additional test data become 

available. Since 2003, seed from several of these orchards has been utilized by federal and 

private landowners for restoration and reforestation. The top families show 40+ percent 

higher survival in short-term greenhouse testing than the most susceptible families (Sniezko 

2004, USDA Forest Service 2006). Field tests have been established on over 30 sites, to 

validate the greenhouse and raised bed results, examine durability of resistance, and test the 

first orchard seed. Early results from these tests are encouraging (Sniezko and others 2006). 

The POC program is one of the fastest moving resistance programs in forest trees (Sniezko 

2006). In many areas, genetic resistance offers the main avenue for restoring POC in 

ecosystems with high mortality as well as keeping it as a component of reforestation for 

public and private landowners. An examination of some of the underlying mechanisms of 

resistance has been completed (Oh and Hansen 2007). At least two types of resistance seem to 

be present, and additional research is needed on the types and number of mechanisms of 

resistance and their inheritance (Sniezko 2004, 2006). Work in the next few years will be 

focused on completing the backlog of parent trees for root dip testing and adding trees to the 

containerized orchards. Increasing the level of resistance from the orchards and further study 

of resistance mechanisms and their inheritance will be undertaken as funding permits. 

Updates of the program are available at http://www.fs.fed.us/r6/dorena/publications/poc. 

Literature Cited 

Oh, E.; E.M. Hansen. 2007. Histopathology of infection and colonization of Port-Orfordcedar by Phytophthora lateralis. Phytopathology 97: 684–693. 

Sniezko R.A. 2004. Genetic resistance in Port-Orford-cedar to the non-native root rot 

pathogen Phytophthora lateralis—2003 Update. In Geils, B.W., ed. Proceedings of the 51st 

Western International Forest Disease Work Conference. Flagstaff, AZ: U.S. Department of 

Agriculture, Forest Service, Rocky Mountain Research Station: 127–131. 

Sniezko R.A. 2006. Resistance breeding against nonnative pathogens in forest trees — 

current successes in North America. Canadian Journal of Plant Pathology. 28: S270–S279. 

Sniezko, R.A.; Kolpak, S.E.; Hansen, E.M.; Goheen, D.J.; Elliott, L.J.; Angwin, P.A. 

2006. Field survival of Phytophthora lateralis resistant and susceptible Port-Orford-cedar 

families. In: Brasier, C.; Jung, T.; Osswald, W., eds. Progress in Research on Phytophthora

Diseases of Forest Trees: Proceedings of the Third International IUFRO Working Party 

S07.02.09. Farnham, U.K.: Forest Research: 104–108. 

 

USDA Forest Service. 2006. Availability of Resistant Port-Orford-Cedar Seed. USDA 

Forest Service. Dorena Genetic Resource Center. Report compiled by Leslie Elliott, 

available at http://www.fs.fed.us/r6/dorena/publications/detail/pub303. 

 
Management

161
162
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

163 

Managing Phytophthora Species in the 

Forest: A Perspective1

Donald J. Goheen2

Introduction 

The main responsibility of the Forest Health Protection organization in the United 

States Department of Agriculture, Forest Service, is to provide assistance on insect 

and disease concerns and issues to federal forest managers and cooperators. The 

Southwest Oregon Forest Insect and Disease Service Center covers an area 

particularly noted for having a large number of management problems associated 

with forest Phytophthora spp., and in fact, we probably have more of these to deal 

with than any similar-sized area in the United States. The six person staff of the 

Southwest Oregon Forest Insect and Disease Service Center spends roughly 60 

percent of its time working on Phythophthora related activities. Major Phytophthora

issues in Southwest Oregon are Port-Orford-cedar root disease, sudden oak death, 

golden chinquapin decline, Phytophthora–caused sugar pine mortality in the Sprague 

Seed Orchard, Phytophthora root diseases at the J. Herbert Stone Nursery, and 

Phytophthora diseases at the Dorena Genetics Resource Center. 

Port-Orford-cedar Root Disease 

Port-Orford-cedar root disease affects Port-Orford-cedar (Chamaecyparus 

lawsoniana), a unique and valuable tree species with a very limited natural range in 

southwestern Oregon and northwestern California. The disease is caused by P. 

lateralis, a non-native pathogen first reported in the Seattle, Washington area on 

nursery stock in 1923. It was introduced into the native range of the host in 1952. 

Long distance spread of P. lateralis involves chlamydospores that are carried in mud 

and soil adhering to machinery, vehicles, animals, and humans. Once the pathogen 

has been introduced into a new area, spread via motile zoospores in water becomes 

important. Port-Orford-cedar root disease is very damaging and causes substantial 

levels of host mortality on certain kinds of sites. Cool wet conditions greatly favor 

infection. High risk sites are the vicinities of streams and ditches, low-lying places, 

and poorly drained areas that are down slope from existing infested areas or below 

roads where new introductions can occur. In attempting to manage Port-Orford-cedar 

root disease, the objective of federal foresters is to maintain Port-Orford-cedar as an 

ecologically and economically significant species on Forest Service and Bureau of 

Land Management lands. Techniques used in management include: excluding vehicle 

entry into yet uninfested areas, road closures (either permanent or temporary), 

roadside sanitation treatments, vehicle washing (especially before traveling from 

infested to uninfested areas), operations planning and scheduling to minimize 

movement of vehicles and equipment from or through infested areas to uninfested 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26–31, 2007, Monterey, California. 2

 USDA Forest Service, Southwest Oregon Forest Insect and Disease Service Center, Forest Health Protection, 

Central Point, OR 97502, USA. 

Corresponding author: dgoheen@fs.fed.us. 
GENERAL TECHNICAL REPORT PSW-GTR-221

164 

areas during all kinds of projects, timing access into stands with Port-Orford-cedar 

during dry warm weather, selection of healthy or treated water sources for use in fire 

suppression and dust abatement, featuring Port-Orford-cedar on sites with 

characteristics unfavorable for establishment and spread of the pathogen, regulating 

harvest of non-timber forest products, public education, and development and use of 

resistant Port-Orford-cedar planting stock (host resistance enhancement appears to be 

particularly promising with Port-Orford-cedar). Management prescriptions involve 

combinations of the above mentioned approaches in integrated strategies. 

Sudden Oak Death 

In Southwest Oregon, P. ramorum kills tanoak (Lithocarpus densiflorus) and, 

occasionally, California black oak (Quercus kelloggi). The pathogen also causes leaf 

and tip damage on a variety of other species, most commonly Pacific Rhododendron 

(Rhododendron macrophylum) and evergreen huckleberry (Vaccinium ovatum). P. 

ramorum was first detected in Oregon in 2001 and now occurs at scattered locations 

within an approximately 6000 hectare area near the coastal town of Brookings. How 

the pathogen was introduced into Oregon is unknown. Since its discovery, the 

objective of forest managers has been to eradicate P. ramorum from Oregon forests. 

This has involved a major cooperative effort by state of Oregon and federal agencies. 

There is an intensive on-going monitoring and survey program that includes aerial 

and ground surveys and water baiting in streams to detect the pathogen. As soon as a 

new infection center is confirmed, it is treated by eliminating all hosts within the 

center and a buffer area. This usually involves cutting, herbicide treatments, and 

burning. Federal and state quarantines are in place to prevent movement of infected 

plant material and soil from the entire area where infections occur. There is also a 

substantial public education program. To date, management treatments have not 

succeeded in eradicating P. ramorum, but they do appear to have been successful at 

keeping the pathogen in a fairly small area and greatly limiting the amount of damage 

it causes relative to that in areas in other regions where no treatments have been 

done. 

Golden Chinquapin Decline 

A very noticeable decline of golden chinquapin (Chrysolepis chrysophylla) has been 

observed in a number of locations in Southwest Oregon mostly since 2000. Affected 

host trees develop root necrosis, substantial cankers on their lower boles, and 

chlorotic crowns. They soon die. Most impacts are noted on hosts growing close to 

roads and in poorly drained locations. Recent work at Oregon State University 

(Saavedra et al. 2007) demonstrated that golden chinquapin decline is caused by P. 

cambivora. The origin of the pathogen on this host in Southwest Oregon is not 

known, but it probably represents another Phytophthora introduction, at least at a 

local level. Though it is a common and widely distributed tree in Southwest Oregon 

and one that may be of substantial importance in providing food for a number of 

wildlife species, golden chinquapin is not a significant timber tree. Concern about 

golden chinquapin decline among forest managers is just beginning to develop. 

Current objectives are to determine the distribution and potential severity of the 

disease, develop an understanding of the ecology of the causal pathogen, and identify 

possible techniques that could be used if management is deemed appropriate. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

165 

Phytophthora-caused Sugar Pine Mortality in the 

Sprague Seed Orchard 

The Sprague seed orchard located near Merlin, Oregon was intended to provide seed 

for regeneration of sugar pine (Pinus lambertiana) on Bureau of Land Management 

and Forest Service lands throughout the northern portion of the species range. A fastgrowing and very valuable tree, sugar pine is the largest of all pines and has a wide 

ecological amplitude. It is greatly desired for management. Trees at the Sprague 

Orchard represent tested families that are resistant to white pine blister rust, caused 

by the fungus Cronartium ribicola an extremely virulent exotic pathogen of fiveneedle pines including sugar pine. Unfortunately sugar pine orchard trees at Sprague 

have experienced substantial mortality due to infection by P. cryptogea which was 

apparently introduced on the site in water used for summer irrigation shortly after the 

orchard was established. The pathogen subsequently was spread widely across the 

orchard in water and via equipment. Approximately 4,000 of the high value orchard 

trees have been killed since 1992. Clearly, the objective of orchard managers is to 

ensure survival of the valuable C. ribicola-resistant sugar pine seed trees, but 

suggested management recommendations have not been rigorously followed. 

Recommendations included water treatment, limiting or suspending summer 

irrigation, avoiding planting of seed trees in low-lying portions of the orchard 

grounds, roguing infected trees, and employing chemical treatments where 

appropriate. These should have been incorporated into management from the time 

that the Phytophthora situation was recognized. The current plan is to phase out the 

Sprague Seed Orchard, transfer the C. ribicola-resistant families to distant uninfested 

sites using seed and cuttings from the Sprague Orchard and exercising great caution 

not to introduce P. cryptogea with them, and accept the disruption that this will cause 

in the timeline of the white pine blister rust resistance program. 

Phytophthora Root Diseases in the J. Herbert Stone 

Nursery 

The J. Herbert Stone Nursery is the main federal bare-root nursery facility for the 

northwestern United States. It supplies seedlings for use in tree planting throughout 

Oregon and Washington. The nursery is located in Southwest Oregon in the town of 

Central Point. A number of Phytophthora spp. have caused significant mortality of 

conifer seedlings, especially Douglas-fir (Pseudotsuga menziesii) and five-needle 

pines, in the nursery in the past. Phytophthora spp. identified at the J. Herbert Stone 

Nursery included P. cryptogea, P. drechsleri, P. megasperma, P. pseudotsugae, P. 

gonapodyites, and P. citricola. In attempting to deal with Phytophthora diseases at 

the nursery, the management objectives are to minimize losses of seedling crops and 

prevent spread of disease to field planting sites on infected seedlings. In the past, 

treatments involved irrigation water chlorination, fumigation of nursery beds, 

avoidance of planting in poorly-drained beds, and monitoring and culling of infected 

seedlings. This combination was quite successful. Unfortunately, the water 

chlorination system has been shut down for safety reasons and fumigation has been 

largely discontinued. Now most treatment involves concentrating planting in welldrained beds, spot-treatments with fungicides, and monitoring. There has not yet 

been a resurgence of Phytophthora spp. at the nursery, but the possibility is a 
GENERAL TECHNICAL REPORT PSW-GTR-221

166 

significant concern. Because the nursery is large (about 120 hectares) and the demand 

for conifer seedlings currently is quite low, nursery managers have leeway to plant 

seedlings on only the least hazardous, best drained portions of the nursery and also do 

not have to plant the same beds repeatedly without crop rotation or fallowing. 

Constant vigilance is practiced in monitoring seedling crops for evidence of disease. 

Phytophthora Diseases at the Dorena Genetic Resource 

Center 

The Dorena Genetic Resource Center is responsible for developing disease resistant 

conifers for federal land management agencies and cooperators throughout Oregon 

and Washington. It has extensive, very important screening and breeding programs 

aimed at developing stock resistant to such diseases as white pine blister rust and 

Port-Orford-cedar root disease. Unfortunately, scattered unexpected occurrences of 

Phytophthora diseases have been detected at the Dorena Center. Several species 

including P. citricola, P. gonapodyidaes, P. pseudotsugae, P. nicotianae, P. lateralis, 

and P. cinnamomi have been identified. The last two are especially worrisome. The 

objectives of the managers of the Genetic Resource Center are to completely 

eliminate impacts of Phytophthora diseases on their resistance testing and breeding 

programs, and prevent loss of test trees, breeding stock, and containerized seed 

orchard trees. So far, Phytophthora diseases have been detected on few trees in very 

limited areas and have been immediately treated when found, but managers recognize 

that with a program like theirs, it is critical to have zero tolerance for any additional 

introductions of pathogenic Phytophthora spp. Current management strategies 

include development of a water treatment plant, stringent sanitation efforts (including 

improvement of growing and storage facilities), strict measures to prevent additional 

introductions on equipment, vehicles, and personnel, and constant vigilance by 

Center employees and Forest Service and Oregon State University pathologists. 

Concluding Comments 

Based on our experience with the above mentioned forest Phytophthora issues, the 

staff of the Southwest Oregon Forest Insect and Disease Service Center has 

developed a list of what we consider to be some of the significant challenges 

associated with management of forest Phytophthora spp. In no particular order they 

include: 

 Many of the Phytophthora diseases that affect forest trees involve nonnative, invasive Phytophthora spp. This has substantial implications 

regarding unexpected, rapidly developing impacts, difficulties in detecting 

and identifying the pathogens when they are first introduced, lack of prior 

knowledge of pathogen biology and ecology, initial lack of knowledge of 

potential management approaches and techniques, and hosts that have not 

evolved with the pathogens and thus have very little genetic resistance. 

Management campaigns against non-native pathogens are most successful 

when detection and response are extremely rapid but this is often more easily 

said than done. Once a new Phytophthora is established, eradication is very 

difficult and expensive. It would be so much preferable to prevent non-native 

species from being introduced into our forests in the first place. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

167 

 Many of our Phytophthora issues have involved various kinds of facilities 

such as nurseries, seed orchards, and breeding centers. Given the large 

concentrations of hosts in such places, Phytophthora diseases have great 

potential to intensify, cause substantial damage, and possibly be spread from 

such facilities to other areas, including the forest if they are not managed 

correctly. Suites of several Phytophthora species have often been found in 

facilities. Facilities can potentially be places where hybridization occurs. 

Facility managers need constant education on the dangers of Phytophthora

spp. (as well as other pathogens). There is room for significant improvement 

in considering Phytophthora risks in the planning of facility location, layout, 

and management regimes. 

 Spread of forest Phytophthora species involves water, roads, and the air, not 

easy avenues to manage. 

 The commitment of administrators and higher level resource managers to 

addressing Phytophthora diseases is frequently less than we might like and 

response is all too often not as rapid as would be desirable. Our managers are 

probably typical of forest managers everywhere. They want to do the right 

thing but they are generally quite conservative and risk averse. Many also 

don’t understand Phytophthora spp. They need to learn more but often feel 

that they don’t have the time. We’ve found that managers can be very 

impressed by well-done risk assessments and impact models. Close 

cooperation among a number of managers or even agencies is important for 

some treatments. The good cooperation that we’ve enjoyed so far with the 

sudden oak death eradication program in Oregon is a model for such projects. 

 The American public owns the public lands managed by the federal 

government in the United States and makes use of them for many, often 

highly diverse activities. Public users can have a significant role in spread of 

some forest Phytophthora spp. (such as P. lateralis). They also have major 

(and sometimes counterproductive) opinions on forest management policies. 

Many in the public have little or no knowledge of Phytopthora spp. Public 

education and information transfer about these pathogens is very important. 

 Costs of Phytophthora spp. management activities are often high (and are 

almost always perceived as very high by managers). 

 Management activities for Phytophthora diseases may appear to conflict with 

other forest objectives. For example, clearing to attempt to eradicate P. 

ramorum may be viewed as negatively impacting some kinds of important 

wildlife habitat. It is essential to communicate well with other resource 

specialists in all Phytophthora management strategies. 

 Use of various chemicals (fungicides, resistance enhancers, herbicides) can 

be very desirable in some kinds of Phytophthora management strategies. 

Unfortunately in the United States, use of chemicals on federal forest lands is 

viewed with suspicion and fear and is actively opposed by sectors of the 

public. What can be done to improve this situation? 
GENERAL TECHNICAL REPORT PSW-GTR-221

168 

 The importance of accurate monitoring and good quality, relatively frequent 

surveys for keeping track of forest Phytophthora-caused diseases cannot be 

overstated. Developing good designs, having well-trained surveyors 

available, and paying for surveys and monitoring efforts can be challenging. 

 Phytophthora spp. can be complex and difficult to identify and work with 

even for trained plant pathologists. Pathologists working with these 

pathogens need to have laboratory facilities. Diagnostics in some cases 

require rather sophisticated laboratory equipment. Extension pathologists 

working with forest Phytophthora spp. are well advised to develop 

relationships with Universities, other research organizations, and 

Phytophthora specialists. We have benefited greatly from our association 

with Oregon State University. 

 There is a great need to support quality research on forest Phytophthora spp. 

There also must be flexibility so that research on newly emerging forest 

Phytophthora issues and newly introduced forest Phytophthora spp. can be 

undertaken when needed. There aren’t enough research facilities that can do 

work on forest Phytophthora spp. 

 Global climate change has the potential to greatly impact the ecology of 

pathogenic forest Phytophthora spp. and affect possible management 

strategies for them. What will be the ramifications of predicted changes? 

How can we prepare ourselves to deal with them? 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

169 

Port-Orford-cedar Family Variation in 

Resistance to Phytopthora lateralis: 

Greenhouse Test of 125 Families1

Richard A. Sniezko,2 Everett M. Hansen,3 Angelia Kegley,2 

Scott E. Kolpak,2 and Paul Reeser3

Abstract 

Artificial inoculation of young Port-Orford-cedar (Chamaecyparis lawsoniana) seedlings in 

containers provides a method of potentially evaluating hundreds of seedling families or rooted 

cuttings over a relatively short time period for resistance to the root pathogen Phytophthora 

lateralis. This method has now been used as a tool for more than ten years in an operational 

screening program to select resistant trees to establish seed orchards. 

The trial reported here evaluates 125 seedling families of Port-Orford-cedar (POC) inoculated 

with P. lateralis in February 2005. The seedlots were a mixture of control crosses (full-sib 

and selfs) and open-pollinated half-sib families from 88 parents (87 unrelated). The seedlots 

represent a mixture of untested parents and previously tested parents. Seedlings were 

approximately 11 months old at time of inoculation, and were grown in 164 cm3

 tubes in soilless medium. Families were arranged in seven blocks in a randomized complete block design, 

generally with seven trees per family in each block. A mixture of two isolates of P. lateralis

was used to inoculate the roots. Seedlings were kept in the greenhouse after inoculation. 

Mortality of seedlings has been assessed thirteen times through January 30, 2007 

(approximately 24 months after inoculation). Dead trees were recorded by date of assessment. 

A seedling was considered ‘dead’ when a stem lesion was evident around the stem above 

ground level following scraping of the bark. The first symptom was often wilted or slightly 

off-color foliage. Mean trial mortality was 54.1 percent at 12 months and 60.3 percent at 24 

months after inoculation. Families ranged in mortality from 0 to 100 percent. Several 

previously well documented resistant and susceptible parents performed as expected, showing 

low, moderate, and high levels of mortality. 

Rates of mortality varied widely for families with high levels of mortality. Of the three 

families with 100 percent mortality, two reached 100 percent mortality within 130 days from 

inoculation, while the third family took 217 days. Traditionally, mortality was assessed for 

only up to 12 months in our trials. However, the longer trial period here appears to separate 

families with moderate levels of mortality and putative major gene resistance from those that 

are ‘slow dying’. There may be at least two types of resistance, and this separation will allow 

us to classify families for selection and breeding. Field trials are underway to ascertain the 

utility of both types of resistance. In addition, future greenhouse trials will examine the 

repeatability of the ‘slow dying’ type of resistance. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 USDA Forest Service, Dorena Genetic Resource Center, 34963 Shoreview Road, Cottage Grove, OR 97424, USA. 3

 Department of Botany and Plant Pathology, Oregon State University, 2082 Cordley Hall, Corvallis, OR, 97331, 

USA. 

Corresponding author: rsniezko@fs.fed.us. 
GENERAL TECHNICAL REPORT PSW-GTR-221

170 

Phytophthora ramorum in Oregon Forests: 

Six Years of Detection, Eradication, and 

Disease Spread1

Alan Kanaskie,2 Everett M. Hansen,3 Ellen Michaels Goheen,4

Michael McWilliams,2 Paul Reeser,3 and Wendy Sutton3

Abstract 

Phytophthora ramorum was first discovered in Southwest Oregon forests in 2001, where it 

was killing tanoak (Lithocarpus densiflorus) and infecting Pacific rhododendron 

(Rhododendron macrophyllum) and evergreen huckleberry (Vaccinium ovatum). At that time 

there were nine infested forest sites totaling 16 ha. Aerial photographs suggest that P. 

ramorum probably was killing tanoak trees at one of these sites as early as 1998. In fall of 

2001 eradication of the pathogen from infested sites began by cutting, piling and burning 

infected plants and all host vegetation within 30 to 100 m. of infected or symptomatic plants. 

Upon completion of burning most sites are planted with non-host or conifer seedlings. After 

one year it was clear that tanoak stumps sprouted prolifically following treatment and that P. 

ramorum occasionally was recovered from these sprouts. Follow-up treatments were 

necessary to destroy residual host material and stump sprouts that potentially harbored the 

pathogen. In 2003 and subsequent years all tanoaks in treatment areas were injected with 

herbicide prior to cutting in order to prevent sprouting (except on USDI-BLM lands where 

herbicide use was banned). Eradication treatments have been completed or are underway on 

approximately 560 ha of forest land, at a cost of $2.6 million dollars. Nearly all eradication 

costs have been paid by federal and state agencies, with much cooperation by landowners. 

There is no compensation to landowners for loss of timber or other values as a result of the 

eradication treatments. 

Early detection surveys are conducted year-round using a combination of aerial surveys, 

ground surveys, and stream water baiting with rhododendron and tanoak leaves. During the 

first 4 years of the eradication effort (2001-2004), the number of new infested sites and 

infected trees decreased each year, suggesting modest success at containment and eradication. 

That trend ended in 2005. In 2006 and 2007 the extent of the disease increased considerably. 

In 2007, six new sites were found outside of the quarantine area, and several of these sites 

were 3 to 4 km from the nearest other infested site (the quarantine area will be enlarged in 

2008). Most of the other new sites were small (less than 0.2 ha) and scattered near the center 

of the quarantine zone. We attribute this disease expansion to consecutive years of unusually 

wet spring and early summer weather which appears to favor long distance spread of P. 

ramorum. All surveys to date have failed to detect the pathogen anywhere in Oregon forests 

except in or very near the 65 km2

 Curry County quarantine area (figs. 1 and 2). The nearest 

known SOD infestation outside of Oregon is 200 km to the south in southern Humboldt 

County, CA. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Oregon Department of Forestry, Forest Health Management, 2600 State Street, Salem, OR 97310, USA. 3 Oregon State University, Department of Botany and Plant Pathology, 2082 Cordley Hall, Corvallis, OR 97331, 

USA. 

4 USDA Forest Service, Forest Health Protection, 2606 Old Stage Road, Central Point, OR 97502, USA. 

Corresponding author: Akanaskie@odf.state.or.us. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

171 

Figure 1—Expansion of sudden oak death in Oregon forests: 2001 2003, 2005, 

and 2007. Black blotches indicate infested areas, all of which have been 

treated in an effort to eradicate the pathogen. Dashed line is the P. ramorum

quarantine boundary. White circles highlight 2007 infestations outside of 

quarantine boundary. Curry County, Oregon. 
GENERAL TECHNICAL REPORT PSW-GTR-221

172 

0

20

40

60

80

100

120

140

160

180

2001 2002 2003 2004 2005 2006 2007

Year

Infected Tanoak

Infested Area (ha)

Hectares or # of Trees

Figure 2—Number of new infected tanoaks and the area of new infestations 

each year in southern Curry County, Oregon. 

Each year most new infested sites occurred very close to previously discovered infested sites. 

Post-treatment surveys indicate that disease is absent on most sites and that the pathogen, 

when it is present, can be recovered only at extremely low frequencies from soil and host 

plants. It is unlikely that new infestations in the adjacent forest occur because the pathogen 

survived the eradication treatments. It is more likely that the new infestations occur because 

the pathogen has already spread to these areas but was not detectable in our surveys. Future 

success of the program will require earlier detection of infected plants, reduction in the time 

between detection and treatment, and larger treatment areas. 

The net effect of the eradication program thus far has been to eliminate the pathogen from 

some sites and greatly slow spread of P. ramorum in Oregon forests. When comparing 

expansion of the Oregon infestation (aggressive eradication program) to a similar area in 

Humboldt county California (no comprehensive control program), it is clear that the disease 

can expand rapidly in the absence of control measures (fig. 3). It is equally clear that 

containment and eradication of P. ramorum from Oregon forests will require a much more 

substantial and aggressive program of early detection and treatment. 

0

500

1000

1500

2000

2500

2001 2002 2003 2004 2005 2006 2007

Year

Oregon Infested Area

Humboldt Cty. Infested Area

Hectares

Figure 3—Comparison of the expansion of sudden oak death infestations in 

Curry County OR and Humboldt County, CA. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

173 

Persistence of Phytopthtora ramorum After 

Eradication Treatments in 

Oregon Tanoak Forests 1

Ellen Michaels Goheen2, Everett Hansen3, Alan Kanaskie4, 

Wendy Sutton3, and Paul Reeser3

Abstract 

Sudden oak death, caused by Phytophthora ramorum, was identified in late July 2001 

in forest stands in Curry County on the Southwest Oregon coast where it was killing tanoak 

(Lithocarpus densiflorus) and infecting Pacific rhododendron (Rhododendron macrophyllum) 

and evergreen huckleberry (Vaccinium ovatum). Affected stands occurred on industrial forest 

land, non-industrial forest land, and federal forest land. Treatments to eradicate the pathogen 

from affected sites were started in the fall of 2001 and consisted at that time of cutting, piling, 

and burning all infected host vegetation and any known Oregon host species within a 15 to 30 

meter buffer around all infected plants. While a number of plant species on the official host or 

associated host lists occured in Oregon forests, only those plant species that had a history of 

being infected in Oregon were treated. Patch size of the initial treatment areas ranged from 0.2 

to 4.5 hectares. Since that time, additional disease centers have been identified and eradication 

treatments have been completed at every site. Some treatments were adjacent to sites treated 

previously while others involved distinct new centers. Size of treated sites has varied widely. 

Over the last five years, treatment methods have been altered to reflect increased 

understanding of host susceptibility and pathogen survival and spread. Additional treatment 

components have included various combinations, where possible, of backpack herbicide 

spraying to kill stump sprouts, stump-top treatments with herbicides to prevent tanoak 

sprouting, injecting all tanoaks greater than 2.5 cm diameter with herbicides to prevent 

sprouting, raking, piling, and burning all Oregon host material, and increasing buffer width to 

100 meters. Some sites have been planted with conifer seedlings while others have not. 

The end result is a mosaic of treatment patch sizes, shapes, and structures including sites 

where 1) Douglas-fir (Pseudotsuga menziesii) logs were removed from the sites and the 

tanoaks and understory species were cut and destroyed in broadcast burns, 2) sites where 10 

to 20 percent of the conifer overstory remains and the treatment area has been replanted with 

conifer seedlings, 3) a site where little change has occurred in an old-growth coast redwood 

(Sequoia sempervirens)/Douglas-fir overstory but more than 90 percent of the midstory and 

understory trees and shrubs were removed, and 4) small (less than 0.2 ha) openings with nonhost mature alder trees left on site. 

Most affected stands in Southwest Oregon occur in the Douglas-fir/Tanoak forest type and 

have an original overstory component comprised predominantly of tanoak and Douglas-fir, 

with lesser amounts of red alder (Alnus rubra), Oregon myrtle (Umbellularia californica), 

cascara (Frangula purshiana), Pacific madrone (Arbutus menziesii), and bigleaf maple (Acer 

macrophyllum). In 2006, an infested site in a Coast Redwood/Tanoak forest type was 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 USDA Forest Service, Forest Health Protection, Central Point, Oregon 97502, USA. 3

Department of Botany and Plant Pathology, Oregon State University, Corvallis Oregon 97331, USA. 

4 Oregon Department of Forestry, Forest Health Management, 2600 State Street, Salem, OR 97310, USA. 

Corresponding author: egoheen@fs.fed.us. 
GENERAL TECHNICAL REPORT PSW-GTR-221

174 

confirmed and subsequently treated. On that site, Douglas-fir and coast redwood make up the 

overstory and tanoak was the predominant component of the midstory prior to treatment. The 

understory species mix in both forest types treated includes substantial amounts of tanoak, 

Pacific rhododendron, Oregon myrtle, and evergreen huckleberry, as well as a number of 

other shrub and herb species. Many of the species in both the overstory and understory 

produce prolific stump sprouts following cutting or burning. 

Treated sites are being monitored for the presence of the pathogen using a variety of 

techniques including stream-water baiting with tanoak and rhododendron leaves, soil 

sampling and baiting using pear and leaf baits, and collecting vegetation samples on site and 

subjecting them to isolation and PCR-based diagnostic tests. While a few of the sites treated 

initially are now evidently free of the pathogen, on many sites P. ramorum, is still present in 

the soil or can be baited from streams flowing through treatment areas. 

There is also a strong interest in monitoring both planted and natural vegetation on these sites. 

This interest stems from the desire to quantify and qualify how vegetation responds to 

treatment protocols, how vegetation responds in the presence of the pathogen, and whether the 

resulting vegetation structure and composition on the sites following treatments is consistent 

with overall landowner objectives. 

A series of variable-radius and fixed area plots designed to sample between 5.0 to 7.5 percent 

of the treated areas were established to describe vegetation condition before and after 

eradication on a selection of sites. Trees greater than 13 cm dbh were accounted for in 

variable-radius plots. Trees less than 13 cm dbh were tallied and percent cover of shrub 

species was estimated on 0.02 ha circular plots. In some cases, pre-eradication conditions 

were determined from plots just outside treated areas but in similar forest types. Plants on 

plots and on transects between plots were examined for symptoms of P. ramorum infection. 

ELISA tests using field diagnostic kits were used to prescreen some symptomatic tissues for 

Phytophthora species infection. Most suspicious samples were sent to the laboratory at 

Oregon State University for identification. 

Vegetation data five years after treatment show little change in overall species composition on 

treated sites, with the obvious exceptions that the overstory tanoak component on treated sites 

was eliminated in all cases, and on two sites, conifers not present before treatment were 

planted after treatment. In situations other than industrial forest lands where merchantable 

Douglas-firs within treated sites were cut and logs were removed to a nearby mill, conifers 

such as Douglas-fir or coast redwood, or non-host hardwoods such as red alder were left 

standing on sites. There were substantial changes in the percent cover occupied by understory 

tanoak, evergreen huckleberry, and Pacific rhododendron after treatment; however, these 

species were not entirely eliminated on any treated site. Tanoak sprouts were sometimes 

missed in post-treatment herbicide sprays. Evergreen huckleberry and Pacific rhododendron 

were not routinely subjected to herbicides and many sprouted back after cutting. Percent 

cover of sword fern (Polystichum munitum) generally increased after treatments. No samples 

collected on plots or along transects between plots were found to be infected by P. ramorum. 

Early eradication prescriptions involved only the cutting and burning of infected and buffer 

host plants. When resprouting tanoaks and evergreen huckleberry plants on several sites were 

confirmed to be infected, herbicide treatment to kill stump sprouts was immediately done. In 

2003, three-meter-diameter plots on five of the treatment areas were located around 43 stumps 

resulting from the cutting of known infected trees. Douglas-fir and redwood seedlings were 

deliberately planted as P. ramorum susceptible baits around each of the stumps on some sites 

while on others, seedlings planted as a part of post-eradication reforestation were used as bait. 

Sprouting tanoak, Pacific rhododendron, and evergreen huckleberry were also monitored for 

infection. These stump-based plots have been revisited and vegetation condition of all host 

plants on each plot has been reassessed several times since the initial plot installation, with the 

most recent evaluation done in January 2007. Since the initial identification of infected tanoak 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

175 

and evergreen huckleberry sprouts, no vegetation on these plots, planted or natural, has been 

found infected. 

In May 2003 soil samples were collected at the base of the selected stumps and baited for the 

presence of P. ramorum. Soil sampling at these stumps has continued through the present on 

most sites. Phytophthora ramorum has been recovered from soil samples periodically since 

2003 at the base of selected stumps on three of the sites, was recovered only in the initial 

sampling on one site, and was never recovered from soil samples on one site (Table 1) 

Table 12Number of samples where Phytophthora ramorum was baited from 

soil collected adjacent to stumps of previously infected trees at five 

eradication treatment sites in Curry County, Oregon 

Sample date Site 5/2003 7/2004 12/2004 6/2005 1/2007 

10 2/8* 0/8 0/7 0/8 0/8 

11 0/5 0/5 0/1 1/4 0/3 

18 1/8 0/8 1/8 3/8 2/8 

33 3/17 0/5 7/16 4/17 8/13 

36 0/5 0/5 Not 

sampled 

0/5 Not 

sampled 

* Number of stumps where P. ramorum was recovered from adjacent soil/Total 

number of stumps sampled at the site 

Baits consisting of Pacific rhododendron and tanoak leaves placed in mesh bags and floated in 

streams running through treated sites are used to monitor the presence of P. ramorum. 

Recovery of P. ramorum from leaf baits in these streams varies from site to site and by 

sampling period. On some sites, P. ramorum can still be recovered from stream water several 

years after initial treatment. However, recovery success is greatest on those sites where the 

pathogen appears to still be active (where new infections adjacent to treated areas have been 

confirmed). No new infections have been observed that would appear to be associated with 

downstream, waterborne movement of the pathogen or other stream related factors. 

After herbicide injection to prevent resprouting of tanoak was incorporated into eradication 

treatment prescriptions on selected sites, monitoring to determine if the pathogen was present 

and sporulting in tanoak canopies and slash pies was instituted. Tanoak and Pacific 

rhododendron leaf baits are floated on distilled water in open plastic bags suspended in 

buckets. Buckets have mesh lids to prevent through-flow of leaves and debris and rainwater is 

captured in the buckets after it flows through the canopy. Over the course of monitoring 

several sites in several years these rainwater capture buckets have been placed under known 

infected green tanoaks, known infected herbicide-treated tanoaks, healthy herbicide-treated 

tanoaks, slash piles with infected leaf material incorporated, under trees on the perimeter of 

treated areas, under healthy tanoaks, and out in the open (no canopy). While inconsistent from 

sampling period to sampling period and site to site, P. ramorum has been recovered in an bait 

buckets in all situations except undwhen buckets are placed under healthy “control” tanoaks 

or out in the open. 

Conclusions from monitoring soil, water, and vegetation response to P. ramorum eradication 

treatments in Southwest Oregon five years after treatment include the following: 

 Phytophthora ramorum can still be recovered from treated sites approximately five 

to six years after treatment. 

 New infections resulting from stream water are not apparent. 

 Most components of the native flora found in Douglas-fir/Tanoak and Coast 

Redwood/Tanoak ecosystems are essentially intact on eradication sites after 
GENERAL TECHNICAL REPORT PSW-GTR-221

176 

treatment, excepting tanoak which has been removed from the overstory and 

midstory of all treated sites. 

 Openings create conditions apparently unfavorable for P. ramorum. No new 

infections have been found on host species regenerating or surviving within treated 

areas. This includes host species in close proximity to known infested stumps or 

where soil remains infested. 

 Landowner objectives for growing conifer species are not compromised. Planted 

Douglas-fir and coast redwood seedlings/saplings associated with known infested 

stumps and in areas where soil remains infested are healthy. 

Vegetation monitoring on treated sites will continue. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

177 

Management of Phytophthora kernoviae

and P. ramorum in Southwest England1

Joan F. Webber2 

Abstract 

Phytophthora ramorum and P. kernoviae are considered to be two of several invasive tree 

Phytophthoras recently arrived in Britain. Both have established to some extent and are found 

in planted ornamental woodland-gardens and woodlands where rhododendrons (mainly 

Rhododendron ponticum) dominate. The infected foliage of rhododendron supports abundant 

sporulation by both these aerial Phytophthoras and there is little doubt that this host has 

played a key part in the spread of these pathogens in the natural environment and subsequent 

infection of trees. Whilst P. ramorum has been found primarily in woodland-gardens, P. 

kernoviae has been found more often in mixed species woodlands with rhododendron as an 

understorey component. A key part of efforts to contain and eradicate both pathogens 

therefore centers on the removal of infected rhododendron because it acts as the primary 

inoculum source on most outbreak sites. 

Much eradication and containment effort has focused on P. kernoviae because it has a very 

confined distribution (almost exclusively in south west England) and has also been found in 

four nurseries. However, the process of rhododendron removal on infested woodland sites can 

be very costly. It has been estimated that full-scale removal from infested woodland sites, just 

in Cornwall alone, could cost £millions. Therefore high priority sites for clearance are 

selected on the basis of several criteria including public access, plant movement (some 

woodland gardens have associated plant retail areas), numbers of infected rhododendron and 

the number of infected trees. Studies at both P. ramorum and P. kernoviae infested sites show 

that both pathogens may persist for many months in naturally infected foliage in the litter 

layer in woodlands. These results suggest that removal of infected rhododendron is just the 

first stage in any attempt to eradicate these pathogens, as they have the potential to persist for 

years and infect any re-growth of rhododendron that may occur. 

Introduction 

Phytophthora ramorum and P. kernoviae are considered to be two of several 

invasive Phytophthoras that have recently arrived in Britain (Brasier and others 

2004) and pose a threat to trees and woodland ecosystems. Their origins are 

uncertain although P. ramorum is now well known as the cause of widespread 

mortality oak mortality in California (Rizzo and others 2005; Frankel, 2008). Since 

2002, P. ramorum has also been reported from twenty European countries, primarily 

in nurseries, where it is usually found infecting the foliage of ornamental plants of 

the genera Rhododendron, Viburnum and Camellia (see European RAPRA project 

on P. ramorum at http://rapra.csl.gov.uk). This is in marked contrast to P. kernoviae

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Forest Research, Alice Holt Lodge, Farnham, Surrey, GU10 4LH, U.K. 

Corresponding author: joan.webber@forestry.gsi.gov.uk. 
GENERAL TECHNICAL REPORT PSW-GTR-221

178 

which has only been reported from two countries: Britain (with records in England, 

Scotland and Wales) and New Zealand (Ramsfield and others 2008). 

The first reports of P. ramorum in Britain came in 2002; initially the only plants 

affected were ornamental species and findings were limited to nurseries. Since then 

however, the pathogen has been found at more than 200 garden/woodland sites, 

mainly infecting Rhododendron ponticum or other rhododendron species and 

cultivars (Defra 2008). If the outbreak is widespread and severe on rhododendron, 

then many spores are produced and nearby trees can become infected and develop 

symptoms of stem bleeding, also known as bleeding canker (Brown and others 

2006). In these circumstances, the tree species most likely to succumb to P. 

ramorum incited bleeding canker is Fagus sylvatica (European beech), although 

other species such as Quercus cerris (Turkey oak), Q. rubra (red oak) and 

Nothofagus obliqua (southern beech) have all proved to be susceptible and become 

naturally infected in the field (Webber 2008). Whilst surveying for P. ramorum in 

south west England in 2003, another new and unrelated species of Phytophthora was 

discovered, later named Phytophthora kernoviae (Brasier and others 2005). It soon 

became apparent that P. kernoviae was an aggressive aerial pathogen which also 

attacked the leaves and shoots of R. ponticum and then sporulated on the leaves. 

Furthermore, just as with P. ramorum, it could spread from rhododendron onto 

mature beech trees causing bleeding stem lesions. However, unlike P. ramorum, the 

majority of P. kernoviae findings have been in the natural or semi-natural 

environment with around 50-60 outbreaks sites identified (Defra 2008) and only a 

handful of findings in nurseries. Thus for both pathogens, the occurrence of 

rhododendron in gardens and woodlands has played an essential part in their spread 

into the natural environment and the subsequent infection of trees. 

Outbreaks of P. ramorum and P. kernoviae 

Climatic analysis, initially using the model CLIMEX (R. Baker, CSL; U.K.: RAPRA 

project http://rapra.csl.gov.uk) and then later revised based on Meentemeyer and 

others (2004), identified the western British Isles as most suitable for the 

establishment of P. ramorum. Essentially the climate of this region which is mild and 

tends to have high levels of precipitation appears to be very conducive for infection 

of aerial plant parts by this pathogen. The same environment also seems to be ideal 

for P. kernoviae with episodes of mild wet weather promoting infection and 

sporulation on susceptible foliar hosts. 

The south west of England, particularly the county of Cornwall, has several features 

which appear ideal for the two species of Phytophthora: this includes high levels of 

precipitation, infrequent frosts and acidic soils which favour the growth of 

rhododendrons. Circumstantial evidence suggests that plant trade and the 

transportation of plants have accidentally introduced both pathogens to this area, and 

the combination of suitable climate and availability of epidemiologically important 

hosts has allowed them both to establish to a limited extent. Since the discovery of P. 

kernoviae in November 2003, it has been found extensively in an area of about 5.5 

square miles in southern Cornwall between the towns of Redruth and Falmouth 

which is now defined as the Phytophthora kernoviae Management Zone or PkMZ 

(Anon. 2004). In addition, a small cluster of outbreaks in southern Wales and also in 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

179 

Argyll, Scotland have been identified, and these areas have similar climatic, soil and 

vegetation characteristics to Cornwall. 

Both Phytophthora pathogens are treated as quarantine organisms although their 

status differs. The risk that P. ramorum poses to the nursery trade and potentially to 

woodlands and other ecosystems within Europe resulted in the European 

Commission decision of 19 September 2002. This put emergency measures in place 

to counter further introductions of P. ramorum and prevent its spread. It requires 

eradication action whenever the pathogen is detected in nurseries, and either 

eradication or containment if it is found in natural or semi-natural environments. In 

Britain, similar measures are applied to P. kernoviae, but as most of the findings have 

been in Cornwall, the efforts at eradication and containment have centred on infested 

woodlands and woodland-gardens in this region. For both pathogens this process 

involves removal and destruction of rhododendron but this does pose a number of 

problems. In many of the woodland gardens where infestations occur and trees are 

infected by P. ramorum or P. kernoviae, the infected rhododendrons are often 

specimen ornamental plants and a valued feature of the gardens. In Cornwall alone 

there are 30-40 of these historic parks and gardens and collectively they cover more 

than 3,500 ha. In contrast, the woodlands which contain infected trees and 

understorey R. ponticum are frequently neglected and unmanaged, and therefore of 

low commercial value, but often have difficult terrain for rhododendron clearance 

activity as well as public access. There are also environmental constraints (no 

burning on site) which adds to the difficulty and expense of disposing of the infected 

material safely. Calculating the cost and impact of eradication action is difficult but it 

has been estimated it could be as much £8-10 million over a five year period to 

eradicate Phytophthora from within the PkMZ alone (Snowden and Thompson 

2005). 

Clearance of Infected Rhododendrons 

The limited distribution of P. kernoviae in particular provides the opportunity to 

contain and possibly even eradiate this pathogen and prevent its spread throughout 

Britain as well as into plant trade pathways. However, in the absence of sufficient 

funding to undertake the complete eradication of all infected rhododendron, clearance 

is currently underway on affected sites that pose the greatest risk of pathogen spread. 

Thus each site is given a priority ranking for clearance based on specific available 

information. Key details which inform the selection of sites include: 

 The amount of rhododendron cover in a woodland or wood-land garden and 

the proportion that is infected. Sites with many rhododendron bushes coupled 

with heavy infestations are considered most likely to act as infection 

reservoirs that allow escape of these invasive pathogens to new locations. 

The occurrence of infected trees on the site is a further indicator that the 

infestation is intense and of longer standing. 

 The value of the site, particularly its status as a heritage garden or ancient 

semi-natural woodland. 

 The potential for both pathogens to transported from the site, possibly 

through plant sales of susceptible species from an on-site retail outlet or by 

plant exchanges. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 180

• Amount of public access to the outbreak area, either as a tourist attraction in 

the case of the heritage woodland gardens or via public footpaths and use for 

dog walking and recreation. Evidence has shown that human activity can 

spread P. ramorum and P. kernoviae (Cushman and others 2008; Webber and 

Rose 2008). 

• The presence of water courses on the site, as water is known to facilitate the 

movement of Phytophthoras. Many streams, water courses or ponds have 

tested positive for these pathogens on infested sites in Britain. 

Sites which meet most or all of these criteria and thereby pose the greatest risk 

(for example, those with heavy infestations combined with high levels of public 

access and plant sales) are scheduled for rhododendron removal as quickly as 

possible. If the whole site cannot be cleared immediately, then areas around 

footpaths and public access are cleared as part of disease management. In 

infested woodland the process not only includes the removal of all infected and 

potentially infected rhododendron bushes, but clearance of all the fallen litter 

which often contains infected rhododendron leaves and could be a source of 

pathogen inoculum in future. In addition, re-growth of R. ponticum often occurs 

from the cut stumps even after herbicide treatment, so repeat herbicide treatments 

are usually needed (Edwards 2006). 

Impact of Rhododendron Removal on Disease 

Development 

In the case of P. kernoviae, extensive monitoring of some infested woodlands within 

the PkMZ has been undertaken before and after clearance of the infected 

rhododendrons. It has concentrated on two small woodland sites, in which 25 percent 

and 17 percent of the total beech tree population has become infected with P. 

kernoviae. The presence of P. kernoviae in soil and litter layers was assessed just 

prior to clearance around the infected trees, as these were the areas with the most 

heavily infected rhododendron. Testing methods involved baiting the samples of litter 

and soil and then isolation on Phytophthora selective medium. One and three years 

after the clearance of the rhododendron understorey, the same locations in the 

woodland were again tested for the presence of P. kernoviae (fig. 1). 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 181 

0

10

20

30

40

50

60

70

80

Pre-treatment

(Sept '04)

 Post-treatment

(Sept '05)

 Post-treatment

Sept 07

Percent Pk positive samples

Litter Soil

Figure 1—Outcome of soil and litter sampling pre- and post- Rhododendron ponticum

removal in a Phytophthora kernoviae infested woodland (September 2004 and 2005 

unpublished data from A. Brown and C. Brasier). 

Removal of infected R. ponticum caused a marked decline in inoculum levels in the 

woodlands (fig. 1) and no further trees became infected although the disease 

progressed on many of the already affected trees. However, even following the 

removal of all infected rhododendrons, P. kernoviae apparently persisted in soil and 

litter, albeit at reduced levels, for at least three years and possibly longer. In addition, 

some of the cut rhododendron stumps continued to sprout with some of the shoots 

becoming infected by P. kernoviae years after the original clearance process. 

Therefore, regular herbicide treatment to kill the stumps and prevent re-sprouting is 

an essential part of any eradication process. 

Persistence of P. kernoviae in soil and leaf litter presumably follows when oospores 

have been formed in infected foliage. Experiments looking at the persistence of both 

P. kernoviae and P. ramorum in naturally infected rhododendron leaves have shown 

that both pathogens are long-lived even when the leaves have been shed. After more 

than year, viable Phytophthora could be detected in 5-10 percent of air suspended 

leaves, and in a much higher proportion of leaves that formed part of the litter layer. 

Monitoring beyond a year was impossible as the leaves break down and become 

comminuted, and are subsumed into the soil layer, but the likelihood is that these leaf 

fragments may still contain long-lived spores; chlamydospores in the case of P. 

ramorum and oospores with P. kernoviae. 

Conclusions

Successful eradication of both P. kernoviae and P. ramorum from infested gardens 

and woodlands will inevitably be a long-term process, because of the ability of both 

pathogens to persist in the environment several years after the main foliar host, 

rhododendron, has been removed. In the addition, the ability of rhododendron to 

regenerate from the remaining stumps following removal (even after herbicide 
GENERAL TECHNICAL REPORT PSW-GTR-221

182 

regenerate from the remaining stumps following removal (even after herbicide 

treatment) coupled with the emergence of new seedlings, emphasises that the process 

of eradication will not be achieved in a single action. However, removal of infected 

rhododendrons markedly reduces the risk of trees succumbing to infection by P. 

kernoviae even when the pathogen continues to persist in the environment. Moreover, 

removal of R. ponticum from woodlands has added benefits apart from combating P. 

kernoviae and P. ramorum. As an introduced plant species it has proved to be highly 

invasive so removal provides biodiversity benefits as well as returning woodlands to 

improved status. 

Literature Cited 

Anon. 2004. The Plant Health (Phytophthora kernovii Management Zone) (England) Order 

2004. The Stationery Office, ISBN0110513878. 

Brasier, C.; Denman, S.; Brown, A.; Webber, J. 2004. Sudden oak death (Phytophthora 

ramorum) discovered on trees in Europe. Mycological Research 108: 1108-1110. 

Brasier, C.M.; Beales, P.A.; Kirk, S.A.; Denman, S.; Rose, J. 2005. Phytophthora 

kernoviae sp. nov., an invasive pathogen causing bleeding stem lesions on forest trees and 

foliar necrosis of ornamentals in the U.K. Mycological Research 109: 853-859. 

Brown, A.; Brasier, C.; Denman, S.; Rose, J.; Kirk, S.; Webber, J. 2006. Tree hosts as 

aerial Phytophthora infections with particular reference to P. ramorum and P. kernoviae at 

two U.K. survey sites. In: Progress in Research on Phytophthora Diseases of Forest Trees 

(Proceedings of the IUFRO conference on Phytophthora pathogens of trees, Freising, 11–17 

September 2004), eds. C. Brasier, T. Jung and W. Oβwald, pp. 122-125. 

Cushman, J.H.; Cooper, M.; Meentemeyer, R.; Benson, S. 2008. Human activity and the 

spread of Phytophthora ramorum. In: Frankel, S.J.; Kliejunas, J.T.; Palmieri, K.M., eds 

Proceedings of the sudden oak death third science symposium. Gen. Tech. Rep. PSW-GTR214, Albany, CA: Pacific Southwest Research Station, Forest Service, U.S. Department of 

Agriculture: 179-180. 

Defra 2008. Consultation on the Future Management of Risks from Phytophthora ramorum

and Phytophthora kernoviae. Department for Environment, Food and Rural Affairs, London. 

www.defra.gov.uk/corporate/consult/phytophthora-ram-kern/

Edwards, C. 2006. Managing and Controlling Invasive Rhododendron. Forestry Commission 

Practice Guide, Forestry Commission, Edinburgh.

Frankel, S.J. 2008. Sudden oak death and Phytophthora ramorum in the USA: a 

management challenge. Australasian Plant Pathology 37: 19-25. 

Meentemeyer, R.; Rizzo, D.; Mark, W.; Lotz, E. 2004. Mapping the risk of establishment 

and spread of sudden oak death in California. Forest Ecology and Management 200: 195-214. 

Ramsfield, T.D.; Dick, M.A.; Beaver, R.E.; Horner, I.J. 2008. Phytophthora kernoviae - of 

Southern Hemisphere origin? This volume. 

Rizzo, D.M.; Garbelotto, M.; Hansen, E.M. 2005. Phytophthora ramorum: integrative 

research and management of an emerging pathogen in California and Oregon forests. Annual 

Review of Phytopathology 43: 309-335. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

183 

Snowdon, P.; Thompson, M. 2005. Phytophthora kernoviae – Economic appraisal of control 

options. Unpublished report. Corporate and Forestry Support, Forestry Commission, 

Edinburgh, U.K. 

Webber, J.F. 2008. Status of Phytophthora ramorum and P. kernoviae in Europe. In:

Frankel, S.J.; Kliejunas, J.;T.; Palmieri, K.M., eds Proceedings of the sudden oak death third 

science symposium. Gen. Tech. Rep. PSW-GTR-214, Albany, CA: Pacific Southwest 

Research Station, Forest Service, U.S. Department of Agriculture: 19-26. 

Webber, J.; Rose, J. 2008. Dissemination of aerial and root-infecting Phytophthoras by 

human vectots. In: Frankel, S.J.; Kliejunas, J.;T.; Palmieri, K.M., eds Proceedings of the 

sudden oak death third science symposium. Gen. Tech. Rep. PSW-GTR-214, Albany, CA: 

Pacific Southwest Research Station, Forest Service, U.S. Department of Agriculture: 195-198.
GENERAL TECHNICAL REPORT PSW-GTR-221

184 

Australasia is at High Risk of a 

Phytophthora ramorum Epidemic1

Daniel Hüberli,2,3 Kylie Ireland,4 Ian Smith,5 Bernard Dell,2 Michael 

Ormsby,6 David Rizzo,7

 Matteo Garbelotto,3 and 

Giles E.St.J. Hardy2

Introduction 

With an increase in global trade of plants and plant products it is paramount for 

Australasia to identify emerging plant diseases, carriers of these diseases, and the role 

of hosts in the transmission of disease. In Australia, a cause for concern and currently 

listed as a category 1 emergency pest pathogen is Phytophthora ramorum. It has a 

wide host range and causes widespread damage in nurseries and private estates across 

Europe and is devastating coastal forest ecosystems of western USA, mainly in 

California (Rizzo and others 2002, Werres and others 2001). 

Several Australasian plant species, including Griselinia littoralis (New Zealand 

broadleaf), Eucalyptus haemastoma (Australian scribbly gum), and Pittosporum 

undulatum (Victorian box), have already been listed as natural hosts of P. ramorum

based on field observations and pathogenicity tests in the USA and Europe (Hüberli 

and others 2006, RAPRA 2007). While P. ramorum has not been detected in New 

Zealand or Australia, a preliminary study has identified ecosystems that could be 

conducive to disease development in Australia (W. Smith unpublished data). It is a 

pathogen that the region cannot afford as the threat and management implications of 

this pathogen on natural ecosystems, agriculture and horticulture may potentially be 

far worse than that currently posed by P. cinnamomi (O’Gara and others 2005). 

The study aims to provide knowledge of potential hosts and therefore carriers of the 

pathogen, provide data for the establishment of robust quarantine practices and 

reduce the risk of an introduction of P. ramorum into Australasia. 

Materials and Methods 

For the New Zealand study, excised branches and leaves of 17 endemic plants and 

three commercial species (Eucalyptus globulus, Pinus radiata and Acacia 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Centre for Phytophthora Science and Management, Biological Sciences, Murdoch University, Murdoch, WA 6150, 

Australia. 

3 Department of Environmental Science, Policy and Management, 137 Mulford Hall, University of California, 

Berkeley, CA 94720, USA. 

4 CRC Plant Biosecurity and Centre for Phytophthora Science and Management, Biological Sciences, Murdoch 

University, Murdoch, WA 6150, Australia. 

5 School of Forest and Ecosystem Science, University of Melbourne, PO Box 137, Heidelberg, VIC 3098, Australia. 6 Biosecurity New Zealand, Ministry of Agriculture and Forestry, PO Box 2526, Wellington, New Zealand. 7

 Department of Plants Pathology, University of California, 1 Shields Ave., Davis, CA 95616, USA. 

Corresponding author: D.Huberli@murdoch.edu.au. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

185 

melanoxylon) as well as two known Rhododendron host species were inoculated with 

P. ramorum during 2005–2006 at the University of California, Berkeley. Branches 

and leaves were either underbark wound or zoospore dip inoculated. A second foliar 

inoculation experiment of seven species, including two Rhododendron spp., was 

conducted using three different zoospore concentrations. Lesions and recovery were 

recorded after six days incubation in humid chambers at 19°C for all experiments. 

The number of sporangia produced on foliage was counted for the first inoculation. 

For the Australian study, an intensive screening of indigenous plants located in 

regions identified as highly conducive to disease using a CLIMAX model will be 

conducted in California. Initial screens will assess susceptibility to the pathogen 

using detached leaves, followed by further work that focuses on whole plant tissue to 

give a more accurate assessment of natural infection potential. Studies of the 

sporulation potential and asymptomatic transmission of these species will also be 

investigated. 

Results and Discussion 

In branch inoculations, Nothofagus fusca and Pinus radiata were identified as 

susceptible (fig. 1), while Fuchsia excorticata was extremely susceptible in foliage 

inoculations (fig. 2). Additionally, F. excorticata was the only species that supported 

high foliar sporulation. Pinus radiata, N. fusca, and F. excorticata, should be added 

to the potential host list for P. ramorum. They should be monitored carefully in New 

Zealand for symptoms in high risk incursion areas as well as within gardens and 

nurseries in the United States and Europe for symptoms and/or sporulation. The 

results from the New Zealand study and those forthcoming from the Australian study 

will be highly beneficial in creating risk maps of any incursion of P. ramorum into 

Australasia. 

0

10

20

30

40

50

60

70

Prumnopitys spicatus 3/3

Dacrycarpus dacrydioides 2/2Dacrycarpus dacrydioides 1/2

Brachyglottis repanda

Prumnopitys spicatus 1/3

Agathis australis 2/2

Dacrydium cupressinum

Agathis australis 1/2

Sophora microphylla

Leptospermum scoparium

Olearia bullata

Nothofagus menziesii

Fuchsia excorticata 1/2

Coprosma robusta

Eucalyptus globulus

Corynocarpus laevigatus

Acacia melanoxylon

Fuchsia excorticata 2/2

Pomaderris prunifolia

Azalea 1/2

Prumnopitys spicatus 2/3

Pseudopanax arboreus

Azalea 2/2

Pinus radiata

Nothofagus fusca

Rhododendron 2/2Rhododendron 1/2

Species

Lesion length (mm)

Figure 1—Mean lesion length ( standard error) of excised branch inoculations 

of endemic and commercial plant species to New Zealand after underbark 

wound inoculation with Phytophthora ramorum mycelium agar plugs. 

Branches were incubated in a misting chamber in the greenhouse for 10 

days. 
GENERAL TECHNICAL REPORT PSW-GTR-221

186 

0

20

40

60

80

100

120

140

160

180

Dacrydium cupressinum 2

Pinus radiata 3Pinus radiata 1

Nothofagus fusca 2

Azalea 3

Dacrydium cupressinum 3

Pinus radiata 2

Nothofagus fusca 3

Dacrydium cupressinum 1

Nothofagus fusca 1

Pittosporum eugenioides 3

Rhododendron 3

Pittosporum eugenioides 1

Rhododendron 2

Pittosporum eugenioides 2

Azalea 2

Rhododendron 1

Azalea 1

Fuchsia excorticata 2Fuchsia excorticata 3Fuchsia excorticata 1

Species

Lesion area (mm2) 

Figure 2—Mean lesion area of excised leaves of endemic and commercial 

plant species to New Zealand after dip nonwounded inoculation with 

Phytophthora ramorum zoospores. Leaves were incubated for 6 days in a 

humid chamber. 

A more detailed account of the New Zealand project can be found in the subsequent 

journal publication: D. Hüberli, B. Lutzy, B. Voss, M. Calver, M. Ormsby and M. 

Garbelotto (2008) Susceptibility of New Zealand flora to Phytophthora ramorum 

and pathogen sporulation potential: an approach based on the precautionary principle. 

Australasian Plant Pathology, in press. 

Acknowledgements 

For the New Zealand project, we thank B. Lutzy, T. Harnik, K. Maeda, A. Smith and 

B. Voss for assistance with inoculations, University of California Botanical Garden at 

Berkeley for the plant material, and for funding support from Biosecurity New 

Zealand, the Betty and Gordon Moore Foundation and the Pacific Southwest 

Research Station USDA Forest Service. For the Australian study, the Department of 

Environment and Water and CRC Plant Biosecurity funded K. Ireland. 

Literature Cited 

Hüberli, D.; Wilkinson, C.; Smith, M.A.; Meshriy, M.; Harnik, T.Y.; Garbelotto, M. 

2006. Pittosporum undulatum is a potential Australian host of Phytophthora ramorum. 

Australasian Plant Disease Notes. 1: 19-21. 

 

O’Gara, E.; Hüberli, D.; Hardy, G. 2005. Phytophthora ramorum: a threat to Australia? 

Australasian Plant Conservation. 13: 22-24. 

RAPRA. 2007. Naturally infected hosts for Phytophthora ramorum [Database]. 

http://rapra.csl.gov.uk/. (11 March 2007). 

 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

187 

Rizzo, D.M.; Garbelotto, M.; Davidson, J.M.; Slaughter, G.W.; Koike, S.T. 2002.

Phytophthora ramorum as the cause of extensive mortality of Quercus spp. and Lithocarpus

densiflorus in California. Plant Disease. 86, 205-214. 

Werres, S.; Marwitz, R.; Man In’T Veld, W.A.; De Cock, A.W.A.M.; Bonants, P.J.M.; 

De Weerdt, M.; Themann, K.; Ilieva, E.; Baayen, R.P. 2001. Phytophthora ramorum sp. 

nov., a new pathogen on Rhododendron and Viburnum. Mycological Research. 105: 1155-

1165.
GENERAL TECHNICAL REPORT PSW-GTR-221

188 

Screening for Resistance to Phytophthora 

cinnamomi in Hybrid Seedlings of 

American Chestnut1

Steven N. Jeffers,2 Joseph B. James,3 and Paul H. Sisco4

Abstract 

American chestnut (Castanea dentata) once was one of the primary hardwood tree species in 

forest ecosystems in the eastern USA. However, in the 1800s, Phytophthora root rot (PRR; 

also known as ink disease), caused by Phytophthora cinnamomi, resulted in widespread death 

of chestnut in the Piedmont region of southeastern states where clay soils are dominant. This 

was followed in the early 1900s by chestnut blight, caused by Cryphonectria parasitica, 

which almost eliminated chestnut from its primary mountain habitat. Since 1989, the 

American Chestnut Foundation (TACF) has been producing hybrid chestnut seedlings by 

crossing Chinese chestnut (C. mollissima) with American chestnut and then backcrossing 

progeny to C. dentata in an attempt to produce American-type chestnut trees resistant to C. 

parasitica. In recent years, hybrid seedlings planted in the field in southeastern states have 

died from PRR before they could be challenged by C. parasitica. Therefore, in 2004, we 

began screening hybrid seedlings for resistance to P. cinnamomi. In 2004 to 2006, hybrid 

seeds from known crosses were obtained from TACF cooperators, and seeds from C. dentata

and C. mollissima were collected in the field. Seeds were stratified and then planted outside in 

April in replicate 568-liter plastic tubs filled with soilless container mix at a field site in 

Oconee Co., SC. Inoculum was produced by growing two isolates of P. cinnamomi, 

originally recovered from chestnut seedlings, on autoclaved rice grains. Seedlings were 

inoculated 12 to 14 weeks after planting. Inocula were combined, mixed thoroughly, and then 

evenly distributed in 1- to 3-cm-deep furrows between rows of seedlings. Seedlings were 

watered as needed throughout the study period, and the container mix in each tub was brought 

to saturation at least once while plants were actively growing. Plants were evaluated for PRR 

symptoms in December when fully dormant. 

Each year, seedlings started dying approximately 3 weeks after inoculation and continued to 

die throughout the summer months; symptoms were typical of PRR. C. dentata seedlings 

consistently were susceptible, C. mollissima seedlings consistently were resistant, and hybrid 

seedlings varied from susceptible to resistant. Resistant seedlings were planted in the field for 

further evaluation. Preliminary results suggest that resistance is incompletely dominant and 

regulated by one gene. Moreover, the genes for resistance to P. cinnamomi and C. parasitica 

do not appear to be linked. Screening efforts have been expanded in 2007 and will continue in 

coming years. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Department of Entomology, Soils, and Plant Sciences; Clemson University; Clemson, SC 29634-0315, USA 3 Chestnut Return; 260 Steve Nix Road; Seneca, SC 29678, USA. 

4

 The American Chestnut Foundation, Southern Appalachian Regional Office; Asheville, NC 28801, USA 

Corresponding author: sjffrs@clemson.edu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

189 

Introduction 

American chestnut (Castanea dentata) once was one of the primary hardwood tree 

species in forest ecosystems in the eastern United States (Freinkel 2007). However, 

in the 1800s, Phytophthora root rot—also known as ink disease and caused by 

Phytophthora cinnamomi—resulted in widespread death of chestnut trees in the 

Piedmont region of the southeastern states where clay soils are dominant (Crandall 

and others 1945; Zentmyer 1980). This was followed in the early 1900s by chestnut 

blight, caused by Cryphonectria parasitica, which almost eliminated chestnut from 

its primary mountain habitat (Anagnostakis 1987; Freinkel 2007). Since 1989, The 

American Chestnut Foundation (TACF) has been producing hybrid chestnut 

seedlings by crossing Chinese chestnut (C. mollissima) with American chestnut and 

then backcrossing progeny to C. dentata in an attempt to produce American-type 

chestnut trees that are resistant to C. parasitica (The American Chestnut Foundation 

2007). In recent years, hybrid seedlings planted in the field in several southeastern 

states have died before they could be challenged by C. parasitica. In 2003, we 

diagnosed Phytophthora root rot as the cause of this seedling mortality (fig 1). 

Therefore, we initiated a project in 2004 to screen hybrid chestnut seedlings for 

resistance to P. cinnamomi—with the objectives of identifying families with high 

levels of resistance and establishing a population of resistant trees for future breeding 

efforts. 

Figure 1—Phytophthora root rot symptoms on hybrid American chestnut 

seedlings. Left, above ground: chlorosis and wilting of foliage; right, below 

ground: dead and decayed roots and a necrotic lesion advancing up from the 

root crown area (arrow) on the lower stem (bark removed). 
GENERAL TECHNICAL REPORT PSW-GTR-221

190 

Materials and Methods 

Trials began in 2004 and have been conducted each year thereafter; results from trials 

conducted in 2004, 2005, and 2006 are summarized here. For each trial, hybrid 

chestnut seeds from known crosses were obtained from TACF cooperators, and openpollinated seeds from C. dentata, C. mollissima, and C. pumila (chinkapin) were 

collected from trees in the field. Seeds usually were received during the fall and early 

winter prior to planting in the spring. Seeds were mixed with moist peat and placed in 

a perforated plastic bag, and bags were stored in a refrigerator to stratify. In early 

April, when the risk of frost was minimal, germinated seeds were washed free of peat 

(fig. 2A) and planted outside in 568-liter plastic tubs filled with soilless container mix 

(Fafard 3B; Conrad Fafard, Inc., Agawam, MA) at Chestnut Return farm—a field site 

in Oconee Co., SC. Seeds from the same family were planted together in a row and 

families were replicated in multiple tubs (fig. 2B); seeds were planted in two tubs in 

2004 and in six tubs in both 2005 and 2006. Plants were watered regularly throughout 

the growing season so that the container mix stayed moist and did not dry out. Each 

year, tubs were top-dressed once early in the growing season with a complete timerelease fertilizer. 

Figure 2—A, Germinated chestnut seeds ready for planting; B, plastic tub filled 

with container mix after planting chestnut seeds—strings identify rows and 

stakes delimit families. 

Hybrid chestnut seedlings were inoculated 12 to 14 weeks after planting with two 

isolates of P. cinnamomi that originally had been recovered from chestnut seedlings 

growing at the field site. Inoculum was produced by growing each isolate axenically 

on autoclaved rice grains (Burns and Benson 2000) at 25°C in the dark for 10 to 14 

days. Equal volumes of rice grains colonized by each isolate were combined and 

mixed thoroughly to produce a composite inoculum. To inoculate plants, a thin layer 

of rice inoculum was evenly distributed in 1- to 3-cm-deep furrows between rows of 

seedlings. Furrows were covered and the tubs were watered thoroughly to prevent the 

inoculum from desiccating. Several weeks after inoculation, the container mix in 

each tub was flooded once for 4 to 6 hours to promote disease development. Plants 

were evaluated for Phytophthora root rot symptoms in late December or early 

January when seedlings were fully dormant. Each plant was removed carefully from 

a tub—taking care to recover as much of the root system as possible. Individual 

plants were rated for symptom severity using a 0 to 3 scale: 

A B 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

191 

0 = healthy, no visible lesions on roots 

1 = lesions on at least one lateral root 

2 = lesions on the tap root 

3 = severe root rot, plant dead 

Surviving seedlings, primarily those rated 0 or 1, were planted in an orchard at 

Chestnut Return farm. These plants were monitored annually for performance in the 

field. 

Results and Discussion 

Each year, seedlings started dying approximately 3 weeks after inoculation and 

continued to die throughout the summer months; symptoms were typical of 

Phytophthora root rot (fig. 1). C. dentata and C. pumila seedlings consistently were 

susceptible to P. cinnamomi and died; C. mollissima seedlings consistently were 

resistant and grew vigorously in the infested soil. Although hybrid seedlings varied 

from susceptible to resistant, most were susceptible and died (1262/1693 = 75 

percent; table 1). The numbers of families and hybrid seedlings evaluated increased 

each year, but the proportion of seedlings surviving with symptom severity scores of 

0 or 1 varied depending on the genetics of the families evaluated (table 1). Over the 

three-year period, 43 families and almost 1700 seedlings were evaluated (table 1). 

Table 1—Numbers of hybrid chestnut families and seedlings evaluated in 

2004, 2005, and 2006 and the frequency of seedlings in four symptom 

severity (SS) classesz

No. seedlings Year Families 

(no.) Evaluated SS = 0 SS = 1 SS = 2 SS = 3 

2004 5 360 21 31 18 290 

2005 15 596 60 34 111 391 

2006 23 737 8 24 123 581 

Total 43 1693 89 89 252 1262 z

 0 = healthy; 1 = lesions on lateral roots; 2 = lesions on tap root; 3 = plant dead 

The strength of resistance in these 43 families was determined by calculating the 

Survival Quotient (SQ), which is expressed as percentage: 

SQ = ([(1 × n0) + (0.5 × n1) +( 0.25 × n2)] / total number of seedlings) × 100 

where n0, n1, and n2 = no. seedlings rated 0, 1, and 2, respectively. 

Of the 43 families evaluated (Table 2), 11 families (26 percent) had no survivors and 

a SQ of 0 percent; 18 families (42 percent) had a SQ between 0.1 and 15.0 percent; 

10 families (23 percent) had a SQ between 15.1 and 30.0 percent; three families (7 

percent) had a SQ between 30.1 and 40.0 percent; and one family had a SQ over 50 

percent (Hyko x JB575, SQ = 56.3 percent). 

Over the three-year period, 189 resistant seedlings were planted in the field for 

further evaluation. To date, 77 of these have survived; these 77 survivors represent 
GENERAL TECHNICAL REPORT PSW-GTR-221

192 

40.7 percent (77/189) of those planted in the field and 4.5 percent (77/1693) of all the 

hybrid seedlings evaluated. Seedlings with a symptom severity rating of 0 or 1 have 

survived better than those with a rating of 2. Consequently, resistance to P. 

cinnamomi was present in some of the hybrid chestnut families that had been selected 

for resistance to C. parasitica. Preliminary results suggest that resistance to P. 

cinnamomi is incompletely dominant and may be regulated by more than one gene. 

Moreover, the genes for resistance to P. cinnamomi and C. parasitica do not appear 

to be linked. Screening efforts have been expanded in 2007 and will continue in 

coming years. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

193 

Table 2—Strength of resistance in 43 hybrid chestnut families screened over three 

years, 2004 to 2006: a survival quotient was computed for each family based on the 

number of seedlings in each of three symptom severity classesz

 

Family Source of Resistance Generation Survival Quotient 

CL-50 Clapper B2F2 0.0 

Andover x BX39 Mahogany BC3 0.0 

HP - A4 (CL248) Clapper B2F3 0.0 

HP - C7 (CL248) Clapper B2F3 0.0 

HP - L (CL248) Clapper B2F3 0.0 

Milliken D5 op Clapper B2F3 0.0 

Milliken D6 op Clapper B2F3 0.0 

VKN x VA307 Clapper B3F1 0.0 

Uxbridge KD-1 x GL356 Graves B3F1 0.0 

Spanish Oak Rd x IL201 Clapper B4F1 0.0 

VKS x CB582 Clapper B4F1 0.0 

Fitchburg KJ19 x BG318 Graves B4F1 0.5 

Swallows x TM616 Graves B3F1 0.5 

Pike Co Marinero x GL367 Clapper B3F1 0.6 

Lincoln, RI-1x QG85 Graves B4F1 1.1 

Fitchberg x GL96 Clapper BC3 1.2 

Sudbury x BE138 Graves BC3 1.4 

Newton CS19 x HE416 Clapper B4F1 2.4 

HP - B4 (CL248) Clapper B2F3 3.1 

rc97-107 (JoScxGR210) x opBC3 Clapper B3F2 3.2 

Uxbridge x GL356 Graves BC3 4.5 

James D-20 x Maddox MS8-12 and 9-12 Clapper B2F3 6.3 

rc97-m (FhSo x opAM) x opBC3 (Gr210) Clapper B4F1 6.5 

ob00-m (JLCe x VA307) x opBC3 Clapper B3F2 8.0 

ob00-025 (TRTCm x AB427) x opBC3 Clapper B3F2 9.1 

hu97-m (Ort x GR137) x opBC3 Graves B3F2 9.3 

CL-149 Clapper B2F2 10.6 

HP - F1 (CL198) Clapper B2F3 12.2 

CL-112 Clapper B2F2 12.9 

Milliken E7 op Clapper B2F3 17.6 

Frye Mtn. x B3_176 Graves B4F1 19.2 

ob99-199 (StT1 x GR210) x opBC3 Clapper B3F2 20.0 

HP - F2 (CL198) Clapper B2F3 20.6 

Sudbury x AB247 Graves BC3 20.7 

Milliken D1 op Clapper B2F3 23.3 

HP - F12 (CL198) Clapper B2F3 24.8 

CL-326 Clapper B2F2 25.0 

Rn88-38 x Hind Douglas Hybrid B2F1 25.0 

Franklin EMC x B2_214 Mahogany B3F1 29.2 

CL-248 Clapper B2F2 32.5 

Boston SB1 x JB575 Mahogany BC3 37.2 

Milliken Tree D1 op Clapper B2F3 37.5 

Hyko x JB575 Mahogany BC3 56.3 

z Survival Quotient (in percent) = ([(1 × n0) + (0.5 × n1) +( 0.25 × n2)] / total number of seedlings) × 100 

where n0, n1, and n2 = no. seedlings rated 0, 1, and 2, respectively 
GENERAL TECHNICAL REPORT PSW-GTR-221

194 

Literature Cited 

Anagnostakis, S.L. 1987. Chestnut blight: the classical problem of an introduced pathogen. 

Mycologia 79:23–37. 

Crandall, B.S.; Gravatt, G.F.; Ryan, M.M. 1945. Root disease of Castanea species and 

some coniferous and broadleaf nursery stocks, caused by Phytophthora cinnamomi. 

Phytopathology 35:162-180. 

Freinkel, S. 2007. American Chestnut: The Life, Death, and Rebirth of a Perfect Tree. 

Berkeley, CA: University of California Press. 284 p. 

Burns, J.R.; Benson, D.M. 2000. Biocontrol of damping-off of Catharanthus roseus caused 

by Pythium ultimum with Trichoderma virens and binucleate Rhizoctonia fungi. Plant Dis. 

84:644-648. 

The American Chestnut Foundation. 2007. http://www.acf.org/. (Date accessed 11 March 

2008). 

Zentmyer, G.A. 1980. Phytophthora cinnamomi and the diseases it causes. Monograph No. 

10. St. Paul, MN: American Phytopathological Society. 96 p. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

195 

Field Evaluation of Cork Oak Seedling 

Viability in a Soil Naturally Infested With 

Phytophthora cinnamomi1

A.C. Moreira,2 J. Albuquerque,3 E. Melo,2 J.M.S. Martins,2 

 R. Tapias,4 and A. Cravador5,6

Abstract 

The present work describes the evaluation of the tolerance or susceptibility of cork 

oak seedlings to Phytophthora cinnamomi infection in the field, and discusses the 

possibility of using their genetic diversity to control the cork oak (Quercus suber) 

decline in areas highly infested with this pathogen in Portugal and Spain. Acorns 

were colleted from trees located in six geographically distinct regions of Portugal 

(Trás-os-Montes, Estremadura, Ribatejo, Alto Alentejo, Baixo Alentejo and Algarve) 

and in two of Andalucía, Spain (Huelva and Málaga), and sown in 2004 in a soil 

naturally infested with P. cinnamomi situated in Algarve, the southern region of 

Portugal. The percentage of acorn germination was evaluated, the mortality was 

assessed and the height of the seedlings was measured twice a year (June/July and 

December) during three consecutive years. The highest percentage of germination 

was observed within families of the Portuguese regions of Alto-Alentejo and 

Estremadura. Mortality in the first year after sowing (2004) reached 12.3 percent and 

doubled during the following two years. Moreover, results show a high variability in 

the percentage of survival after germination between half-sib seedlings, which was 

not found to be correlated with geographical nor maternal origin. In 2006, seedlings 

with disease symptoms or already dead were harvested and P. cinnamomi was 

detected in 85 percent of them. Seedling height in December 2006 was significantly 

different between families of same maternal origin and between regions. Those from 

Alto-Alentejo, Baixo-Alentejo and Algarve produced the highest seedlings at the end 

of the third year. In summary, both maternal and regional origin significantly 

influences both acorn germination and seedling growth, indicating a distinct 

susceptibility to P. cinnamomi among maternal genotypes. 

Introduction 

Cork (Quercus suber L.) and holm (Q. ilex subsp. rotundifolia Lam.) oak trees are 

two evergreen species with high economic importance for the Iberian Peninsula. 

“Montado” is an agro-silvo-pastoral system created by man, populated mainly by 

cork and holm oak trees and peculiar to southern Iberian Peninsula. It faces a serious 

ecological, socio-economic and landscaping problem due to the oak decline that is 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Estação Agronómica Nacional, Departamento de Protecção de Plantas, INIAP 2784-505 Oeiras, Portugal. 3

 Associação de Produtores Florestais da Serra do Caldeirão, Barranco do Velho, Salir, Portugal. 4

 Departamento de Ciencias Agroforestales, Universidad de Huelva, Carretera de Palos de la Frontera s/n, 

21819 Palos de la Frontera, España. 

5

 Universidade do Algarve, Faculdade de Engenharia dos Recursos Naturais, Campus de Gambelas, 8005-

139 Faro, Portugal. 

6

 IBB/CGB-UTAD apartado 10103, Vila Real 5001, Vila Real, Portugal. 

Corresponding author: acmarcellino@yahoo.com.br.
GENERAL TECHNICAL REPORT PSW-GTR-221

196 

threatening the stability of the entire ecosystem. Phytophthora cinnamomi Rands has 

been associated with extensive decline and death of these species (Brasier and others 

1993 a, b, Moreira and Martins 2005, Sánchez and others 2002, Sánchez and others 

2003, Tuset and others 1996). In Portugal, the isolation frequency of P. cinnamomi

from cork and holm oak roots and associated soils has increased in the last decade, 

namely in Alentejo region (from 35percent to 90 percent, non published results). This 

soil-borne plant pathogen has been introduced in Europe, where it is known since the 

early 19th century (Brasier 1993, Zentmeyer 1980); it causes the death of the fine 

roots, reducing the capacity of absorption of water and nutrients, leading to plant 

decay and death (Moreira and Martins 2005, Robin and others 1998). 

The control of this pathogen is very difficult, as it presents a large host range and 

may persist in soil for long periods as resistant structures (Moreira and Martins 2005, 

Zentmeyer 1980). Several methods have been proposed to control P. cinnamomi.

Chemical treatments were tested in Spain (Fernández-Escobar and others 1999, 

Navarro and others 2004) and are being used in some areas of Portugal to treat adult 

trees, but their long-term efficiency has not yet been clearly established. 

Resistance to P. cinnamomi has been reported in some tree species (Butcher and 

others 1984; Stukely and Crane 1994). The possibility of using cork oak plants more 

tolerant or resistant to P. cinnamomi infection can be an important tool to the 

reforestation of highly infested areas. Tree survival in several dieback affected sites 

suggests the existence of genetic variability in this species that may be used to select 

more tolerant individuals. 

The present work reports the results of a preliminary experiment designed to assess 

the diversity of response of cork oak plants to P. cinnamomi infection in field 

conditions and to evaluate their resistance or tolerance potential. 

Material and Methods 

Acorns from 20 individual trees were collected at several places in each of six 

geographically distinct regions of Portugal (Trás-os-Montes, Estremadura, Ribatejo, 

Alto Alentejo, Baixo Alentejo and Algarve) and in two of Andalucía, Spain (Huelva 

and Málaga) (fig. 1). Trees were selected imposing at least 100 m distance from one 

another. Acorns were collected during November and December 2003. Seeds were 

preserved at low temperature (4 oC) until they were sown. Sowing was carried out in 

January 2004 in Barranco do Velho, Algarve (south of Portugal), in a loamy soil 

naturally infested with P. cinnamomi where all the maquis vegetation was cut. None 

of the extant adult cork oak trees, healthy, diseased or already dead, was uprooted 

during the period of observation. The field was divided in ten 160 m2 plots. In each 

plot sowing was carried out in such a way that acorns collected from each tree were 

represented in each plot and randomly distributed. Acorns were sown in rows spaced 

1 m from each other, and at 1 m intervals along the rows. Soil of six plots (1 through 

6; see fig. 2) was mechanically ripped to improve root system development and waterflow; the other four plots, located in an area with a high slope, were left untreated to 

prevent soil erosion. The percentage of germinated acorns was assessed six months 

after sowing. Seedling mortality and their stem height were recorded twice a year 

(June or July and December) during three consecutive years. During 2006 and 2007, 

eighty seedlings having contrasting dieback symptoms and development were 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

197 

removed to observe the root system and to detect the presence of the pathogen. The 

roots were cut into 1 cm sections and plated onto selective medium (PARBHy; see 

Robin 1991). Recovery of the pathogen was assessed after three and five day 

incubation at 24 oC. 

Figure1—Geographical origin of the tested acorns. 

Figure 2—Distribution of the experimental plots. Solid lines: counter curves 

(equidistance = 25 m); dotted lines: seasonal brooks. 
GENERAL TECHNICAL REPORT PSW-GTR-221

198 

Results and Discussion 

Results showed that germination was highly dependent on the family and geographic 

origin (contingency analysis, P≈0 in both cases). Acorns from populations of Alto 

Alentejo and Estremadura showed the highest percentage of emergence, those from 

Trás-os-Montes, Ribatejo and Huelva the lowest (fig 3A, Table 1). 

Table 1—Differences of percentage germination of acorns and 

cumulated mortality (three years) of seedlings in half-sibling populations 

according to geographical origin 

Origin Germination Mortality Origin Germination Mortality

Alto Alentejo 0.76 0.19 Baixo Alentejo 0.61 0.23

Estremadura 0.73 0.29 Ribatejo 0.60 0.27

Algarve 0.62 0.28 Trás-os-Montes 0.53 0.27

Málaga 0.62 0.22 Huelva 0.47 0.28

Location and soil treatment of the experimental plots seemed to influence the 

germination and the growth – plots 1 through 4 allowed a higher percentage of seed 

germination and a better development of the seedlings than plots 7, 8 and 9 (extreme 

differences ranging from 33 percent to 81 percent; Table 2). Eventually, mechanical 

soil preparation and other local factors (such as slope, exposition, distance to the 

seasonal brook, uneven distribution of the inoculum in the soil) may have contributed 

to the lack of homogeneity of the ten plots. The shallow and rocky soil nature of 

these plots certainly contributes to a lower germination rate and may be a 

contributing factor to P. cinnamomi infection (Brasier 1993; Moreira and Martins 

2005). To test the significance of this constraint, the plots were ordered by the overall 

percentage of germination, and separated in two groups as in Table 2. A Spearman’s 

correlation was calculated between the ranks of each half-sibling in both groups of 

plots. The correlation coefficient was found to be highly significant (P(rs)<0.01). 

Thus, it can be concluded that, despite the very high differences in percentage 

germination among plots, the relative position of each half-sibling respecting to the 

others was maintained. 

Table 2—Percentage of acorn germination 

in ten experimental plots 

Plot Germination Plot Germination 

 1 0.81 5 0.58 

 3 0.79 10 0.56 

 2 0.78 8 0.51 

 4 0.69 7 0.47 

 6 0.61 9 0.33 

By the end of autumn 2004 (first year) global mortality was 12.3 percent and 

decreased during the two subsequent years (6.3 percent in 2005 and 6.0 percent in 

2006; see details in fig 3B). The observed mortality followed the same trend in all 

three years of observation, its incidence seeming to be randomly distributed by the 

half-siblings, and also by their geographic origin (contingency analysis, not 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

199 

significant). As observed for germination, the heterogeneity of the plots seemed not 

to interfere with the comparative mortality: Spearman’s correlation was also highly 

significant when applied to the comparison of the ranks of the percentage of mortality 

in half-siblings at the end of the three years of observations. The evolution of 

mortality can be understood having in mind the lower than normal precipitation that 

occurred in the years 2003-2004 and 2004-2005 (570 and 370 L·m-2, respectively; 

normal: 800-1000 L·m-2 per year). In 2005 the precipitation was lower than in 2004, 

and this may have influenced survival and development of the seedlings, in spite of 

their having been irrigated twice each summer. During 2006 precipitation values 

were normal, mortality decreased to 6 percent in that year. 

Seedling height in December 2006 was significantly different among half-sibling 

populations and among regions (P<0.001; fig 3C). Acorns from Alto Alentejo, Baixo 

Alentejo and Algarve gave origin to the tallest seedlings, which were significantly 

different in height from those from Trás-os-Montes and Ribatejo (Table 3). This 

suggests that, as for germination, seedling development is connected with 

geographical origin. 

Alto Alentejo

Algarve

Baixo Alentejo

Estremadura

Huelva

Málaga

Ribatejo

Trás-os-Montes

0

20

40

60

Shoot length (cm)

 2004

 2005

 2006

C

0.00

0.05

0.10

0.15

0.20

0.25

Mortality

B

0

20

40

60

80

Germination (%)

A

Figure 3–Germination (A), mortality (B) and growth (C) of cork oak acorns 

according to their provenance and sown in a P. cinnamomi naturally infested 

soil. 

Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

201 

Field observations from 2004 to 2006 showed a high variability on germination and 

on growth within families. Ten seedlings represented each family, which is a very 

small sample to be representative. The families listed in Table 4 were those with 

bigger shoots (>44 cm average) and higher survival rates (> 8 out of 10 plants). 

However, there was a continuous variation of those two measures within each halfsib family, as would be expected in segregating descents. 

Table 4—List of families that include the best-developed individuals (2003-

2006) 

Average growth (cm) Family Origin Survival in 10 

plants Min Max Mean 

015 AL 9 41 104 60 

045 AA 9 12 62 44 

011 AL 8 28 70 46 

041 AA 8 34 68 55 

044 AA 8 22 74 50 

054 AA 8 23 60 44 

059 AA 8 17 63 50 

047 AA 8 16 80 50 

030 BA 8 30 76 53 

In 2006 and 2007 eighty seedlings representing 57 half-sib families and all levels of 

development were harvested to assess infection. Phytophthora cinnamomi was 

recovered from the roots of 85 percent of cork oak seedlings, irrespective of their 

development; 25 percent showed non aerial symptoms and a good development, and 

50 percent showed dieback and poor development; all recently dead seedlings (10 

percent) were infected. In 15 percent of seedlings P. cinnamomi was not detected. 

The soil selected for this experiment was highly infested with P. cinnamomi and its 

inoculum was largely distributed, even in places where almost all the adult cork oak 

trees disappeared (e.g., plots 2, 3 and 4). It was previously shown that many shrub 

species growing in cork oak stands are infected, contributing to the inoculum build 

up. Cistus populifolius, for instance, which is very abundant in the area, is a species 

very susceptible to the infection (Moreira and Martins 2005). This suggests that 

maquis species and cork oak adult trees may provide the initial P. cinnamomi 

inoculum to the seedling infection affecting oak reforestation. 

Plots 7, 8 and 9 showed the worst results on germination and development of the 

seedlings. They were located on a hilltop with a very shallow and rocky soil that was 

not mechanically prepared. This is in agreement with previous observations 

identifying hilltops as places favoring infection by P. cinnamomi (Brasier 1993, 

Moreira and Martins 2005). This result suggests that mechanical soil preparation, soil 

variability, and the plot location may have had an influence on the emergence 

success. 
GENERAL TECHNICAL REPORT PSW-GTR-221

202 

Results here reported illustrate the response diversity to the infection, which can be 

interpreted as resulting from the genetic diversity of cork oak species (Coelho and 

others 2006). A similar conclusion was drawn from a study conducted under 

controlled conditions by Tapias and others (2007). 

Maternal and geographical origin significantly influences both acorn germination and 

seedling development, indicating a potential to exploit the differential susceptibility 

to P. cinnamomi in future breeding programs. 

Acknowledgments 

This study was supported by the PO-AGRO 8-Programme for Experimental 

Development and Demonstration from Ministério da Agricultura, Pescas e 

Desenvolvimento Rural (Project 416). 

References 

Brasier, C.M.; Moreira, A.C.; Ferraz, J.F.P.; Kirk, S. 1993a. High mortality of cork oak in 

Portugal associated with Phytophthora cinnamomi. Pp. 461-462. In Luisi N.; Lerario P.; 

Vannini A.(eds.), Recent advances in studies on oak decline. Proceedings of an International 

Congress. Bari, Itália, 13-18 September, 1992. 

Brasier, C.; Robredo, F.; Ferraz, J.F.P. 1993b. Evidence for Phytophthora cinnamomi 

involvement in Iberian oak decline. Plant Pathology 42: 140-145. 

Butcher, T.B.; Stukely, M.J.C.; Chester, G.W. 1984. Genetic variation in resistance of 

Pinus radiata to Phytophthora cinnamomi. For. Ecol. Manage. 8: 197-220. 

Coelho, A.C.; Lima, M.B.; Neves, D.; Cravador, A. 2006. Genetic diversity of two 

evergreen oaks (Quercus suber L. and Q (ilex) rotundifolia Lam.) in Portugal using AFLP 

markers. Silvae Genetica 55 (3): 105-118. 

Fernández-Escobar, R.; Gallego, F.J.; Benlloch, M.; Membrillo, J.; Infante, J.; Pérez de 

Algaba, A. 1999. Treatment of oak decline using pressurized injection capsules of antifungal 

materials. Eur. J. For. Path. 29: 29-38. 

Moreira, A.C.; Martins, J.M.S. 2005. Influence of site factors on the impact of 

Phytophthora cinnamomi in cork oak stands in Portugal. For. Path. 35 (3): 145-162. 

Navarro, R.M.; Gallo, L.; Sánchez, M.E.; Fernández, P.; Trapero, A. 2004. Efecto de la 

fertilización fosfórica y la aplicación de fosfitos en la resistencia de brinzales de encina y 

alcornoque a Phytophthora cinnamomi Rands. Investigación Agraria. Sistemas y Recursos 

Forestales. 13: 550-558. 

Robin, C.1991. La maladie de l’encre du chêne rouge (Quercus rubra L.) causée par 

Phytophthora cinnamomi Rands: Perspectives pour amélioration génétique de la résistance, 

thèse de l’Université de Bordeaux II, 140 pp. 

Rodriguez-Molina, M.C.; Blanco-Santos, A.; Palo-Núñez, E.J.; Torres-Vila, L.M.; 

Torres-Álvarez, E.; Suárez-de-la-Cámara, M.A. 2005. Seasonal and spatial mortality 
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patterns of holm oak seedlings in a reforested soil infected with Phytophthora cinnamomi. 

For. Path. 35: 411-422. 

Sánchez, M.E.; Caetano, P.; Ferraz, J.; Trapero, A. 2002. Phytophthora disease of 

Quercus ilex in southwestern Spain. For. Path. 32: 5-18. 

Sánchez, M.E.; Sánchez, J.E.; Navarro, R.M.; Fernández, P.; Trapero, A. 2003.

Incidencia de la podredumbre radical causada por Phytophthora cinnamomi en masas de 

Quercus en Andalucía. Bol. San. Veg. Plagas 29: 87-108. 

Stukely, M.J.C.; Crane, C.E. 1994. Genetically based resistance of Eucalyptus marginata to 

Phytophthora cinnamomi. Phytopathology, 84: 650-656. 

Tapias, R.; Moreira, A.C.; Cravador, A.; Fernández, M.; Alcuña, M.M.; Sáenz, A.; 

Domingos, A.C.; Melo, E. 2007. Variability of tolerance/resistance of Quercus suber L. 

seedlings to Phytophthora cinnamomi Rands. Survival and growth evaluation. 

SUBERWOOD2005 Conference. New challenges for integration of cork oak forests and 

products. Huelva, 20-22 October 2005. 

Tuset, J.J.; Hinarejos, C.; Mira, J.L.; Cobos, J.M. 1996. Implicación de Phytophthora 

cinnamomi Rands en la enfernedad de la “seca” de encinas y alcornoques. Boletín de Sanidad 

Vegetal, Plagas 22: 491-499. 

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GENERAL TECHNICAL REPORT PSW-GTR-221

204 

National Best Practice in the Management 

of Phytophthora cinnamomi for Biodiversity 

Conservation in Australia1

Emer O’Gara,2 Kay Howard2, Tim Rudman3 and 

Giles E.St.J. Hardy2

Abstract 

Disease in natural ecosystems of Australia, caused by the introduced plant pathogen 

Phytophthora cinnamomi, is listed as a key threatening process under the Australian 

Government Environment Protection and Biodiversity Conservation Act 1999 (EPBC Act). 

The Act requires the Australian Government to prepare and implement a threat abatement 

plan for nationally coordinated action to mitigate the harm caused by P. cinnamomi to the 

Australian environment particularly threatened flora, fauna species and ecological 

communities. 

The ‘National Threat Abatement Plan for Dieback Caused by the Root-Rot Fungus 

Phytophthora cinnamomi’ (NTAP) was released in 2001, and was reviewed and revised in 

2006. The NTAP is designed to promote a common understanding of the national threat P. 

cinnamomi poses to biodiversity in Australia. 

The Centre for Phytophthora Science and Management is a key stakeholder in the 

management of Phytophthora in the State of Western Australia. The Centre has also played a 

key role in undertaking some of the most significant projects of the National Threat 

Abatement Plan, and undertook the review and revision of the Plan in 2006. This has placed 

the Centre in a unique position to critique the mechanisms for management of P. cinnmomi in 

Australia, and the impact that the three-tiered (Commonwealth or Federal, State and Local) 

system of government has on developing, attaining and maintaining ‘best practice’ 

management. This paper examines national and state management frameworks and discusses 

the outcomes for biodiversity conservation in Australia. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Centre for Phytophthora Science and Management, Murdoch University, South St, Murdoch, Western Australia 

6150, Australia. 

3 Vegetation Conservation Section, Biodiversity Conservation Branch, Department of Primary Industries & Water, 

GPO Box 44, Hobart, Tasmania 7001, Australia. 

Corresponding author: e.ogara@murdoch.edu.au. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

205 

Development of U.K./EU/EPPO Pest Risk 

Analyses for Phytophthora kernoviae, P. 

ramorum and P. lateralis1

Claire E. Sansford2

Abstract 

Pest Risk Analysis (PRA) is an internationally recognised, structured process of 

determining whether plant pests and pathogens (known generically as ‘pests’) that are 

normally absent from a country or area could enter, establish and cause an economic or 

environmental risk that is deemed unacceptable. If required, PRA is also used to help 

identify potential phytosanitary measures to reduce the identified risk to an acceptable 

level. The WTO Sanitary and Phytosanitary Agreement requires that a scientific 

assessment of the risk posed by a plant pest is undertaken before any phytosanitary 

measures are set. Such measures must be the minimum needed to reduce the risk without 

unnecessarily impacting on trade. Before this process can commence an organism must be 

identified as being a unique taxonomic entity. In recent years, new plant diseases have 

emerged in the natural environment that have not been immediately connected with a 

single identifiable cause. Subsequently these diseases were found to have been caused by 

new species of Phytophthora. PRAs have been produced and developed over several years 

for two of these: Phytophthora kernoviae and Phytophthora ramorum. Both pathogens 

have been identified as posing a risk to the environment, private and managed gardens and 

woodlands as well as to the ornamental plant trade in the U.K./EU/EPPO region. As a 

result of the PRAs, emergency legislation has been implemented in the U.K. and EU 

respectively for these pathogens to allow action to be taken against them wherever they 

are found. This is due for review in 2008. Following a trend of increasing environmental 

risks posed by new Phytophthora species that can be moved in the ornamental plant trade, 

an EPPO Panel produced a PRA for an ‘old’ Phytophthora, P. lateralis. This PRA resulted 

in a recommendation for regulation in the EPPO region to protect the ornamental plant 

trade alone, as no special environmental areas were identified as being at risk in the 

region. There are more ‘old’ Phytophthora species that are known to pose 

environmental/economic risks and there are likely to be more ‘new’ species emerging. 

With the globalisation of the plant trade these are increasingly likely to be moved into new 

areas. Identifying these threats before they become a reality is a challenge but there is now 

a heightened awareness of the likely increase in circulation of Phytophthora species in 

trade. The PRA framework can be used to determine the risk potential and to 

communicate the risk and the risk management options to policy makers, who have the 

option to consider implementing appropriate preventative measures in a timely way. 

Pest Risk Analysis—the FAO and the WTO 

Pest Risk Analysis (PRA) is a structured process by which the identification and 

assessment of the risk of entry, establishment and spread of exotic plant pests 

(including pathogens) is determined and the potential economic and environmental 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Central Science Laboratory (CSL), York, U.K., YO41 1LZ. 

Corresponding author: c.sansford@csl.gov.uk. 
GENERAL TECHNICAL REPORT PSW-GTR-221

206 

damage arising from their introduction in a given country or area is assessed. PRA 

became formally recognised by the Food and Agriculture Organisation (FAO) 

Secretariat to the International Plant Protection Convention (IPPC) in 1996, by the 

publication of guidelines (FAO, 1996) for the PRA process. PRA is defined as ‘the 

process of evaluating biological or other scientific and economic evidence to 

determine whether a pest should be regulated and the strength of any phytosanitary 

measures to be taken against it’ (FAO, 2007). At the outset of the development of 

standards for PRA, consideration of the risks posed to the environment by pest risk 

analysts was limited, with most PRAs produced concentrating on the risks to crop 

plant species.

Subsequent to the publication of PRA guidelines (FAO, 1996) more detail on what 

elements need to be considered when preparing a PRA were agreed by the Interim 

Commission on Phytosanitary Measures (ICPM) in April 2001, with a supplement to 

include an analysis of environmental risks being approved in April 2003 (and one to 

account for risks from Living Modified Organisms in April 2004), leading to the 

publication of the International Standard for PRA for Quarantine Pests (FAO, 2004). 

The analysis of risks posed by plant pests to the environment and biological diversity 

within this standard includes full consideration of those that might threaten 

uncultivated/unmanaged plants, wild flora, habitats and ecosystems contained in the 

PRA area. 

PRA is the fundamental process which underpins the setting of phytosanitary 

regulations and is prescribed by the World Trade Organisation Sanitary and 

Phytosanitary Agreement (WTO, 1995) for this purpose, as well as the IPPC (IPPC, 

1997). A fundamental principal when phytosanitary regulations are set, is that the 

measures that are imposed should be the minimum necessary to protect plant health 

whilst continuing to facilitate, or at least ensure, minimal disruption to trade. 

Pest Risk Analysis Schemes and Processes 

Different national, regional and international schemes have evolved since the 

publication of the first international standard for PRA (FAO, 1996) with the most 

recent being based upon the framework presented in FAO (2004). At the regional 

level, the European and Mediterranean Plant Protection Organisation (EPPO) have 

published a detailed decision support scheme (EPPO, 2007), with shorter schemes 

being developed by some of the 49 EPPO member countries. 

FAO, 2007a (a revision of FAO, 1996) lists the main reasons (initiation points) for 

preparing a PRA as: 

1. A request is made to consider a pathway that may require phytosanitary 

measures 

2. A pest is identified that may justify phytosanitary measures 

3. A decision is made to review or revise phytosanitary measures or policies 

4. A request is made to determine whether an organism is a pest 

Initiation point 2 is triggered when a pest is detected that poses an immediate or 

potential danger, which under the IPPC (IPPC, 1997) has to be reported, usually to 

the National Plant Protection Organisation (NPPO). This is the main initiation point 

for producing PRAs for new and emerging plant pathogens. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

207 

The PRA process commences with gathering as much information as possible to 

enable the pest risk analyst to answer a series of questions which will determine 

whether the pest fulfils the definition of a quarantine pest, defined as: ‘A pest of 

potential economic importance to the area endangered thereby and not yet present 

there, or present but not widely distributed and being officially controlled’ (FAO, 

2007). This is followed by a determination of the risk of entry, establishment and 

economic/environmental impact culminating in an assessment of the overall risk, and 

a decision as to whether and which phytosanitary measures to apply (pest risk 

management). At each stage of the PRA the analyst is required to identify areas of 

uncertainty in their responses. For pathogens new to science, the level of uncertainty 

is likely to be great, especially in the determination of the risk of establishment, the 

likely impact, as well as the efficacy of measures that could be taken to contain or 

eradicate the pathogen. Where the magnitude of the threat and the level of 

uncertainty are both great, it may be necessary to commission research, the results of 

which can be used to develop the PRA, thus reducing the uncertainty. The revision of 

a PRA in this way may result in a change to the initial phytosanitary policy. 

Meanwhile, in the short-term, once an organism has been identified as a potential 

risk, there is a requirement for good sampling, detection and diagnostic techniques to 

be developed. These can be used by the NPPO as a first barrier to entry. This joinedup process is effective in developing our understanding of the risk but also in 

developing risk management strategies to deal with interceptions and outbreaks of 

plant pests. 

Emerging Pathogens and the Plant Trade—a Challenge 

for the Pest Risk Analyst

Recently, pest risk analysts have had to consider the risks posed by previously 

unidentified pathogens new to science. This is a challenge, as without full taxonomic 

identity it is difficult to proceed with a PRA. FAO (2004) requires that ‘the identity of 

the pest should be clearly defined to ensure that the assessment is being performed 

on a distinct organism, and that biological and other information used in the 

assessment is relevant to the organism in question. If this is not possible because the 

causal agent of particular symptoms has not yet been fully identified, then it should 

have been shown to produce consistent symptoms and to be transmissible. The 

taxonomic unit for the pest is generally species...’.etc. 

Defra Statistics

(http://statistics.defra.gov.uk/esg/publications/bhs/2006/default.asp) show a 

significant increase in imports of various ornamental plants into the U.K. between 

1995 and 2005. For example ca. £6.4 million worth of trees in 1995 increased to ca. 

£64 million in 2005; ca. £5 million ‘cuttings, slips and other young plants’ increasing 

to ca. £16 million. This increase in trade (which has occurred around the world) has 

led to new opportunities for plant pathogens to be moved to new areas or countries. 

For those previously undescribed pathogens, without prior knowledge of their 

existence and if symptom development in plant material is discrete, there is a risk that 

they can enter new areas or countries undetected and establish away from their 

centres of origin. The flora present in the new area may differ from that in the area of 

origin of the pathogen. It is feasible therefore that these ‘new’ pathogens could infect 

and cause disease in a wider range of plant species than was previously available to 
GENERAL TECHNICAL REPORT PSW-GTR-221

208 

them, since there will be no inherent resistance in many of the potential host plants. 

This is especially significant for Phytophthora species which can have a very wide 

host range. On entering a new area, there are many opportunities presented to them to 

increase their host range and to perpetuate themselves there for the foreseeable 

future. 

The emergence of previously unknown Phytophthora species in the ornamental trade, 

e.g. Phytophthora ramorum, or, with the potential to be moved in such a trade e.g. 

Phytophthora kernoviae, both of which pose a threat to the environment, has proved 

challenging for pest risk analysts and their colleagues. In the U.K. there has been 

strong scientific co-operation between Government Agencies including the U.K. 

Central Science Laboratory (CSL) and Forest Research (FR); funding bodies (Defra, 

Forestry Commission (FC), U.K. levy bodies, the European Commission (EC)), 

policy makers, and inspectors in Defra and the FC, as well as the equivalent 

European and North American organisations, to develop a programme of research 

that could be used to enhance our understanding of these new species in order to 

progress the PRAs, particularly for P. ramorum. This has been essential to inform 

policy makers on the risks and strategies that could be deployed against these new 

species. 

Phytophthora ramorum

Phytophthora ramorum was first described as a new species of Phytophthora in 2001 

(Werres and others 2001) and is now known to occur in Europe, the USA and Canada 

where it is subject to official control. Phytophthora ramorum is considered to be an 

exotic pathogen introduced separately to both the USA and Europe from an unknown 

area, or areas, of origin, speculated but not proven to be Asia. The history of its 

identification and the risk it poses to the environments of Europe and North America 

ties-in with an increasing awareness of the risks posed by plant pathogens that can be 

moved in the plant trade. Prior to being formally named and described, the pathogen 

was first observed infecting rhododendron and viburnum in Germany and 

rhododendron in the Netherlands in 1993; however, these observations were not 

formally reported at the time (Werres and others 2001). Separately, in 1995, tree 

death was first observed on tanoak (Lithocarpus densiflorus) in California, USA. The 

causal agent was unknown, but since then, substantial mortality of tanoak as well as 

several Quercus (oak) species became apparent in California and the phenomenon 

became known as ‘sudden oak death’. Investigations into the cause finally led to the 

isolation of a new Phytophthora species in California in July 2000. Although no 

connection had been made between the European and North America scenario, 

because of the massive scale of tree mortality in the USA and the fact that a unique 

causal agent had finally been identified, an assessment of the risk that this new 

Phytophthora posed to the U.K. was produced in September 2000 (Brasier 2000). 

This assessment did not propose specific phytosanitary measures as it was undertaken 

using the EPPO risk assessment scheme (pre-dating the EPPO PRA scheme, thus not 

including the third stage of PRA, i.e. risk management). This risk assessment 

concluded that the pathogen had potential to establish in the U.K., possibly entering 

on nursery stock, and that there was a significant risk posed to (at least) U.K. native 

and exotic oaks. Although there was much uncertainty identified, the author felt that 

urgent research was required to address this; meanwhile suggesting that a decision be 

taken on the status of the pathogen before more information became available. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

209 

In January 2001, Brasier determined that the new Phytophthora species causing 

sudden oak death in California and that which had been isolated from rhododendron 

in the Netherlands and Germany (and viburnum) was the same species. EPPO added 

the pathogen to their Alert List (an early warning, without a full PRA) in January 

2001. A second PRA was produced by the U.K. in April 2001 (published June 2001; 

Jones and Sansford 2001) with an accompanying Datasheet (unpublished). The PRA 

highlighted the risks to the U.K., EU and EPPO region, again identifying 

uncertainties and research needs, but also recommending surveys in the EU/EPPO 

region to determine distribution as well as consideration of phytosanitary measures 

including controls on imports of known susceptible hosts and their products into and 

within the EU/EPPO region from areas/countries where the pathogen has been found, 

to try to prevent entry. It was recommended that consideration should be given to 

continuation of EPPO Alert listing and to making the pathogen an EU/EPPO 

quarantine pest. As a result of the PRA, in the summer of 2001, Defra’s Plant Health 

and Seeds Inspectors (PHSI) for England and Wales commenced limited surveys for 

the as yet unnamed Phytophthora. 

After the pathogen was formally named as P. ramorum in October 2001 (Werres and 

others 2001) a third formal U.K. PRA was published in January 2002 (Jones 2002) 

with an accompanying Datasheet (unpublished). In February 2002, as a result of the 

ongoing survey work, Defra’s Plant Health and Seeds Inspectors (PHSI) submitted 

one symptomatic plant of Viburnum tinus to CSL from a garden centre in southern 

England, and it was confirmed to be infected with P. ramorum (Lane and others 

2003). This was the first U.K. record of P. ramorum. 

Following on from the PRA work, U.K. (England) legislation aimed at P. ramorum

was enacted in May 2002 (Anon. 2002a). This was somewhat earlier than EC 

legislation which came into force in September 2002 (Anon. 2002) based largely on 

U.K. actions. The U.K. (England) legislation required that susceptible plant material 

(listed as 11 genera at this stage) entering England from the USA must be 

accompanied by a Phytosanitary Certificate (PC) with an Additional Declaration 

(AD) that it originated in an area free from P. ramorum. Post-entry controls were also 

required. The U.K. (England) legislation was revoked and replaced in November 

2002 (Anon. 2002b) reflecting the first EC requirements (Anon., 2002). (NB: 

Generally speaking, once enacted, EC legislation is usually adopted by individual EU 

Member States in domestic legislation; thus the Devolved Administrations of the 

U.K. (England, Wales, Scotland and Northern Ireland) each have their own 

legislation). The EC legislation broadened controls on imports of susceptible material 

not just from the USA, but also from other non-EU countries and had requirements 

for controls on movement of susceptible plants within the EU, as well as controls on 

outbreaks, and a requirement for EU Member States to conduct surveys to be 

reported back to the EC by December 2003. One other European PRA was prepared 

by the Netherlands in October 2002 (de Hoop and others 2002) to ensure that 

phytosanitary measures arising from the new EC legislation to be taken in that 

country were technically justified. 

Meanwhile, to address the uncertainties in the U.K. PRA, the U.K. research 

programme for P. ramorum commenced in the spring of 2002. The programme dealt 

initially with the immediate needs, which were to develop an effective diagnostics 

capability and to undertake host range testing, both of which were needed to support 

the inspection and surveillance efforts for interceptions and outbreaks. The long-term 
GENERAL TECHNICAL REPORT PSW-GTR-221

210 

research effort was aimed at developing our understanding of the risk of 

establishment and spread. This was done by laboratory and field studies of the biotic 

and abiotic factors affecting sporulation, dispersal, germination, infection processes, 

survival and population behaviour. Some work on climatic matching was undertaken 

to develop the prediction of the risk of establishment. One area that was not fully 

developed was an examination of the economic implications in terms of the direct 

effects that the pathogen would have in the long-term. An evaluation of risk 

management strategies was also done by laboratory and field experiments. All of the 

information generated was intended to be used to develop the PRA and to inform the 

policy makers of the risk and the management options. Details of Defra-funded 

research can be found at http://www.defra.gov.uk/planth/pramorum6.htm. 

Accounting for ongoing research results, literature, and findings of P. ramorum in the 

EU and North America, the PRA (and Datasheet) were updated again and the revised 

PRA was published in March 2003 (Jones and others 2003) and revised further and 

published again in October 2003 (Sansford and others 2003). This last revision predated the first tree findings in the U.K. and the Netherlands in late October 2003. A 

full update of the Datasheet was prepared during the following year (Sansford and 

Brasier 2004) and a draft PRA, but prior to completion, the first U.K. findings of the 

new pathogen Phytophthora kernoviae (also in October 2003, see below) and the 

expansion in its host range over the following year led to changing priorities and the 

PRA work for P. ramorum was put on hold. 

EC legislation for P. ramorum was amended in April 2004 (Anon. 2004) accounting 

for changes in host range and assessed risk and again in March 2007 (Anon. 2007). 

Current measures still require official surveys to be reported back to the EC at the 

end of the year, and broadly-speaking, import and internal movement controls of 

rhododendron, viburnum and camellia (the three most commonly affected traded 

genera in the EU) with statutory action to be taken on findings. 

In 2007 a full update of the U.K. Datasheet accounting for the results of the full U.K. 

research programme, key aspects of the EU and USA research programme, as well as 

EU and North American survey findings was prepared (Woodhall and Sansford 

2007) to re-examine the risks to the U.K. and to suggest risk management options in 

preparation for a U.K. policy review for P. ramorum in 2008. The pathogen is now 

known to affect more than 70 plant genera in 33 different families. Its origin is still 

unknown and it is still only recorded in North America and the EU where it is found 

both outdoors and in the nursery trade which is where the majority of U.K. findings 

have been made. CSL/FR studies in the U.K. undertaken to date support the view that 

rhododendron foliage is acting as the primary inoculum source for infection of tree 

stems in the U.K. leading to bleeding cankers (summarised in Woodhall and Sansford 

2007). Clearance of invasive Rhododendron ponticum in affected woodlands has 

reduced inoculum loads and reduced the risk to trees; this is one of a number of risk 

management options for current and future consideration in the U.K. In the EU, only 

the U.K. and the Netherlands have reported trees with bleeding cankers caused by P. 

ramorum and these are all associated with infected rhododendron. In contrast to the 

USA, tree mortality is minimal. Phytophthora ramorum is likely to continue to be 

locally damaging in the U.K., especially in managed ornamental gardens with 

established susceptible plants, or woodlands infested with rhododendron, especially 

R. ponticum, and coastal valleys in the south and west of the U.K. The current U.K. 

PRA for P. ramorum is at http://www.defra.gov.uk/planth/pra/sudd.pdf 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

211 

A PRA to examine the risks from P. ramorum to trees, woodland ecosystems and 

other environmentally important habitats (e.g. heathlands) in the EU and the hardy 

ornamental nursery industry as well as to public gardens will be finalised in 2008. 

This PRA will be the culmination of a three year EU-funded research project 

‘RAPRA’ (http://rapra.csl.gov.uk/) which aimed to provide scientific information to 

produce a comprehensive risk assessment for Europe as well as risk management 

options. The consortium that has undertaken the work to complete the PRA 

comprises 8 organisations in 5 European countries and one in the USA. The RAPRA 

Project commenced in January 2004. Work has been done on pathogen distribution 

and spread, host range, epidemiology, and mating types as well as economic impact 

and risk management. The RAPRA PRA will be used to inform the review of EC 

legislation in 2008. 

North American risk assessments/analyses undertaken by various Government 

Agencies can be found at 

http://www.cnr.berkeley.edu/comtf/html/pest_risk_assessments.html#EU

Phytophthora ramorum continues to pose a threat to the managed and unmanaged 

environment (woodlands, gardens, possibly heathland), the timber and ornamental 

plant trade and the tourism industry (i.e. associated with historic gardens) in the U.K., 

EU, North America and to other countries where it is yet to be reported or introduced. 

Phytophthora kernoviae 

A climate-matching model using the CLIMEX programme (Sutherst and Maywald 

1985) was run by CSL in March 2003 (R. Baker, CSL, U.K., personal 

communication, 2003) to identify which areas of the U.K. were most at risk from P. 

ramorum. This work was based upon identifying climatic similarities between the 

U.K. and Oregon/California where P. ramorum occurs. The outcome, was that based 

upon a more similar match with Oregon alone (compared to California), the south 

and west of the U.K. were identified as potentially at high risk of P. ramorum 

establishing there. This information was used to inform the Defra PHSI surveys for 

P. ramorum required under emergency EC (and U.K.) legislation since 2002 (Anon. 

2002, 2004, 2007). Surveillance by Defra PHSI was intensified in Cornwall and as a 

result of this a new and unknown Phytophthora species was isolated by CSL from 

symptomatic rhododendron (R. ponticum and Rhododendron spp.) from an 

established woodland area adjoining a commercial nursery in Cornwall in late 

October 2003. Concurrently, FR isolated a similar Phytophthora from a large 

bleeding canker on a mature beech tree (Fagus sylvatica) and from an adjacent 

rhododendron with foliar symptoms adjacent to the tree at a second woodland site 

located 23km from the first site. The pathogen was causing widespread foliar 

necrosis and shoot dieback of the rhododendrons. By December 2003 the isolates 

were considered to be the same organism and all samples submitted to CSL by the 

PHSI since that date for testing for P. ramorum were also tested for it. 

Having identified a new problem, a joint CSL/FR PRA for Phytophthora taxon C sp. 

nov. (the first informal name for the pathogen) was completed (February 2004) 

(Sansford and others 2004) examining the risks that the pathogen posed to the U.K. 

As with the first PRA for P. ramorum, there was much uncertainty because this was a 
GENERAL TECHNICAL REPORT PSW-GTR-221

212 

new species of Phytophthora, but clearly the pathogen posed a risk to at least beech 

and rhododendron and had potential to spread geographically as well as to other as 

yet unidentified hosts. It was also considered to be an exotic introduction but its 

origin was unknown; consequently management of entry pathways was difficult. The 

PRA recommended destruction of infected plants along with further surveys to 

determine the true distribution of the pathogen both in the U.K. and the EU. Hostrange testing was suggested to determine which other species were at risk and to 

inform future inspection and surveillance work. Accurate diagnostic methods were 

developed by CSL to differentiate the pathogen from P. ramorum. Long-term 

research akin to that being undertaken for P. ramorum was also recommended to 

understand the epidemiology of the pathogen and to develop control strategies. Up to 

September 2004 approximately 5000 samples submitted by the PHSI to CSL for 

testing for P. ramorum were also tested for P. taxon C sp. nov. The pathogen was 

found on a wider range of hosts and sites; new findings emerged in south Wales and 

the first nursery finding occurred in Cheshire. The PRA was updated several times to 

account for these findings culminating in September 2004 (Sansford and others 

2004a). Emergency legislation aimed at containing the pathogen within a specified 

area of the south-west of England with a view to eradication was implemented in 

2004 (Anon., 2004a). Named in 2005 as P. kernoviae (Brasier and others 2005) CSL 

and FR have mainly concentrated on undertaking host-range testing and studying the 

efficacy of rhododendron clearance (the main source of inoculum) as a way of 

reducing the risk the trees, with limited epidemiological studies. Of particular 

significance was the 2006 report of its presence in New Zealand on cherimoya 

(Annona cherimola) since at least 2002 (Anon., 2006; NAPPO, 2006; EPPO, 2006) 

which now dates back to at least the 1950s. Ramsfield and others (2007) reported1

that an unknown Phytophthora known as the Tokoroa Phytophthora is in fact P. 

kernoviae and was first found in the 1950s, under stands of symptomless Pinus 

radiata (radiata pine). This has identified a potential past/future pathway for entry to 

the U.K. and other countries. A new PRA was completed in November 2007 

(Sansford 2007) accounting for all new information since September 2004 to support 

a U.K. policy review in 2008. n examining the risk of entry, detailed CSL records of 

imports of ornamentals into the U.K. from New Zealand permitted under a 

derogation from a prohibition up until 1987 shows large quantities of known 

susceptible hosts (magnolia, rhododendron, Pieris) entering the U.K. between 1977 

and 1985. Imports continued after 1985 but no details were documented as they were 

no longer banned, provided they were accompanied by a PC. Whilst none of these 

ornamentals have been recorded as hosts in New Zealand no surveillance of nurseries 

has been reported there. P. kernoviae has a more limited host-range than P. 

ramorum; by January 2008 it was known to affect 16 genera in 10 families. Unlike P. 

ramorum it has been found on very few (3) U.K. nurseries with most findings being 

outdoors in the south-west of England, south Wales and one (eradicated) location in 

the north-west of England. As with P. ramorum, CSL/FR studies in the U.K. support 

the view that rhododendron foliage is acting as the primary inoculum source for 

infection of tree stems in the U.K. leading to bleeding cankers (summarised in 

Sansford, 2007). Clearance of invasive R. ponticum will reduce the inoculum load in 

woodlands and gardens for P. kernoviae (as well as P. ramorum). The 2007 PRA is 

now being updated to account for the first finding in Scotland and the first finding of 

the pathogen on an environmentally important species Vaccinium myrtillus (bilberry) 

both of which occurred in December 2007. Phytophthora kernoviae continues to pose 

 

1

 Not reported in the abstract; reported in the platform presentation. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

213 

a threat to the managed and unmanaged environment (woodlands, gardens, possibly 

heathland), the timber and ornamental plant trade and tourism (i.e. associated with 

historic gardens) in the U.K., as well as New Zealand. There is a risk to other 

countries where it is yet to be reported or introduced. 

Phytophthora lateralis 

Understanding that existing (‘old’) Phytophthora species can also move in trade and 

have potential to pose new risks, co-operation at the regional level under the auspices 

of EPPO has facilitated the development of the first ever EPPO PRA for 

Phytophthora lateralis and recommendations for appropriate regulation, this time to 

protect the ornamental trade. 

EPPO added P. lateralis to their Alert List in January 2001 as a result of a literature 

search for ‘sudden oak death’ which elicited various papers reporting significant tree 

mortality on Chamaecyparis lawsoniana (Port-Orford-cedar) in the U.S. in forests as 

well as in nurseries and gardens. The pathogen is assumed to have been introduced to 

North America in 1923 from an unknown origin (Roth and others 1972, Kliejunas 

and Adams 1981, Erwin and Ribeiro 1996). Phytophthora lateralis is considered to 

be an exotic introduction to North America from an unknown source (Hansen and 

others 2000). France and the Netherlands produced draft (unpublished) PRAs in 1999 

and 2001 respectively in response to findings in France, and the U.K. prepared a draft 

Datasheet in early 2006 which was provided to EPPO to support their first ever 

EPPO ‘Performing PRA’ panel who also met in early 2006. Phytophthora lateralis

was isolated and identified from C. lawsoniana in 1996 and 1998 in different parts of 

France (Hansen and others 1999); its introduction to France is suspected to have been 

from North America but this could never be confirmed. It was suggested that this 

probably stemmed from a single infestation of young, potted, greenhouse-propagated 

cedars in a commercial nursery. Introduction to the Netherlands was first noticed in 

2004 when a survey was conducted of 350 nurseries with C. lawsoniana. 

Phytophthora lateralis was isolated from the stem bases of C. lawsoniana plants 

from one isolated nursery but the origin of the pathogen was unknown, especially as 

there were no associated imports of the affected plant material and propagation 

material originated from the affected nursery (Meffert 2005). Both the French and 

Dutch outbreaks are considered to be eradicated. The EPPO Panel considered that P. 

lateralis has been introduced occasionally to new areas from unknown origins but 

that it was not established in the EPPO region. The Panel agreed that there was a risk 

of entry (based upon the French and Dutch outbreaks) although imports of the main 

host, C. lawsoniana into EU countries (within the EPPO region) is banned, as with 

the exception of specific derogations for bonsai, the importation of plants of 

Chamaecyparis from countries outside of Europe is prohibited under EU legislation 

(Anon. 2000). Soil from North America is also prohibited. Entry is most likely on 

roots of Taxus brevifolia (Pacific yew, the other main host) and possibly other 

ornamental plants from affected areas of North America (or from the unknown origin 

of the pathogen). Entry may also be possible as a contaminant of growing media or 

soil associated with non-host ornamental species. 

The probability of establishment in the EPPO region was evaluated as high (host 

plants are cultivated in the PRA area, some parts of the PRA area have very 

favourable climatic conditions, nursery production practices are favourable to the 

pathogen) and economic impacts would mainly arise from losses of host plants on 
GENERAL TECHNICAL REPORT PSW-GTR-221

214 

specialist nurseries. Environmental impacts were thought likely to be low because the 

main hosts (C. lawsoniana, Chamacyparis spp. and T. brevifolia) are not key 

components of natural ecosystems in the PRA area. We agreed that P. lateralis 

fulfilled the criteria of a quarantine pest. Having identified the main pathways of 

entry, the Panel proposed risk management options. Phytophthora lateralis was 

moved from the EPPO Alert List to the EPPO A1 list of pathogens as a result of the 

EPPO PRA. 

The U.K. PRA (Woodhall and Sansford 2006) which followed the draft Datasheet 

recommended that consideration be given to EU surveys of susceptible hosts to 

ensure country freedom and, if deemed appropriate, listing P. lateralis as a II/AI 

quarantine pest for the U.K./EU with specific requirements for the relevant pathways. 

This PRA and the proposed measures are subject to consultation though the Defra 

website. 

Discussion and Conclusions 

PRA is a structured process that allows us to evaluate risks from exotic pests, to 

identify uncertainties, to recommend suitable phytosanitary measures and to ensure 

that phytosanitary legislation enacted to manage the risk is re-evaluated in the light of 

new knowledge resulting from research targeted at addressing the uncertainties and 

other sources of information. In the U.K., the link between pest risk analysts, 

researchers, funders, inspectors and policy makers is a strong one. Under the current 

international phytosanitary framework, led by the Secretariat of the IPPC and 

required by the WTO SPS Agreement (WTO 1995) there is no process by which 

phytosanitary measures can be enacted against an unidentifiable cause. Brasier 

(2005) is critical of this system and states (Brasier 2007) that ‘international plant 

health protocols are list-based, allowing regulation only of named organisms’. 

However, it is possible to regulate at the genus level provided the organism is 

identified as being unique (FAO 2004). Thus the first PRAs for P. ramorum and P.

kernoviae were for ‘an unknown Phytophthora’ and emergency legislation was 

enacted in the U.K. for both organisms as quickly as possible following PRA 

production. Our experience with these pathogens has led to a joined-up and proactive 

approach to tackling new phytosanitary problems as far as the legislative process 

allows. 

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Containment and Eradication of 

Phytophthora cinnamomi in Native 

Vegetation in South-Western Australia 

and Tasmania1

William A. Dunstan,2 Timothy Rudman,3 Bryan L. Shearer,4 Nicole 

A. Moore,2,5 Bernard Dell,2 Colin Crane,4 Sarah Barrett,5 and 

Giles E.St.J. Hardy2

Abstract 

The aim of our experiments was to develop protocols that can be used to contain and eradicate 

spot infestations of P. cinnamomi that, if untreated, are likely to threaten extensive areas of 

native vegetation or areas of high conservation value. Treatment regimes were guided by two 

assumptions: 1) within the selected sites, transmission of the pathogen is by root-to-root 

contact, and 2) the pathogen is a weakly competitive saprotroph. In Western Australia (WA), 

treatment and control plots were set-up along an active disease front within scrub-heath 

vegetation dominated by Banksia spp. Treatments, applied sequentially and in combination, 

included: 1) destruction of the largest plants within disease free vegetation forward of the 

disease front; 2) destruction of all plants to create a ‘dead zone’; 3) installation of physical 

root barriers and subsurface irrigation for the application of fungicide/s; 4) surface 

applications of fungicides selective against oomycetes (triadiazole and metalaxyl-M), and 5) 

surface injection and deep (± 1 m) treatments with Metham-sodium. In a separate experiment 

in Tasmania (TAS), combined treatments including vegetation removal, Ridomil and 

Metham-sodium and root barriers, or Ridomil and root barriers alone, were applied to 

experimental plots within active disease centres in Eucalyptus-Banksia woodland. 

In the WA experiment, P. cinnamomi was not recovered (by soil baiting) from plots after 

treatment with Ridomil and metham-sodium. In the TAS experiment, similar results were 

achieved with combined treatments (vegetation removal + Ridomil + metham sodium) but in 

plots treated with Ridomil alone, recoveries of P. cinnamomi increased after initially showing 

a significant reduction in recoveries. 

Introduction 

Phytophthora cinnamomi, and disease caused by it, is listed as one of five key threatening 

processes affecting biodiversity in Australia. The area of native vegetation affected by P. 

cinnamomi exceeds many hundreds of thousands of hectares in Western Australia (> 635 000 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Centre for Phytophthora Science & Management, School of Biological Sciences & Biotechnology, DSE, Murdoch 

University, Perth 6150, Western Australia, Australia. 

3

 Biodiversity Conservation Branch, Resource Management & Conservation, Department of Primary Industries & 

Water, 134 Macquarie St., Hobart 7001, Tasmania, Australia. 

4 Science Division, Department of Environment & Conservation, 50 Hayman Rd, Como 6152, Western Australia, 

Australia. 

5 Nature Conservation Section, Department of Environment & Conservation, 120 Albany Highway, Albany 6330, 

Western Australia, Australia. 

Corresponding author: w.dunstan@murdoch.edu.au. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

219 

ha; Department of Environment & Conservation 2006), Victoria and Tasmania, and tens of 

thousands of hectares in South Australia (Environment Australia 2001). Although infestations 

within the conservation estate are widespread and frequently extensive, large areas remain 

free of the pathogen and may be protectable. The control and management of P. cinnamomi in 

natural ecosystems raises considerable challenges in terms of managing the impact of the 

pathogen in diverse plant communities (Hardy and others 2001). Apart from the application of 

control measures designed to prevent human vectoring of the pathogen, there has been little 

testing and operational use of control measures in native vegetation apart from the use of 

phosphite (Hardy and others 2001). Phosphite has been shown to slow, but not stop, the 

autonomous spread of P. cinnamomi in native vegetation (Shearer and others 2004). 

The aims of the experiments were to contain and eradicate P. cinnamomi within 

natural infestations in native vegetation. To achieve the aims a combination of 

methods were applied sequentially to experimental plots within sites naturally 

infested with P. cinnamomi. Methods and the rationale for their employment 

included: 1) localised destruction of vegetation to deny food resources to the 

pathogen, 2) physical root barriers, to prevent root intrusion into pathogen infested 

soil, particularly by plants with extensive lateral root systems, and 3) direct attack on 

the pathogen with fungicides and fumigant/s, starting with oomycete specific 

compounds and progressing to more robust treatments. The vulnerable or endangered 

grass-tree Xanthorrhoea braceata occurs within the site experimental site in 

Tasmania. A fungicide + root barriers only treatment was included in the experiment 

with the aim of assessing its potential as a non-destructive method of protecting 

individual plants at risk of destruction by P. cinnamomi. 

Methods and Material 

Experimental Sites 

Tasmania (TAS) 

The experimental site (146.62oE, 41.16oS) is located within Narawntapu National 

Park on the north central coast of Tasmania. The soil is an Aeric (or Semiaquic) 

Podosol (after the Australian soil classification of Isbell 2002), derived from 

Quaternary sands that are greater than 3 m deep. The vegetation is classified as 

Eucalyptus amygdalina coastal forest and woodland (Harris and Kitchener 2005), 

with scattered Eucalyptus viminalis and an understorey of Banksia marginata and 

scattered to dense Xanthorrhoea australis in heath comprised principally of species 

within the families epacridaceae and papilionaceae. The climate is temperate. 

Estimates for the mean maximum in the hottest month is ca. 21oC, mean minimum in 

the coldest month ca. 5oC, and mean annual rainfall is ca. 800 mm. 

Western Australia (WA) 

The experimental site (118.72oE, 34.57oS) is located near Cape Riche. The soil is a 

Rudosol, derived from fine (< 0.8 mm) aeolian Quaternary sands that are greater than 

2.5 m deep. The vegetation is a scrub-heath dominated by Banksia spp., principally 

B. attenuata and B. baxteri, with an understorey that includes a diverse range of other 

woody shrubs from species within the families myrtaceae, proteaceae, epacridaceae 

and papilionaceae. Mean annual rainfall is ca. 490 mm. 

Isolates of P. cinnamomi from both sites were all of the A2 mating type. 
GENERAL TECHNICAL REPORT PSW-GTR-221

220 

Site treatments 

Western Australia

Treatment and control plots (n = 7 each) were set-up along an active disease front, 

with each plot covering 10 m of disease front, and separated from adjacent plots by a 

buffer of not less than 5 m. Treatments, applied sequentially and in combination, 

included: 1) Destruction of the largest plants (principally Banksia spp.) to a distance 

of 10 m on the disease free side of the disease front, by felling and application of 

glyphosate to stumps (May 2006). 2) Removal of all plants to a distance of 4 m from 

the disease front, by slashing and applications of herbicides (triclopyr and glyphosate 

(Aug-Sep 2006). 3) Installation of HDPE root barriers (90 cm deep x 1 mm thick) to 

ca. 80 cm depth and, subsurface irrigation for the application of fungicide/s (Mar 

2007). 4) Surface applications of Terrazole (triadiazole, 10 g/m2

 a.i.; Jun and Aug 

2006). Applications of Ridomil 25G (metalaxyl-M, 2.5 g/m2 a.i.; Jun and Aug 2007). 

5) Surface injection, at 15 x 25 cm injection point spacings and to ca. 20 cm depth 

with Metham-sodium (90 g/m2

; Jun 2007). 6) Deep (± 1 m) treatments with 

Metham-sodium, 0.5l/m2

 applied via 40 mm PVC vertical tubes installed at 1 m 

centres, between the disease front and root barrier (Sep 2007). 

Tasmania 

Experimental plots (5 m x 5 m) were selected from areas showing signs of recent 

pathogen activity within an extensive area of infestations. The experiment was an 

unbalanced design, with four plots receiving all treatments (vegetation removal + 

fungicide + fumigation), four plots were treated with fungicide only (after minor 

clearance of organic litter and woody debris), and seven plots were untreated. Root 

barriers (120 cm deep x 1 mm thick) were installed at 80-90 cm deep around all 

treated plots (complete treatments and fungicide only) with the aim of preventing 

reinfestation of plots by root borne P. cinnamomi. Six of the 11 larger species of 

terrestrial mammals known to occur within the park could be considered as potential 

vectors of P. cinnamomi because of their digging habits. Given the large number of 

animals present, and the significant amount of soil disturbance caused by them, 

exclusion fencing was installed around each plot. Vegetation removal, barrier and 

fence installation, fungicide application, and injection with metham-sodium, were 

completed in Apr 2007. Initial treatments were followed by a further fungicide 

treatment (Aug 2007) and deep application of metham-sodium (Sep 2007). Ridomil 

25G (2.5 g/kg Metalaxyl-M) was applied at 100 g/ m2

. Surface injection with 

Metham-sodium (90 g/m2 ) to 15- 20 cm deep was at 150 cm x 250 cm spacings. 

Deep treatment to ± 1 m with Metham-sodium, (0.75 l/m2

) was applied via 40 mm 

PVC vertical tubes installed at 1 m spacings. The calculated potential mass of 

methylisothiocyanate (MITC; the active product from the hydrolysis of methamsodium) applied to the plots was 180 g/m2

. 

Assessment 

In the WA experiment, soil sampling was systematic and stratified in two ways: (1) 

soil depth; sampled at 0-25 cm, 25-50 cm, 80-100 cm and 1.5 m, and (2) distance 

from disease front (as surveyed in Feb 2006); sampled at 0.5 m and 2.5 m from the 

disease front, with three sample points at each distance. In the TAS experiment, plots 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

221 

were sampled systematically at five points, and at three depths per sampling point (0-

25 cm, 25-50 cm, and 80-100 cm). Soil samples with roots (120-150 g wet wt.) were 

flooded and baited with Lupinus angustifolius seedlings. Seedling radicles were 

plated onto NARPH medium (Hüberli 2000), and P. cinnamomi was identified by 

colony form and micro morphology. 

Results 

Recoveries of P. cinnamomi from the WA experiment are shown in Figure 1. A trend 

in increasing recoveries of P. cinnamomi in untreated plots, at 2.5 m from the original 

disease front (figs 1A-D), showed that the pathogen was active and the infestation 

was expanding. Surface treatments with terrazole had no significant effect on 

recoveries of P. cinnamomi (recoveries at 0.5 m in treated plots, figs 1A and B). 

There were no recoveries of P. cinnamomi from any of the treated plots after ridomil 

treatments and surface injection with metham-sodium (fig 1C), or after the additional 

deep treatment with metham-sodium (fig 1D). There were no recoveries from the 0.5 

m mark in treated plots in the last two assessments (figs 1C and 1D), in contrast to 

the previous harvest where recoveries of P. cinnamomi at the same distance in treated 

plots were similar to untreated controls (fig 1B). 
GENERAL TECHNICAL REPORT PSW-GTR-221

222 

Figure 1—Effect of treatment on recovery of Phytophthora cinnamomi, Cape 

Riche, Western Australia. Untreated plots (controls, + P. cinnamomi) n = 7, 

treatments (+ vegetation clearance + fungicide + fumigation) n = 7. n = 21 for 

each histogram bar. A. After first Terrazole application and partial vegetation 

clearance (Jul 2007). B. After second Terrazole application and completion of 

vegetation clearance (Dec 2006). C. After ridomil treatments and surface 

injection with metham-sodium. (July 2007). D. After deep treatment with 

metham-sodium (Nov 2007). Distances (m) are from the active disease front 

(surveyed Feb 2006). NR = no recovery of P. cinnamomi. 

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

 untreated, 0.5 m

untreated, 2.5 m

treatments, 0.5 mtreatments, 2.5 m

treatment, distance from disease front, and soil horizon

0-25 cm

25-50 cm

100 cm

150 cm

A

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

untreated, 0.5 muntreated, 2.5 m

treatments, 0.5 mtreatments, 2.5 m

treatment, distance from disease front, and soil horizon

0-25 cm

25-50 cm

100 cm

150 cm

C

NR NR

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

untreated, 0.5 muntreated, 2.5 m

treatments, 0.5 mtreatments, 2.5 m

treatment, distance from disease front, and soil horizon

0-25 cm

25-50 cm

100 cm

150 cm

B

NR

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

untreated, 0.5 m

untreated, 2.5 m

treatments, 0.5 mtreatments, 2.5 m

treatment, sampling distance from disease front, and soil horizon 

0-25 cm

25-50 cm

100 cm

150 cm

D

NR NR
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

223 

Results from four assessments from the TAS experiment are shown in Figure 2. 

Recoveries of P. cinnamomi in the pre-treatment assessment showed a similar 

distribution of the pathogen between plots and within the soil profile to 1 m (fig 2A). 

In plots treated with Ridomil alone, recoveries of P. cinnamomi were reduced 

significantly (p < 0.001), with a trend for increased recoveries with soil depth (fig 

2B). In fungicide only treated plots, P. cinnamomi was recovered at all assessments 

and post-treatment deaths of X. australis had also occurred. Phytophthora 

cinnamomi was recovered at low frequency at 1 m in treated plots after the first 

treatment (ridomil + metham-sodium surface injection; fig 2B) but no further 

recoveries were made from any of the complete treatment plots after further 

application of ridomil and deep treatment with metham sodium (figs 2C-D). 

Figure 2—Effect of treatment on recovery of Phytophthora cinnamomi, 

Narawntapu National Park, Tasmania. Control (untreated) plots n = 7, 

fungicide only n = 4, complete treatments n = 4. n = 20 for each histogram 

bar in fungicide only and complete treatments, and n = 35 in each histogram 

bar in controls, Error bars are one standard error from the mean. A. 

Assessment before treatment, April 2007. B. After initial treatment, June 

2007. C. October 2007, after second fungicide application, and deep 

treatment with metham-sodium. D. December 2007. 

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

control fungicide only complete treatments

treatment and soil horizon

0-25 cm

25-50 cm

100 cm

A

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

control fungicide only complete treatments

treatment & soil horizon

0-25 cm

25-50 cm

100 cm

B

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

control fungicide only complete treatments

Treatment and soil horizon

0-25 cm

25-50 cm

100 cm

C

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

control fungicide only complete treatments

Treatment and soil horizon

0-25 cm

25-50 cm

100 cm

D
GENERAL TECHNICAL REPORT PSW-GTR-221

224 

Discussion 

Physical root barriers at both sites remain untested. From the results in the WA 

experiment so far, the pathogen has been stopped short of the barriers by the 

combination of other treatments. The rate of progress of disease over the last 4-5 

years is estimated to be 1-2 m/yr, too slow to expect deaths of susceptible plants 

beyond root barriers, if the system has failed. A separate experiment is in progress, 

designed to test the efficacy of root barriers alone. The WA experiment was not a fair 

test of the efficacy of terrazole because there was almost certainly inadequate rainfall 

after both applications of the fungicide to enable its infiltration into the soil profile. 

Rainfall at the site over any 24 h period within the critical period after either 

application of Terrazole was not more than 6 mm. In the WA experiment, incidental 

observations on unintended treatment effects included: 1) A decline in plant health, 

including deaths, of individual plants in some understorey species where large plants 

had been removed, that was probably caused by increased exposure. 2) In treated 

plots, an increase in soil moisture within the zone where vegetation was completely 

removed. Increased soil moisture is likely to be more favourable to the survival of 

chlamydospores (Weste and Vithanage 1979) but, given the soil type, would be 

unlikely to enhance lateral movement of the pathogen within the soil profile. 3) Soil 

erosion by wind and water within the ‘dead zone’ of treated plots has been minimal 

(after > 1.5 yr), but could be a problem in the longer term. 

Based on recoveries of the pathogen in both experiments, a combination of 

vegetation destruction, ridomil and metham-sodium applications shows promise as a 

method to at least contain, and possibly eliminate, the pathogen within small 

infestations. In neither experiment was fumigation with metham-sodium alone used 

as a treatment. Therefore, we cannot make any conclusions about the efficacy of 

metham alone, or metham x metalaxyl interactions. Metham-sodium was used alone 

in an attempt to eradicate P. cinnamomi from plots within infested eucalypt forest by 

Weste and others (1973), with some success, where Phytophthora cinnamomi was 

not recovered from two of three sites for at least 18 months after treatment, but 

reinfestation from adjacent infested forest occurred at one site. Vegetation removal 

and metalaxyl was used, among other treatments, in an eradication experiment in 

Banksia woodland by Hill and others (1995). Very large reductions in recoveries of 

P. cinnamomi were achieved at 0.1-0.4 m soil depth for up to at least 20 months after 

treatment, however the pathogen was always recovered at later assessments (up to 20 

months). Persistence of metalaxyl (half-life 10-82 d; Davison and McKay 1999) and 

MITC (2 percent of applied amount after 24 d; Zhang and Wang (2002)in agricultural 

soils is low, but further assessments will be required to determine whether the 

pathogen has been eliminated or has only been suppressed by residual fungicide/s. 

From long-term monitoring of P. cinnamomi in native vegetation (Victoria, 

Australia), Weste (2003) did not recover the pathogen in four of six sites after 30 

years, and observed regeneration of highly susceptible species. If the autonomous 

spread of the pathogen can be arrested, and adequate measures to prevent vectoring 

of the pathogen off-site by animals and people can be implemented, then there is 

some prospect for limiting damage and recovery of vegetation to its original state. In 

both experiments we chose sites where a combination of characteristics that included 

topography, soil type, vegetation (species composition and structure), and climate, 

were favourable to achieving positive results. However, extensive areas within southwestern Australia, south-eastern Australia and Tasmania have similar site 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

225 

characteristics to those used in both experiments, therefore the methods described 

could be applied on a broader scale, with modification to suite individual sites. 

Acknowledgements 

This project was supported by the Department of Environment and Water Resources 

(Commonwealth of Australia). We wish to acknowledge the following for assistance 

in the field and laboratory: Tasmania. Brigid Morrison, Mark Dobrowolski, Tim 

Wardlaw, Mike Ikolowski, Anthony Timmerman, Gary Sutton, Judy Westbrook, 

Alena Hrasky, Andrew Ford, Mark Hedditch, Dave Widger. Western Australia. John 

Watson, Sarah Barrett, Greg Freebury, Deon Utber, Joanna Young, Rodney 

Armistead, Chen Yinglong, Daniel Huberli, Nari Williams, Dianne White, Joshua 

Gilovitz, Trudy Paap, Peter Scott, Georgia Scott, Christine Davis, Jessica Davis, 

Bryoni Palmer, Sarah Jackson, Nathalie Long. 

Literature Cited 

Davison, E.M.; McKay, A.G. 1999. Reduced persistence of metalaxyl in soil associated with 

its failure to control cavity spot of carrots. Plant Pathology. 48: 830-835. 

Department of Environment and Conservation. 2006. Phytophthora Dieback Atlas. 

Department of Environment and Conservation: Kensington, Western Australia. 39 p. 

Environment Australia. 2001. Threat Abatement Plan for Dieback Caused by the Root-rot 

Fungus Phytophthora cinnamomi. Environment Australia, Commonwealth Government of 

Australia, Canberra. 

Hardy G.E.S.; Barrett S.; Shearer B.L. 2001. The future of phosphite as a fungicide to 

control the soil borne plant pathogen Phytophthora cinnamomi in natural ecosystems. 

Australasian Plant Pathology. 30: 133-139. 

Harris, S.; Kitchener, A. 2005. From forest to fjaeldmark: Descriptions of Tasmania’s 

vegetation. Tasmania Department of Primary Industries, Water and Environment, Hobart. 

Hill, T.C.J.; Tippett, J.T.; Shearer, B.L. 1995. Evaluation of three treatments for 

eradication of Phytophthora cinnamomi from deep leached sands in south-western Australia. 

Plant Disease. 79: 122-127. 

Hüberli, D.; Tommerup, I.C.; Hardy, G.E.StJ. 2000. False-negative isolations or absence 

of lesions may cause mis-diagnosis of diseased plants infected with Phytophthora cinnamomi. 

Australasian Plant Pathology. 29: 164-169. 

Isbell, R.F. 2002. The Australian Soil Classification. Revised Edition. CSIRO Publishing, 

Collingwood, Victoria. 

Shearer, B.L.; Crane, C.E.; Fairman, R.G. 2004. Phosphite reduces disease extension of a 

Phytophthora cinnamomi front in Banksia woodland, even after fire. Australasian Plant 

Pathology. 33: 249-254. 

Weste, G.; Vithanage, K. 1979. Survival of chlamydospores of Phytophthora cinnamomi in 

several non-sterile, host-free forest soils and gravels at different soil water potentials. 

Australian Journal of Botany. 27: 1-9. 
GENERAL TECHNICAL REPORT PSW-GTR-221

226 

Weste, G. 2003. The dieback cycle in Victorian forests; a 30-year study of changes caused by 

Phytophthora cinnamomi in Victorian open forests, woodlands and heathlands. Australasian 

Plant Pathology. 32: 247-256. 

Weste, G.; Cooke, D.; Taylor, P. 1973. The invasion of native forest by Phytophthora 

cinnamomi. Post-infection vegetation patterns, regeneration, decline in inoculum, and 

attempted control. Australian Journal of Botany. 21: 13-29. 

Zhang, Y.; Wang, D. 2007. Emission, distribution and leaching of methyl isothiocyanate and 

chloropicrin under different surface containments. Chemosphere. 68: 445-454. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

227 

Does Abiotic Stress on a Plant Influence 

Phosphite Protection to Phytophthora 

cinnamomi?1

Daniel Hüberli,2 Trudy Paap,2 Nicole A. Moore,2,3 Karyn Gower,2

Nathalie Long,2,4 Sarah Barrett,3 Greg Freebury,3 Ted Spadek,5

Bernard Dell,2 and Giles E.St.J. Hardy2

Introduction 

Large areas of indigenous forests, Banksia woodlands and heathlands in Australia are 

devastated by Phytophthora dieback disease caused by Phytophthora cinnamomi

(Weste 1994). In southwestern Australia, some 50 percent of the 5710 plants endemic 

to the region are susceptible (Shearer and others 2004a). Phosphite has been shown to 

be effective in controlling this pathogen’s impact on a wide range of plant species 

across different families (Hardy and others 2001). 

Recently, disease extension was reduced after phosphite treatment even after fire 

(Shearer and others 2004b). However, very little is known about the influence of a 

plant’s physiological status at the time of phosphite application on the subsequent 

efficacy of phosphite treatment to control Phytophthora dieback disease. The key 

seasonal stresses in an Australian ecosystem of fire and flooding are explored. 

Materials and Methods 

Adenanthos cuneatus (resprouter), Banksia attenuata (resprouter) and B. baueri

(reseeder) are all susceptible to P. cinnamomi and are responsive to phosphite 

treatment. These species were selected within four plots in an area of the Stirling 

Range National Park (Western Australia) that was scheduled for a fuel-reduction 

burn. Treatments of the plots were: 1) phosphite spray without fire, 2) phosphite spay 

with fire, 3) no phosphite spray without fire, and 4) no phosphite spray with fire. 

Phosphite treatment was applied six weeks before the fire or nine months post-fire 

when all resprouter species had sufficient foliage. Periodic measurements during the 

experiment include leaf water potential, leaf gas exchange, lesion lengths on 

inoculated stems, and phosphite concentration in leaves, stems, lignotubers and roots. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Centre for Phytophthora Science and Management, Biological Sciences, Murdoch University, Murdoch, WA 6150, 

Australia. 

3 Nature Conservation Section, Department of Environment & Conservation, 120 Albany Highway, Albany, WA 

6330, Australia. 

4 Université des sciences Montpellier II, Place Eugène Bataillon, 34 095 Montpellier Cedex 5, France. 5

 Chemistry Centre (Western Australia), 125 Hay Street, East Perth, WA 6004, Australia. 

Corresponding author: D.Huberli@murdoch.edu.au. 
GENERAL TECHNICAL REPORT PSW-GTR-221

228 

Waterlogging trials were conducted in the greenhouse using B. attenuata and B. 

baxteri (reseeder). The response of these plants and subsequent recovery from 

waterlogging was examined. In the main trial, a phosphite spray treatment was 

applied before (Experiment 1) and after (Experiment 2) one waterlogging event of 

either 3 or 10 days (8 days for Experiment 2) duration. Plant physiology traits, lesion 

development and phosphite concentrations in leaf, stem and root were taken 1 week, 

1 month and 4 months after the phosphite treatment. 

Results and Discussion 

Preliminary data for the fire experiment show that phosphite was present in the roots 

in the pre- and post-fire spray treatments. These data also suggest that lesions were 

reduced in all three species treated with phosphite. Further work is ongoing in this 

experiment. 

For the waterlogging experiments, phosphite in plant tissue was at similar levels for 

each species and was not affected by waterlogging in Experiment 1, Week 1 harvest 

(fig 1), but lesions on B. baxteri stems were not reduced in treated plants as they were 

for B. attenuata. Photosynthesis and water potentials were reduced for waterlogged 

B. attenuata especially at the ten day waterlogging treatment, but were not impacted 

in waterlogged B. baxteri. Leaf water potentials, leaf gas exchange, lesion lengths on 

inoculated stems, and phosphite concentration in leaves, stems, and roots measured at 

one and four months for Experiment 1 and all measurement periods for Experiment 2 

will be presented in a future publication. 

Figure 1—Mean ( standard error) phosphite concentrations in plant tissue 

and lesion lengths in stems 1 week after inoculation with Phytophthora 

cinnamomi of Banksia attenuata and B. baxteri seedlings that had been 

sprayed or not (controls) 1 week prior to the inoculation. Plants were treated 

with phosphite 1 week after waterlogging for 0, 3 and 10 days. 

0

100

200

300

400

500

038

Phosphite (μg/g)

Leaf

Stem

Root

0

100

200

300

400

500

038

Waterlogging (days)

Phosphite (μg/g)

Leaf

Stem

Root

Plants dead 

0

20

40

60

80

100

120

038

Lesion length (mm)

No Phosphite

Phosphite

0

20

40

60

80

100

120

038

Waterlogging duration (days)

Lesion length (mm)

No Phosphite

Phosphite

Lesions 

Banksia attenuata Banksia baxteri 

100

Phosphite concentration 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

229 

Acknowledgements 

We thank O. Fry, S. Jackson, T. Johnson, J. O’Brien, B. Palmer, G. Scott, and P. 

Scott for assistance in the field and laboratory. The project 19.2.3 was funded by the 

Department of Environment and Water. 

Literature Cited 

Hardy, G.E.St.J.; Barrett, S.; Shearer, B.L. 2001. The future of phosphite as a fungicide to 

control the soilborne plant pathogen Phytophthora cinnamomi in natural ecosystems. 

Australasian Plant Pathology. 30: 133-139. 

Shearer, B.L.; Crane, C.E.; Cochrane, A. 2004a. Quantification of the susceptibility of the 

native flora of the South-West Botanical Province, Western Australia, to Phytophthora 

cinnamomi. Australian Journal of Botany. 52: 435-443. 

Shearer, B.L.; Crane, C.E.; Fairman, R.G. 2004b. Phosphite reduces disease extension of a 

Phytophthora cinnamomi front in Banksia woodland, even after fire. Australasian Plant 

Pathology. 33: 249-254. 

Weste, G. 1994. Impact of Phytophthora species on native vegetation of Australia and Papua 

New Guinea. Australasian Plant Pathology. 23: 190-209.
GENERAL TECHNICAL REPORT PSW-GTR-221

230 

In Vitro Activity of Plant Crude Extracts and 

In Situ Protective Effect of Phlomis 

purpurea Against Phytophthora cinnamomi1

Dina Neves,2 Paula Caetano,3 Marília Horta3, Nelson Sousa3, Lídia 

Dionísio3, Naresh Magan,4 Alfredo Cravador3,5 

Abstract 

The effect of ethanol extracts of Arbutus unedo (Ericaceae), Helichrysum stoechas

(Compositae) and Phlomis purpurea (Lamiaceae) was evaluated against mycelial 

growth of Phytophthora cinnamomi. All plant extracts exhibited anti-P. cinnamomi

activity. The most effective extract was from roots of P. purpurea, with a maximum 

percentage of inhibition of 78.3 percent at 1 mg/ml. P. purpurea was shown to 

significantly protect the susceptible Quercus ilex L. (holm oak) from P. cinnamomi

infection, in a glasshouse trial. This spontaneous plant reduces the soil inoculum

potential suggesting a possible supressive ability to reduce the ocurrence of root 

diseases caused by P. cinnamomi. 

P. purpurea, is endemic to southern Iberian Peninsula and Morocco and is present in 

the understorey of Q. suber and Q. ilex habitat. The present results suggest that it 

could be a potential candidate to control P. cinnamomi, but further research is 

required, including field trials. 

Introduction 

Cork oak and holm oak trees are essential components in the agro-ecosystems of the 

Iberian Peninsula (montado in Portugal, dehesa in Spain) supporting rich wildlife 

populations and simultaneously used by farmers to grow cereals, graze animals and, 

as far as cork is concerned, harvest cork. Cork production in the Iberian Peninsula 

accounts for 80 percent of mean annual world production (DGRF 2006, APCOR 

2006). 

Today, these ecosystems are threatened by a decline disease whose main agent has 

been identified as the soil born phytopathogen Phytophthora cinnamomi (Brasier and 

others 1993, Moreira 2001, Sánchez and others 2002, 2005, Tuset and others 1996).

Despite the ecological and socio-economic importance of these systems, efficient 

control measures are still missing. To control this disease, the use of synthetic 

fungicides has had limited success and the use of genetically modified organisms is 

still controversial (Kremer and Motavilli 2004, Motavalli and others 2004, Priest 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Universidade do Algarve, Instituto Transfronteiriço Universitário para a Ciência, a Cultura e o Ambiente 

(ITUCCA), Campus de Gambelas, 8005-139 Faro, Portugal. 

3

 Universidade do Algarve, Campus de Gambelas, Faculdade de Engenharia dos Recursos Naturais, 8005-139 Faro, 

Portugal. 

4

 Institute of Biosciences and Technology, Cranfield University, Silsoe, Bedford, MK45 4DT, U.K. 

5 IBB/CGB-UTAD apartado 10103, Vila Real 5001, Vila Real, Portugal. 

Corresponding author: dneves@ualg.pt. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

231 

2000). Moreover, in the last few years it has become evident, as a result of public 

awareness and environmental laws, that new and safer alternatives to synthetic 

pesticides are needed. The plant world can provide alternatives to existing chemicals 

as a source of biological natural chemicals extracted or delivered from them (Bais 

and others 2002, Molina-Torres and others 2004). In particular, the use of resistant 

native flora to control P. cinnamomi could be an environmentally friendly approach. 

Several plants from the Q. suber and Q. ilex habitat were surveyed and Phytophthora 

cinnamomi was not isolated from Helichrysum stoechas and Phlomis purpurea

(Moreira-Marcelino 2001, Moreira and Martins 2005). These plant species could be a 

potential control of P. cinnamomi reducing the amount of P. cinnamomi inoculum by 

producing anti-P. cinnamomi compounds. 

In the present study, we examined the activity of 3 plant extracts on P. cinnamomi

mycelial growth and determined whether those plants were or not P. cinnamomi

hosts. The protective effect of Phlomis purpurea towards Q. ilex was also 

determined. 

Methods and Materials 

Plants and Phytophthora Isolates 

The plants used to prepare the extracts were collected in Q. suber stands in Algarve, 

south of Portugal (Table 1). 

Table 1—Species, collection site, date of collection and plant material used to 

prepare the extracts 

Plant Collection site Date Plant material 

Arbutus unedo Barranco-do-Velho November, 2005 Leaves 

November, 2005 Whole plant (with 

dried flowers) 

January, 2006 Whole plant 

Helichrysum 

stoechas 

Barranco-do-Velho 

June. 2006 Whole plant (with 

flowers) 

Phlomis purpurea Moncarapacho January, 2006 Whole plant 
GENERAL TECHNICAL REPORT PSW-GTR-221

232 

Plant material for pathogenicity tests and in situ protective effect study of Phlomis 

purpurea against Phytophthora cinnamomi was obtained from nurseries (Table 2). 

Table 2—Provenence and age of plants used for pathogenicity tests and in 

situ protective effect study of Phlomis purpurea against Phytophthora 

cinnamomi 

Species Nursery Age Experiment 

“Viveiros Florestais 

Monte Gordo (VFMG)” - 

Portugal 

Arbutus unedo 10 months old 2.1, 2.2 

“Viveiros Florestais de 

Huelva (VFH)” - Spain 

11 months old 2.3 

Helichrysum 6 months old 2.1, 2.2 

stoechas 

“Viveiros Monte-Rosa 

 (VM-R)”- Portugal 6 weeks old 2.3 

“Dir. Reg. Agric. do 

Algarve (DRAAG)” - 

Portugal 

4 and 2 months 

old 

Phlomis purpurea 2.1, 2.2 

“Viveiros florestais de 

Huelva” - Spain 

7 months old 2.3 

Quercus ilex “VFMG”- Portugal 10 months old 2.2 

“Viveiros florestais de 

Huelva” – Spain 

11 months old 2.3 

Quercus suber Aliança Florestal - 

Portugal 

7 months old 2.2 

The two P. cinnamomi isolates used in the present work, PA37 and PA45, belong to 

the mycological library of the Laboratory of Molecular Biotechnology and 

Phytopathology of the “Universidade do Algarve” (UAlg). Their origin, site and 

isolation date are described in Table 3. Both are A2 mating type. 

Table 3—Identification of Phytophthora cinnamomi isolates used in the assay. 

Isolate Site Date of isolation Origin 

PA37 Lagos May 1995 Soil from Quercus suber

PA45 S. Brás de Alportel May 2000 Soil from Q. suber

Plant extracts 

The plant extraction protocol was adapted from Carini and others (2001). The dry 

mass of the plant part used (roots, leaves, flowering tops) were chopped with a 

grinding-master (Moulinex® 1,2,3). Fifty grams of the chopped leaves or flowering 

tops were suspended in 350 ml of 70 percent aqueous ethanol or methanol 

(containing 0.1 percent ascorbic acid to prevent oxidation) and shaken for 72 hours 

(Güllüce and others 2004). Roots were shaken for seven days (Molina-Torres and 

others 2004). Ethanol was eliminated by evaporation (Heidolph 94200, Bioblock 

Scientific I) under reduced pressure at 35°C and, in the case of H. stoechas, the 

aqueous residue washed with methylene chloride (1:1, v:v), to eliminate free 

aglycons and other non phenolic substances. The extract was concentrated under 

vacuum (Savant, SC 110A) to almost dryness to obtain a brownish sticky suspension 

and weighted to constant weight. The crude extract was dissolved in water or 35 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

233 

percent aqueous ethanol and filtered through a 0.45 m nitrocellulose filter 

(Millipore). The extraction yield for H. stoechas was 16 percent whereas for P. 

purpurea was 15 percent. 

Assessment of foliar and root symptoms 

At the end of each infestation experiment, plants were removed from pots, soil was 

removed from roots and the latter washed with tap water and kept in water until they 

were evaluated (in the same day).The severity of aerial and root symptoms was 

assessed for each plant on a 0-4 scale basis (0= without symptoms, 4= dead plant). 

The aerial part of the plant was evaluated according to the percentage of leaves with 

yellowing, necrosis or defoliation and the root part by the amount of roots and 

percentage of necrotic roots. Plants were kept with roots wrapped in a wet absorbent 

paper in the dark at 4oC, until needed, but no longer than 12 days (Streito and others 

2002). 

Phytophthora cinnamomi Re-isolation 

Segments of roots from infested and control plants were plated on NARPH [Corn 

meal agar – CMA (17 gL-1) amended, after autoclaving (at 0.7 Kp/cm2

 for 15 min.) 

and cooled down to 45 oC, with nystatin (0.03 gL-1), ampicillin (0.25 gL-1), 

rifampicin (0.10 gL-1), pentachloronitrobenzene - PCNB (0.10 g/l) and Hymexazol 

(197.2 lL-1)] medium to re-isolate P. cinnamomi. Segments of roots were washed 

into running tap water for 1-2 hours, aseptically and transversally cut into small 

segments (5-8 mm long) on a sterile filter paper. The exposed cortex was directly 

plated (six to eight root groups), using sterile forceps, on NARPH medium. For each 

plant and isolate 5 Petri dishes were used and sealed with parafilm. The plates were 

incubated at 24oC in the dark for 2-3 days. Colonies isolated from the tissues were 

classified on the basis of cultural characteristics and identified by the morphology of 

vegetative and reproductive structures under a light microscope (Sánchez and others 

1998). Percentage of P. cinnamomi recovering was calculated. Samples from which 

the oomycete was not recovered were processed until being recovered or up to three 

times. 

In vitro Activity of Plant Crude Extracts 

To evaluate the activity of various plant extracts on P. cinnamomi growth, 

preliminary assays were performed to optimize the technique. These included the 

comparison of P. cinnamomi growth using two different isolates, with different 

shaking times, two different solvents and root versus aerial part extract (leaves or 

leaves and flowers). All the experiments were done using the following procedure: a 

mycelial agar plug (5 mm in diameter) of each isolate was cut from an actively 

growing colony and placed on Potato Dextrose Agar (PDA) culture media (Park 

2003). PDA culture media (Difco) was prepared according to the manufacturer’s 

instructions. The plant extracts were incorporated into the PDA culture media, prior 

to inoculation: after autoclaving, PDA was placed in a water bath at 45oC before 

addition of plant extracts to avoid thermal degradation. The mixture was 

homogenised in a magnetic stirrer. Twelve millilitres of culture media, was poured 

into 90 mm Petri dishes and allowed to solidify. Control Petri dishes received the 

same amount of culture media and the corresponding amount of solvent. Petri dishes 

were sealed with parafilm, put into plastic bags, grouped according to the plant 

extract and incubated, in the dark, at 24 oC. Length and width of colonies were 

measured every day until colonies reached the edge of the plate (about 10 days). 
GENERAL TECHNICAL REPORT PSW-GTR-221

234 

These assays were performed in triplicate per isolate and product (plant extract) and 

were conducted three times. At the end of the incubation period, the 50 percent 

effective concentration (EC50) that caused an inhibition of 50 percent of the mycelial 

growth, when compared with the control, was determined. The EC90 was also 

calculated. 

The percentage of inhibition of each tested substance was calculated after subtracting 

the diameter of each agar plug (5mm) from the diameter of each colony, as follows: 

inhibition (percent) = 100 [(Ut-Tr)/Ut], whereby, Ut = mean value for untreated 

control and Tr = mean value for media supplemented with plant extracts (Mekuria 

2005). 

Effect of Plants Extracts from Helichrysum stoechas and Phlomis 

purpurea on Phytophthora cinnamomi Mycelial Growth: Experiment 

1.1. 

Plant extracts used were from Helichrysum stoechas (root and aerial part) and 

Phlomis purpurea (root and aerial part) in ethanol. Aerial part (leaves and flowers for 

H. stoechas and just leaves for P. purpurea) were shaken for 72 hours and roots for 7 

days. The effect of those extracts, at 1.0, 5.0 and 10.0 mgml-1, on P. cinnamomi

(isolates PA37 and PA45) mycelial growth was assessed. 

Efficiency of Plant extracts from Helichrysum stoechas, Phlomis 

purpurea and Arbutus unedo on Phytophthora cinnamomi Growth: 

Experiment 1.2. 

Phytophthora cinnamomi, isolates PA37 and PA45 were inoculated in PDA media 

supplemented with A. unedo (leaves), H. stoechas (leaves and flowers) and P. 

purpurea (roots) ethanol extracts. The concentrations used were 1 and 5 mgml-1. 

Mycelial growth was measured. 

Efficiency of Three Concentrations of Helichrysum stoechas Plant 

Extracts on Phytophthora cinnamomi Growth: Experiment 1.3. 

Helichrysum stoechas (leaves and/or flowers) ethanol extract at 1.0, 5.0 and 10.0 

mgml-1 was tested on growth of isolates PA37 and PA45. 

Efficiency of Two Concentrations of Phlomis purpurea Plant Extracts 

on Phytophthora cinnamomi Growth: Experiment 1.4. 

Phytophthora cinnamomi, isolates PA37 and PA45 were inoculated in PDA media 

supplemented with P. purpurea (roots) ethanol extract at 1 and 5 mgml-1. 

Pathogenicity Tests 

Inoculum preparation 

Axenic cultures of PA37 and PA45 were grown in the dark at 24oC on V8A. After 4-

5 days, 5 mm agar plugs were taken from the edge of individual colonies and 

transferred into a 9 mm Petri dish containing V8 broth (V8b). Isolates were grown 

for 4 weeks at 24oC, to allow the production of abundant infective structures. 

Inoculum suspensions were prepared by mixing the content of six agar plates, 

previously washed and agar free, with 600 ml of sterile deionised water in a blender 

(Philips twister) for 3 min at high speed. Phytophthora cinnamomi suspensions for 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

235 

plant infestations were prepared in water because they are easier to prepare, and are 

as effective as culture media inoculated with the isolates (Sánchez and others 1998, 

2005). 

Inoculation—Experiment 2.1.

Inoculation of Arbutus unedo, Helichrysum stoechas and Phlomis purpurea using P. 

cinnamomi isolates PA37 and PA45 was performed by placing 5 mm agar plugs from 

the edge of individual colonies on the top of the elongation zone (around 5 mm above 

the root tip) and wrapped by foil. Control plants received a V8A plug. Plants were 

incubated at 24.5oC. Segments of roots were cut 1 cm above the infested point and 

inoculated every 24 h for 96 hours. After 96 h all agar plugs were removed to 

NARPH to check viability. 

Infestation—Experiments 2.2. and 2.3. 

For infestation, plants were taken out from their containers and the root ball was 

homogeneously covered with the inoculum of PA37 and PA45 isolates before 

transferring them into a four litres plastic pot (13 x 13 x 30 cm) containing potting 

soil (PlantaFlor, Humus verkausfs, GmbH, D-49377 Vechta, Germany). One hundred 

millilitres of inoculum suspension in water was added per plant. All controls were 

treated in the same way, except for the addition of inoculum which was substituted 

by water. To avoid contamination controls were treated first. Replicate pots per 

species, per isolate and controls were gathered in plastic trays (40 x 30 x 24 cm) and 

placed in a glasshouse. To avoid cross contamination, infested and control pots were 

placed in different trays. The plants were sprayed and watered every day for one 

week, to avoid stress. After one week, every tray, including control ones, were filled 

with tap water and plants submitted to soil waterlogging two days per week. The 

water level in the trays was maintained at 5 cm below the soil surface, by adding 

water each time that plants were submitted to waterlogging, as described (Sánchez 

and others 1998). Maximum and minimum temperatures were recorded every day. 

Two experiments (2.2. and 2.3.) were carried out: 

Experiment 2.2 

Six replicates of each plant species (except for P. purpurea of which only three plants 

per isolate were available due to rupture of stock at the nursery) were inoculated with 

a 100 ml suspension of PA37 isolate and another six with the PA45 one. This 

experiment was kept in the glasshouse during spring till mid summer for 11 weeks 

with an average temperature of 28.1oC (average of maximum and minimum 

temperature was 36.1oC and 20.1oC respectively). The last two weeks were very hot, 

with maximum temperatures around 40oC. The experiment was, then ended. 

Experiment 2.3 

A second pathogenicity test was carried out differing from the precedent one for the 

number of replicates (10 plants per species, including Phlomis purpurea), the 

exclusion of cork oak plants and the inoculum (used as a mixture of PA37 and PA45 

isolates). This experiment was kept in the glasshouse during autumn till the end of 

winter, for 16 weeks with an average temperature of 20.3oC (average of maximum 

and minimum temperature was 28.0oC and 12.5oC respectively). 
GENERAL TECHNICAL REPORT PSW-GTR-221

236 

In situ Protective Effect of Phlomis purpurea Against Phytophthora 

cinnamomi

A mixture of stock pure cultures, at identical quantities, of PA37 and PA45 

isolates was used. 

Experiment 2.4.: Influence of Phlomis purpurea on the Infection of 

Quercus ilex Plants by P. cinnamomi

Inoculum preparation and infestation were done by the same procedures as described 

above. Three agar plates of each isolate (total six) were used. Groups of ten replicate 

pots were infested with the mixture of P. cinnamomi isolates (positive control), P. 

purpurea alone, Q. ilex alone and one Q. ilex seedling planted between two P. 

purpurea plants. Negative controls consisted of identical replicate groups to which 

sterile distilled water was added. All pots were gathered in plastic trays and placed in 

a glasshouse. The average temperature was 20.3oC (average of maximum temperature 

was 28.0oC and average of minimum temperature 12.5oC). The experiment lasted for 

16 weeks. 

Soil Inoculum Potential 

After plant removal from pots, soil from i) P. purpurea alone, ii) Q. ilex grown alone, 

and iii) Q. ilex seedling grown between two P. purpurea was analysed for P. 

cinnamomi inoculum population. Two and half grams of soil, taken out from several 

places of a total mass of soil of ca 650 g, were placed in an Erlenmeyer containing 50 

ml of 0.2 percent sterile agar solution and homogenised with a magnetic stirrer. Once 

the mixture was homogeneous, 1 ml was plated in 20 Petri dishes containing NARPH 

culture media (2.2.1). Plates were incubated in the dark at 24.5oC. After 24 hours, 

plates were washed with running tap water to eliminate the soil. Plates were 

incubated in the dark at 24.5oC for another 2 to 3 days (D’Souza and others 2004, 

Rodríguez and others 2004). P. cinnamomi population was expressed as colony 

forming units per gram of soil (c.f.u./g soil). 

Statistical Analysis 

Results were analysed using “Statistix for windows version 7”. The mean values 

were compared with those from control plates by Fisher’s protected LSD (Least 

Significant Difference) test (Steel and Torrie 1985). 

Results and Discussion 

In vitro Activity of Plant Crude Extracts 

Ethanol extract from leaves and flowers of H. stoechas significantly inhibited 

(p<0.05) mycelial growth when compared to the root extract (Plate 1, fig 1). H. 

stoechas root extract slightly stimulated growth at 1 and 5 mgml-1. For this reason, H. 

stoechas aerial parts were selected for further tests. On the contrary P. purpurea root 

extract inhibited significantly (p<0.05) mycelial growth when compared to the leaf 

extract (Plate 2, fig 2). For this reason, P. purpurea root extract was chosen for 

further tests. 

Phlomis purpurea root extract caused a maximum percentage of P. cinnamomi

inhibition of 93.9 percent, while H. stoechas leaf and flower caused 69.1 percent 

inhibition and A. unedo leaves 61.7 percent, all at 5 mgml-1. The three means were 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

237 

also significantly different from each other. There was no significant difference 

between replicates. As ethanol extract from A. unedo leaves caused the least 

inhibitory effect on P. cinnamomi growth and because it is a host for this oomycete, it 

was decided to exclude A. unedo and focus on the other two plant extracts. 

Helichrysum stoechas extracts at concentrations of 1.0, 5.0 and 10.0 mgml-1 showed 

maximum inhibitory effects of 38.8, 76.6 and 86.7 percent respectively, against both 

isolates. The effective concentration of H. stoechas extract that inhibited mycelium 

growth by 50 percent (EC50) at day 9 was 7.4 mgml-1 and EC90 was 13.4 mgml-1. 

Phlomis purpurea root extracts at concentrations of 1 and 5 mgml-1 caused 

significantly different mean maximum percentage of inhibition─ 78.3 and 93.1 

percents respectively. The EC50 and EC90, at day 9, were 4.1 mgml-1 and 8.0 mgml-1, 

respectively. 

In culture media supplemented with plant extracts, it was often found a reduction in 

colony density, which was probably due to the toxic effect of the plant extracts. 

Although being the commonest method used, the agar supplemented with the 

compound or extract to be tested may not be the most suitable, because it does not 

take into account the colony density. An alternative method could be to grow the 

pathogen in a liquid medium followed by determination of dry mass. 

Phlomis purpurea is very rich in saponins (both leaf and root produces froth) which 

are glycosidic compounds characterized by their detergent-like properties. Many 

saponins are recognized by their ability to haemolyse erythrocytes (Hostettmann and 

others 1991). Saponins in plants are considered as having the function of 

phytoantecipins, a group of compounds with constitutive or preinfectional defence 

properties, or of phytoalexins, assuring postinfectional defences against pathogens 

(Hammerschmidt 1999). Saponins are responsible for the resistance of Avena sativa

(oat) roots to the fungus Gaeumannomyces graminis (Hostettmann and others 1991). 

However, in the present study, the leaves did not have anti- P. cinnamomi activity, 

but the roots did. Probably, other compounds are involved in the inhibition of 

mycelial growth, since P. cinnamomi contains few membrane sterols, the target of 

saponins (Kamoun and others 1999). These authors observed that oomycetes are 

unaffected by saponins. To understand what compound caused such a strong 

inhibition it is important to know the extract composition. 
GENERAL TECHNICAL REPORT PSW-GTR-221

238 

3ODWH2Growth of Phytophthora cinnamomi (A and C: PA37; B and D: PA45 

isolates) on Potato Dextrose Agar (PDA) medium with 0±10 mgml1

Helichrysum stoechas root (A and B) extract after 5 days growth and leaves 

and flowers (C and D) extract after 10 days growth. 

0 mgml-1 1 mgml-1 5 mgml-1 10 mgml-1

A 

B 

C 

D 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

239 

-20

0

20

40

60

80

100

Mycelial growth 

inhibition (%)

PA 37 root PA 37 aerial

parts

PA 45 root PA 45 aerial

parts

1 mg/ml 5 mg/ml 10 mg/ml

Figure 1—Growth inhibition percentage of mycelial Phytophthora cinnamomi

(isolates PA37 and PA45), on Potato Dextrose Agar (PDA) supplemented 

with 1, 5 and 10 mgml-1 of Helichrysum stoechas ethanol root and aerial parts 

extract. 

0,0

20,0

40,0

60,0

80,0

100,0

Mycelial growth 

inhibition (%)

PA 37 root PA 37 aerial

parts

PA 45 root PA 45 aerial

parts

1 mg/ml 5 mg/ml

)Lgure 2—Growth inhibition percentage of mycelial Phytophthora cinnamomi

(isolates PA37 and PA45), on Potato Dextrose Agar (PDA) supplemented 

with 1 and 5 mgml-1 of Phlomis purpurea ethanol root and aerial parts extract. 
GENERAL TECHNICAL REPORT PSW-GTR-221

240 

Plate 2—Growth of Phytophthora cinnamomi (A and C: PA37; B and D: PA45 

isolates) on Potato Dextrose Agar (PDA) medium with 0-10 mgml-1 Phlomis 

purpurea leaf (A and B) and 0-5 mgml-1 root (C and D) extracts after 5 days 

growth. For the root extract assay the 10 mgml-1 plates are not present due to 

insufficient root extract to perform the experiment. 

A 

B 

C 

D 

0 mgml-1 1 mgml-1 5 mgml-1
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

241 

Pathogenicity tests 

Phytophthora cinnamomi was recovered from A. unedo root fragments of 

plants infested with the isolate PA37 after 24 h but not after 48, 72 and 96 h, 

while recovery was always observed from roots of infested with isolate PA45. 

Phytophthora cinnamomi was recovered neither from any of H. stoechas or P. 

purpurea roots infested with this pathogen nor from the control root fragments 

at 24 h, 48 h, 72 h and 96 h. After 96 hours all agar plugs were viable and P. 

cinnamomi recovered from the root’s first centimetre. 

The plants did not show major foliar symptoms except Q. ilex and to a lesser extend 

Q. suber. However the severity of root symptoms was greater than control in all 

plants, except for H. stoechas and P. purpurea. 

The general comparison of means of severity of foliar and root symptoms by isolates 

indicates that isolate PA37 is not significantly different from PA45. When comparing 

means of root symptoms by species, Q. suber and Q. ilex are not significantly 

different from each other, show necrosis of feeder roots and the amount of roots is 

reduced, but are significantly different from A. unedo and H. stoechas. There is no 

significant difference between H. stoechas infested with either PA37 or PA45 isolate 

and the control. Phlomis purpurea was not included in the general statistical analysis 

because there was only a small number of replicates (3) for each isolate; therefore, a 

separate one-way ANOVA test was performed which revealed that there is no 

significant difference between infestation with PA37, PA45 and the control. 

Phytophthora cinnamomi was re-isolated from roots of Q. suber, Q. ilex and A. unedo

but not from the roots of H. stoechas and P. purpurea, confirming precedent 

observations by Moreira-Marcelino (2001). 

At the end of the pathogenicity trials the plants did not show major foliar symptoms, 

except for an A. unedo plant that died. In a trial in which Q. suber was not included 

the general comparison of means of severity of foliar and root symptoms by species 

showed that, A. unedo and Q. ilex are not significantly different from each other, but 

both are significantly different from H. stoechas and P. purpurea. Concerning root 

symptoms, P. purpurea presented the least average value, significantly different from 

H. stoechas. Arbutus unedo and Q. ilex infested with P. cinnamomi showed higher 

root severity symptoms when compared with the control plants. Phytophthora 

cinnamomi was re-isolated from roots of Q. ilex and A. unedo that were grown in soil 

infested with P. cinnamomi but not from the roots of H. stoechas and P. purpurea. 

Although there was a significant difference in root symptoms between plants grown 

in infested soil and control plants (except for P. purpurea), the same did not occur for 

the aerial/foliar symptoms. It is, usually, necessary to have a very extensive root 

lesion to observe the aerial symptoms (Erwin and Ribeiro 1996). However, this 

oomycete was not re-isolated from infested roots of H. stoechas and P. purpurea in 

any of the conducted experiments. 

Plants were submitted to waterlogging twice per week. This condition favours the 

pathogen growth and has a negative effect on plant health (Burgess and others 1999; 

Davison 1988; Sánchez and others 1998, 2002, 2005; Stolzy and Sojka 1984; 
GENERAL TECHNICAL REPORT PSW-GTR-221

242 

Zentmyer 1980), by a reduction in O2 supply and in the nutrient uptake. The low 

level of root symptoms showed by the control compared with the infested plants, in 

all experiments, confirms that flooding was not responsible for the symptoms showed 

by infested plants as previously demonstrated by Sánchez and others (2005). 

In situ Protection Trial 

At the end of the protection trial carried out in situ neither Q. ilex nor P. purpurea

aerial parts showed symptoms of disease. Roots of infested Q. ilex seedlings 

presented high severity symptoms significantly different from those of infested 

seedling surrounded by two infested P. purpurea plants. The association of P. 

purpurea with Q. ilex plants significantly reduced the root symptom severity on the 

susceptible Q. ilex. P. purpurea showed no significant difference either in aerial or 

root symptoms between control and infested plants. 

Phytophthora cinnamomi was re-isolated from 100 percent of Q. ilex infested roots, 

but only from 36.8 percent of Q. ilex infested roots that have grown surrounded by P. 

purpurea plants, meaning that the association of P. purpurea to Q. ilex growing in a 

close environment reduced the infection of Q. ilex roots. Like in previous 

experiments P. cinnamomi was not re-isolated from P. purpurea roots. 

Phytophthora cinnamomi was not recovered from soil associated with P. purpurea. 

The inoculum potential was significantly reduced in soil where P. purpurea plants 

grew next to Q. ilex seedlings when compared with soil associated to Q. ilex grown

alone. 

These results confirm the high potential of P. purpurea to control this pathogen. Q. 

ilex was firstly chosen because this species is, apparently, more susceptible to P. 

cinnamomi than Q. suber and consequently presented symptoms quicker, so 

glasshouse results could be achieved sooner. A trial with Q. suber is presently being 

prepared. 

Studies by D’Souza and others (2004, 2005) revealed that Acacia pulchella and A. 

extensa have a high potential for biological control of P. cinnamomi. However, in 

Portugal the genus Acacia is considered invasive and a programme to reduce or even 

eliminate some Acacia species is underway (Marchante and others 1999, Invader 

Project 2002). This problem is not unique, and a biological control must be carefully 

designed and performed with endemic species to avoid an imbalance in the 

ecosystem. 

In our experiments necrosis was never seen in P. purpurea roots. In fact, P. purpurea

roots were abundantly infested with P. cinnamomi mycelium and sections (4 μm 

thickness) observed by light microscopy─no P. cinnamomi was observed in the 

internal root tissues of P. purpurea (Medeira and Maia, pers. comm., 2007). Rarely, 

the oomycete colonised some epidermal cells and in this case signs of degradation of 

this pathogen were visible (Ibidem). These observations confirm that P. purpurea is 

resistant to P. cinnamomi infection. A similar behaviour was observed in A. pulchella

when inoculated with 10-20 zoospores: hyphae associated with necrotic cortical cells 

were lysed, but some penetrated the stele 16 h after inoculation. However, root 

necrosis and wilting were absent (Weste and Cahill, 1982). 

Bais and others (2002) identified rosmarinic acid (RA) in the root exudates of hairy 

root cultures of Ocimum basilicum (sweet basil) elicited using cell wall extracts 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

243 

(CWE) from P. cinnamomi. Upon elicitation, the production of RA was enhanced 

2.67-fold compared with the untreated control. RA showed to have activity against a 

wide range of soil born microorganisms, including P. drechsleri, P. megasperma and 

P. parasitica. In the same study, 250 M of RA provoked clumping and breakage of 

P. drechsleri hyphae. Widmer and Laurent (2006) stated that plants containing 

caffeic acid and RA, inhibit zoospore germination of the three Phytophthora spp. 

studied, namely P. palmivora, P. megakarya and P. capsici. Phenolic acids, like RA, 

exist constitutively in members of the Lamiaceae and Boraginaceae families. Both 

Ocimum and Phlomis are genera of the Lamiaceae family which comprises more than 

7000 species. It will be interesting to find out whether P. purpurea roots also exude 

RA. Phenolic acids have known antimicrobial properties, so it has been hypothesized 

that these compounds may play the role of phytoantecipins (Dixon 2001; VanEtten 

and others 1994), and phytoalexins as referred to above (Bais and others 2002).

Studies on the action of P. purpurea extracts on P. cinnamomi sporangia formation, 

zoospore release and germination and chlamidospore formation are presently 

underway. 

Literature Cited 

APCOR 2006. Produção suberícola. http://www.realcork.org/artigo.php?art=289 (25 April 

2007). 

Brasier, C.; Robredo, F.; and Ferraz, J. 1993. Evidence for Phytophthora cinnamomi

involvement in Iberian oak decline. Plant Pathology 42: 140-145. 

Bais, H.; Walker, T.; Schweizer, H.; Vivanco, J. 2002. Root specific elicitation and 

antimicrobial activity of rosmarinic acid in hairy root cultures of Ocimum basilicum. Plant 

Physiology and Biochemistry 40: 983-995. 

Burgess, T.; McComb, J.A.; Colquhoun, I.; Hardy, G. 1999. Increased susceptibility of 

Eucalyptus marginata to stem infection by Phytophthora cinnamomi resulting from root 

hypoxia. Plant Pathology 48: 796-806. 

Davison, E. 1988. The role of waterlogging and Phytophthora cinnamomi in the decline and 

death of Eucalyptus marginata in Western Australia. GeoJournal 17: 239-244. 

Direcção Geral dos Recursos Florestais [DGRF]. 2006. Resultados preliminares do 

inventário florestal nacional- Julho 2006. http://www.dgrf.minagricultura.pt/v4/dgf/pub.php?ndx=2563 (29 Dec 2006). 

Dixon, R. 2001. Natural products and plant disease resistance. Nature 411: 843-847. 

D’Souza, N.; Colquhoun, I.; Shearer, B.; Hardy, G. 2004. The potential of five Western 

Australian native Acacia species for biological control of Phytophthora cinnamomi. 

Australasian Plant Pathology 52: 267-279. 

D’Souza, N.; Colquhoun, I.; Shearer, B.; Hardy, G. 2005. Assessing the potential for 

biological control of Phytophthora cinnamomi by fifteen native Western Australian jarrahforest legume species. Australasian Plant Pathology 34: 533-540. 

Güllüce, M.; Adiguzel, A.; Ogutcu, H.; Sengul, M.; Karaman, I.; Sahin, F. 2004.

Antimicrobial effects of Quercus ilex L. extract. Phytotherapy research 18: 208-211. 
GENERAL TECHNICAL REPORT PSW-GTR-221

244 

Hammerschmidt, R. 1999. Phytoalexins: What have you learned after 60 years? Annual 

Review Phytopathology 37: 285-306. 

Hostettman, K.; Hostettman, M.; Marston, A. 1991. Saponins. In: Methods in plant 

biochemistry. Edited by Dey, P., Harborne, J. & Banthorpe, D. London: Academic press: 435-

471. 

Invader Project 2002. Avaliação do potencial de recuperação de ecossistemas invadidos por 

Acacia e metodologias para controlar a invasão.

http://www.ci.uc.pt/invasoras/index.php?menu=63&language=pt&tabela=geral (27 May 

2007). 

Kamoun, S.; Huitema, E.; Vleeshouwers, V. 1999. Resistance to oomycetes: a general role 

for the hypersensitive response? Trends in plant science 4: 196-200. 

Kremer, R.J.; Motavalli, P. P. 2004. The environmental impact of transgenic crops on soil 

biological processes and functions. Journal of Environmental Quality 33 (3): 805. 

Marchante, H.; Campelo, F.; Freitas, H. 1999. Ecologia do género Acacia nos ecossitemas 

dunares portugueses. In: Proceedings of the 1o encontro sobre invasoras lenhosas, 16-18 

November Gerês, Portugal. 

Mekuria, T.; Steiner, U.; Hindorf, H.; Frahm, J. P.; Dehne, H.W. 2005. Bioactivity of 

bryophyte extracts against Botrytis cinera, Alternaria solani and Phytophthora infestans. 

Journal of Applied Botany and Food Quality 79: 89-93. 

Molina-Torres, J.; Salazar-Cabrera, C.J.; Armenta-Salinas, C.; Ramiréz-Chávez, E. 

2004. Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: 

Affinin and reduced amides. Journal of Agricultural and Food Chemistry 52: 4700-4704. 

Moreira-Marcelino, A. 2001. Aspectos da interacção entre Phytophthora cinnamomi e a 

doença do declínio em Quercus suber e Q. rotundifolia. Thesis (PhD). Algarve University. 

Moreira, A.; Martins, J. 2005. Influence of site factors on the impact of Phytophthora 

cinnamomi in cork oak stands in Portugal. Forest Pathology 35: 145-162. 

Motavalli, P.; Kremer, R.; Fang, M.; Means N. 2004. Impact of genetically modified crops 

and their management on soil microbially mediated plant nutrient transformations. Journal of 

Environmental Quality 33: 816–824. 

Pires, N. 2005. Primeiras fases de infecção de raízes de Quercus suber L. (sobreiro) e 

Quercus ilex spp rotundifolia (Lam.) o. Schwartz (azinheira) por Phytophthora cinnamomi.

Thesis (MSc). Algarve University. 

Priest, S.H. 2000. U.S. public opinion divided over biotechnology? Nature Biotechnology 18: 

939–942. 

Sánchez, M. E.; Andicoberry, S.; Trapero, A. 2005. Pathogenicity of three Phytophthora

spp. causing late seedling rot of Quercus ilex ssp. Ballota. Forest Pathology 35: 115-125. 

Sánchez, M. E.; Caetano, P.; Ferraz, J.; Trapero, A. 2002. Phytophthora disease of 

Quercus ilex in south-western Spain. Forest Pathology 32: 5-18. 

Sánchez, M. E. ; Ruiz, D. ; Pérez, A. ; Blanco, M.A.; Trapero, A. 1998. Occurence and 

etiology of death of young olive trees in southern of Spain. European Journal of Plant 

Pathology 104: 347-357. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

245 

Steel, R.;Torrie, J. 1985. Bioestadística: principios y procedimientos. Bogotá: Colombia, 

McGraw-Hill. 

Stolzy, L.H.; Sojka, R.E. 1984. Effects of flooding on plant disease. In: T.T. Kozlowski. 

Flooding and plant growth, Orlando, FL, Academic Press: 221-264. 

Streito, J.; Jarnouen, G.; Villartay, G.; Tabary, F. 2002. Methods for isolating the alder 

Phytophthora. Forest Pathology 32: 193-196. 

Tuset, J.J.; Hinarejos, C.; Mira, J.L.; Cobos, J.M. 1996. Implicación de Phytophthora 

cinnamomi Rands en la enfermedad de la “seca” de encinas y alcornoques. Boletin Sanydad 

Vegetal, Plagas 22: 491-499. 

VanEtten, H.; Mansfield, J.; Bailey, J.; Farmer, E. 1994. Two classes of antibiotics: 

phytoalexins versus “phytoantecipins”. The Plant Cell 6: 1191-1192. 

Weste, G.; Cahill, D. 1982. Changes in root tissue associated with infection by Phytophthora 

cinnamomi. Journal of Phytopathology 103: 97-108. 

Widmer, T.; Laurent, N. 2006. Plant extracts containing caffeic acid and rosmarinic acid 

inhibit zoospore germination of Phytophthora spp. pathogenic to Theobroma cacao. European 

Journal of Plant Pathology 115: 377–388. 

Zentmyer, G.A. 1980. Phytophthora cinnamomi and the diseases it causes. Monograph 10. St 

Paul, Minnesota, American Phytopathological Society Press. 
GENERAL TECHNICAL REPORT PSW-GTR-221

246 

Investigating the Potential of Disease 

Control Practices for Integrated 

Management of Phytophthora kernoviae on 

Mature Magnolias in the United Kingdom1

S. Denman,2 S.A. Kirk,2 and A. Whybrow2 

Abstract 

Phytophthora kernoviae (Pk) a newly invasive Phytophthora species was first discovered in 

native woodlands and heritage gardens in Cornwall, U.K., in November 2003. Essentially it is 

an extremely aggressive pathogen of woodland Rhododendron ponticum causing leaf 

necrosis, shoot tip dieback and defoliation of infected plants. It also but it also attacks inner 

bark of European beech (Fagus sylvatica) causing lethal bleeding cankers. The pathogen 

forms inoculum on infected rhododendron foliage from where it infects susceptible tree stems 

in close proximity. However, in heritage gardens a range of exotic plant species are foliage 

hosts for Pk: Drimys, Magnolia, Michelia and Pieris, as well as rhododendron. 

Loss of exotic plant species in heritage gardens has negative socio-economic effects for local 

communities. Not only are the gardens an important source of tourist revenue in south-west 

England, but cultural and horticulturally valuable plants are housed there. For example part of 

the ‘National Magnolia Collection’ occurs on sites in Cornwall, as do a number of ‘Champion 

trees’. Some of these specimens are susceptible to attack by P. kernoviae; disease reduces 

aesthetic value but can also result either in death or removal of infected plants as directed by 

plant health regulation orders. Many of these plants are well established and loosing them not 

only changes the structure and composition of gardens, but some are irreplaceable because 

they are unique genotypes that are rare or even extinct in their natural habitats. Gardeners are 

finding it difficult to maintain the traditional constitution and ambience of the gardens and are 

concerned that the impacts of the disease may ultimately be felt in decreased numbers of 

visitors. 

Management to reduce and possibly eliminate disease on culturally valuable specimens in 

gardens is reliant on scientific data. A key aspect of integrated management is removal of 

primary sources of inoculum, i.e. rhododendron, without which other aspects of an integrated 

approach cannot be considered. Chemical intervention to reduce sporulation and prevent 

disease advance onto new tissue is a management tool worth investigating on deciduous trees. 

However, prior to this it is essential to establish if the pathogen survives and overwinters on 

infected shoots, and whether new infections are initiated from these overwintering infections, 

in order to select appropriate chemicals and optimise treatments. This study monitored the 

spread and intensity of disease and determined whether the pathogen survived in first and 

second year shoots of Magnolia naturally infected with Pk. To do this symptom expression 

was monitored and recorded over 20 mo and isolation used to confirm the presence of Pk. A 

bimodal pattern of symptom expression occurred with peaks in spring (May-June) and 

autumn (October-November). The pathogen overwintered on the fury outer bud scales, in 

buds, and in leaf scars only in the current year’s tissue, which was infected in autumn. Leaf 

debris also harboured P. kernoviae over winter. In spring infection was first evident in 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Forest Research, Alice Holt Lodge, Farnham, Surrey, GU10 4LH. 

Corresponding author: sandra.denman@forestry.gsi.gov.uk. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

247 

blossom and new foliage developing from buds. Further investigation of sanitation practices 

and chemical applications for disease management are merited on valuable deciduous trees in 

gardens. 
GENERAL TECHNICAL REPORT PSW-GTR-221

248 

Population Genetic Analyses of Four 

Phytophthora Species Recently Described 

in California Forests Reveal 

They Are All Introduced1

M. Garbelotto,2 R. Linzer,2 W. Monahan,2 and S. Bergemann2 

Abstract 

Four Phytophthora species have recently been described in California natural ecosystems: 

Phytophthora ramorum, P. ilicis, P. pseudosyringae, and P. cinnamomi. The first three 

species are of recent description, while the last one is a well-known pathogen associated with 

agricultural crops in the Western USA and in many areas world-wide. P. ramorum and P. 

cinnamomi are both causing significant mortality in native California hosts, while the other 

two species are associated with limited mortality of affected plant hosts. We have used both 

AFLP and microsatellite analyses to unravel the population genetic structure of all four 

species. All display a population structure typical of introduced species, independent of the 

symptoms they are causing. This finding indicates that as long as an ecological niche is 

available, aggressive and non- aggressive microbes can be introduced in novel environments. 

While the origin of P. ilicis remains a mystery, a perfect match was found between P. 

ramorum and P. cinnamomi isolates from the wild with isolates from agricultural situations. 

In the case of P. pseudosyringae, California isolates were nested within European isolates 

suggesting that the California population originated from Europe. It should be noted that these 

analyses evidentiate possible pathways of introduction, but not the actual origin of these 

exotic microbes. An analyses of hundreds of California bay laurel and tanbark oak leaves 

collected during a time-span of over one hundred years and deposited in the Jepson’s 

herbarium at UC-Berkeley indicated that the symptoms caused by P. ramorum, P. ilicis, and 

P. pseudosyrinage were absent in historical collections, also supporting the exotic origin of 

these three pathogens. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 University of California, Berkeley, Berkeley CA, USA. 

Corresponding author: matteo@nature.berkeley.edu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

249 

IUFRO Fourth Meeting of Working Party 

7.02.09, Phytophthoras in Forest and 

Natural Ecosystems: Meeting Summary1

Susan J. Frankel2

On behalf of the Fourth Phytophthoras in Forest and Natural Ecosystems local 

organizing committee and co-chairs Everett Hansen, Clive Brasier, and Giles Hardy, 

I thank you for your contributions to this Working Party meeting. The past week has 

stimulated much thought and discussion, thanks to the 100 participants from 15 

countries, 48 papers and 31 posters, two field trips, and two evening overview 

sessions. 

The following questions, explored throughout the week, highlight some of the critical 

issues raised. 

1. What is the relationship between Phytophthoras in nurseries and forest 

Phytophthora diseases? What are the implications of the detection of P. kernoviae, 

P. pseudosyringe, P. nemorosa, and other forest Phytophthora species in nurseries 

and orchards? Many forest Phytophthoras have been detected on rhododendron 

nursery plants. Which species could use rhododendron nursery plants as a pathway 

into forests? How can we identify the potentially damaging species before they 

escape? 

2. How can Phytophthora species be detected earlier in forests? Can we detect them 

before they reach outbreak levels, while they are still eradicable? 

3. How do we define Phytophthora species? How important is the division of 

Phytophthoras into species? Rather than focusing on differences between species, 

could we learn more by focusing on the common biology of most Phytopthora 

species? 

4. Phytophthora europaea has been recovered from oak forests in Europe, isolated 

from California bay laurel leaves and streams in California, and from Christmas tree 

plantation soils in Michigan (Balci and others 2007). Can other Phytophthora species 

adapt to such varied niches in different ecosystems? 

5. Ioos’ revision of the phylogeny of Phytophthora alni based on his genetic analysis 

(this volume) raises many questions. How well can Phytopthora species hybridize? 

Are there other “species clusters” that we are not yet aware of? If not, why not? 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 USDA Forest Service, Pacific Southwest Research Station, 800 Buchanan Street, West Annex, Albany, CA 94710, 

USA. 

Corresponding author: sfrankel@fs.fed.us. 
GENERAL TECHNICAL REPORT PSW-GTR-221

250 

6. How can we integrate Phytophthora genetic investigations with natural history or 

management studies? The gap between molecular biologists and field ecologists 

seems to be growing. How can we bring these specializations together? 

7. How important is designation and support of a forest Phytophthora model 

species? Which forest Phytophthora species would make the ideal model species? 

What selection criteria should be used? 

8. What roles do chlamydospores and sporangia play in Phytophthora survival and 

dispersal? Could chlamydospores be more important in dispersal than previously 

thought? 

9. In laboratory inoculations, Vettraino and Denman each report (this volume) an 

inverse relationship between P. ramorum lesion size and sporulation, with smaller 

lesions generating greater spore production. What are the implications of this finding 

for regulatory inspections? Could inconspicuous lesions yield significant inoculum 

for new infections? 

10. Removing infected trees and creating a buffer zone around the infested area is 

currently the only method used to attempt Phytophthora eradication from forest 

environments. In areas where tree removal is undesireable for environmental or 

social reasons, what other treatments could be used? 

11. Phytophthora species seem ubiquitous in water courses. Are water courses 

Phytophthora species’ primary habitat? What roles do Phytophthora species play in 

rivers and streams? Genetic analysis confirms that the genus Phytophthora is most 

closely related to brown algae and diatoms, and is evolutionarily distant from Fungi. 

Does this classification have implications for our understanding or management of 

Phytophthora species? 

12. Ramsfield and others (this volume) report that P. kernoviae may be native to the 

Southern Hemisphere. What actions should a country take when they determine a 

damaging Phytophthora is resident in their soil? 

13. How do we engage scientists in other disciplines, the public, policy-makers, 

politicians, and others to raise awareness and funding to prevent further adverse 

impacts caused by forest Phytophthora species? 

Literature Cited 

Balci, Y.; Balci, S.; Eggers, J.; MacDonald, W. L.; Juzwik, J.; Long, R.P.; Gottschalk, 

K.W. 2007. Phytophthora spp. associated with forest soils in eastern and north-central U.S. 

oak ecosystems. Plant Dis. 91:705-710. 

 
Posters

251
252
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

253 

White Oak Decline and Mortality in 

Southern Ohio1

Yilmaz Balci,2 Robert P. Long,3 Selin Balci,2 Daniel Balser,4 and 

William L. MacDonald3

Abstract 

Extensive white oak (WO = Quercus alba L.) decline and mortality has been observed since 

2002 in oak forests across southern Ohio. The mortality was especially severe at the 3800 ha 

Scioto Trail State Forest in southern Ross County. A series of stressors including drought 

(1998 and 1999) and insect defoliators (forest tent caterpillar, Malacosoma disstria; common 

oak moth, Phoberia atomaris; half-wing geometer, Phigalia titea) adversely affected tree 

health through 2004. WO crown dieback and mortality initially developed as single trees or 

small groups of trees on lower slope and bottomland sites. In 2006, WO mortality was 

assessed in seven severely affected bottomland stands using 3 to 7 800 m2 plots at each site 

(total n=34). Standing dead WO basal area ranged from 9 - 18 m2 ha-1, about 57 percent to 

84 percent of total WO basal area. Sapling regeneration (stems <10 cm dbh and > 1.4 m in 

height) is dominated by maples (Acer saccharum, A. rubrum) that are poised to fill canopy 

gaps. Random plots (n=103) were established in declining (n= 59) or non-declining (n=44) 

sites to evaluate soils and roots for the presence of Phytophthora spp. Among the species, P. 

cinnamomi was frequently isolated in 69 percent (41 sample trees) of soils from declining 

trees and 57 percent (25 sample trees) of soils from non-declining trees. However, a 

significant association was not detected between the presence of Phytophthora and 

deteriorating crown status. The average dry weight of WO roots (< 5 mm diameter) at lower 

slopes was significantly less than those located on upper slopes. The inoculum level was 

significantly higher in lower elevations than on higher elevations. Excess moisture in 2003 

and 2004 may have favored development of Phytophthora spp. or other root pathogens and 

may have contributed to the WO decline in lower slopes. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Division of Plant and Soil Sciences, West Virginia University, Plant and Soil Sciences, Morgantown, WV, USA. 3 USDA Forest Service, Northern Research Station, 359 Main Road, Delaware, OH, USA. 4 Division of Forestry, Ohio Department of Natural Resources, 2045 Morse Rd Building H-1 Columbus, OH, USA. 

Corresponding author: Yibalci@mail.wvu.edu. 
GENERAL TECHNICAL REPORT PSW-GTR-221

254 

Detection and Diagnosis of Phytophthora 

ramorum With Specific Hybridization Real 

Time PCR Probes1

Lassaad Belbahri,2 Gautier Calmin,2 Stephan Wagner,3 Eduardo 

Moralejo,4 Steve Woodward,5 Tomasz Oszako,6 and 

François Lefort2

Abstract 

Sudden oak death, caused by Phytophthora ramorum, poses a serious threat to native 

American oaks, and is also present in Europe where it has been discovered in numerous 

European ornamental plant nurseries. Its proven aggressiveness against plants in the Fagaceae 

and Ericaceae and the damage it has caused in North America lead to assign it a quarantine 

pathogen. It has also been listed in Europe in the biosecurity group 3, usually mostly devoted 

to severe pathogens for humans and animals. The timely and accurate detection of P. 

ramorum is a critical aid in the study of the epidemiology and biology of this pathogen. As a 

regulated organism, the availability of a sensitive and reliable assay is essential when 

attempting to achieve early detection of the pathogen. In this work, new specific 

hybridisation probes for a real time PCR amplification method were found to be rapid, robust, 

and laboursaving, and proved suitable for routine use in a molecular diagnostic laboratory. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Laboratory of Applied Genetics and Plant Health, Institute Earth Nature and Landscape, Univ. of Applied Sciences 

of Western Switzerland, 150 Route de Presinge 1254 Jussy, Switzerland. 3 Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Horticulture, 

Messeweg 11/12, 38104, Braunschweig, Germany.

4 Instituto Mediterráneo de Estudios Avanzados, IMEDEA (CSIC-UIB), Miquel Marquès 21, 07190, Esporles, 

Balearic Islands, Spain.

5 University of Aberdeen, School of Biological Sciences, Plant and Soil Science, Cruickshank Building, St. Machar 

Drive, Aberdeen AB24, 3UU, Scotland, U.K. 6 Forest Research Institute, Dept. of Forest Phytopathology, Sekocin Las, 05-090 Raszyn, Poland. 

Corresponding author: lasaad.belbahri@hesge.ch.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

255 

Occurrence of Novel Phytophthora Taxa in 

Hungarian Alder Forests1

Lassaad Belbahri,2 József Bakonyi,3 Zoltán Nagy,3 Gautier 

Calmin,2

 András Koltay,4 Steven Woodward,5 and François Lefort2

Abstract 

Numerous formally or informally designated new Phytophthora taxa have been recently 

described from natural and semi-natural ecosystems using the advantage of molecular 

techniques. Of the novel taxa, P. alni has been identified in Hungary. However, during 

surveys for the incidence of this pathogen, we also obtained three additional Phytophthora 

isolates from alder root and soil. They could not be identified accurately based solely on 

morphological characteristics. Therefore, our aim was to carry out a complex morphological, 

physiological and phylogenetic analysis to reveal their correct taxonomical and phylogenetic 

positions. The phylogenetic work included sequence analyses of the rDNA ITS, the nuclear a 

and β-tubulin, translation elongation factor 1 alpha and actin coding genes as well as the 

mitochondrial NADH dehydrogenase subunit 1 and cytochrome c oxidase 1 and 2 genes. The 

results indicated that the three studied isolates represented two different taxa, designated as 

Phytophthora sp. 1 and Phytophthora sp. 2, each belonging to Waterhouse’s group V. 

Morphological differences between them were observed in the dimension of oogonia, 

oospores and sporangia as well as the colony pattern on carrot agar. Multigene sequence 

analysis revealed that Phytophthora sp. 1 possessed an internal transcribed spacer identical to 

that of the informally named P. taxon Forestsoil, whereas Phytophthora sp. 2 represented 

another novel taxon that has not been identified elsewhere before. A thorough morphological 

and physiological characterisation as well as a detailed phylogenetic analysis in relation to 

other Phytophthora spp. will be presented. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Laboratory of Applied Genetics, School of Engineering of Lullier, University of Applied Sciences of Western 

Switzerland, 150 route de Presinge, 1254 Jussy, Switzerland.

3 Plant Protection Institute, Hungarian Academy of Sciences, Herman Ottó út 15, 1022 Budapest, Hungary.

4 Hungarian Forest Research Institute, Frankel L. u. 42-44, 1023 Budapest, Hungary. 5 Plant and Soil Science, School of Biological Sciences, University of Aberdeen, St. Machar Drive, Cruickshank. 

Building, Aberdeen, AB24 3UU, Scotland, U.K. 

Corresponding author: lasaad.belbahri@hesge.ch.
GENERAL TECHNICAL REPORT PSW-GTR-221

256 

Phytophthora polonica sp. novo Isolated 

From Polish Alder Stands in Decline1

Lassaad Belbahri,2 Tomasz Oszako,3 Gautier Calmin,2 Eduardo 

Moralejo,4

 J.A Garcia,4 E. Descals,4 and François Lefort2

Abstract 

In a survey of Phytophthora associated with alder decline in Poland, several isolates of a 

homothallic Phytophthora sp., which could not be assigned to other taxa including P. alni 

subspecies, were consistently recovered from rhizosphere soil samples. Their morphology 

and pathogenicity, as well as sequence data for three nuclear regions (ITS rDNA, EF-1α and 

β-tub) and a coding mitochondrial DNA region (nadh1), were examined. The new 

Phytophthora species is characterized by moderate to slow growth on carrot agar at 20  ̊C, 

high optimal (ca. 30  ̊C) and maximum (ca. 38  ̊C) growth temperatures. It forms catenulate, 

often lateral, hyphal swellings, large chlamydospores in agar media and in soil extract, 

persistent. Sporangia are ovoid to ellipsoid and non-papillate. Large oogonia with paragynous 

and sometimes amphigynous antheridia were observed. From pathogenicity tests on alder 

twigs and on a few Iberian trees, P. polonica seems to be a poor inner bark colonizer. Despite 

being pathogenic to fruits in wound inoculations, no plant diseases attributable to P. polonica 

have appeared in Poland. In a phylogenetic analysis using either Bayesian inference or 

Maximum Likelihood methods P. polonica falls within clade10 “sensu Cooke and others 

(2000)”, together with P. insolita and in clade 8 “sensu Kroon and others (2004)” of the 

Phytophthora genus. This new species was named Phytophthora polonica Belbahri L, 

Moralejo E & Lefort F. sp. nov. 

Literature Cited 

Cooke, D.E.L.; Drenth, A.; Duncan, J.M.; Wagels, G.; Brasier, C.M. 2000. A molecular 

phylogeny of Phytophthora and related oomycetes. Fungal Genet Biol 30: 17–32. 

Kroon, L.P.N.M.; Bakker, F.T.; van den Bosch, G.B.M.; Bonants, P.J.M; Flier, W.G.

2004. Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear 

DNA sequences. Fungal Genet Biol 41: 766–782. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2 Laboratory of Applied Genetics and Plant Health, Institute Earth Nature and Landscape, Univ. of Applied Sciences 

of Western Switzerland, 150 Route de Presinge 1254 Jussy, Switzerland. 3 Forest Research Institute, Dept. of Forest Phytopathology, Sekocin Las, 05-090 Raszyn, Poland. 4 Instituto Mediterráneo de Estudios Avanzados, IMEDEA (CSIC-UIB), Miquel Marquès 21, 07190, Esporles, 

Balearic Islands, Spain. 

Corresponding author: lasaad.belbahri@hesge.ch.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

257 

Production of Viable Oospores by 

Phytophthora ramorum1

Xavier Boutet2 and Anne Chandelier2

Abstract 

A method was developed to improve the production of gametangia by Phytophthora ramorum

in vitro. The agar type, the nutrient source and the genotype of the strains involved in the 

pairing are important factors in the pathogen’s potential for sexual reproduction. Oospore 

viability was assessed with a coloration method using tetrazolium bromide. Most of the 

oospores were stained in purple, corresponding to spores in dormancy. A few germinated 

oospores were subcultured for further characterization. Our results suggest that new strains 

could be generated through breeding. This observation should be considered during pest risk 

analysis. 

Introduction 

Phytophthora ramorum (Werres, De Cock, Man in’t Veld) is a heterothallic species. 

Initial pairing studies revealed that all European isolates were of A1 type while all 

American isolates were of A2 type (Werres and others, 2001). In 2003, a Belgian 

isolate was identified as a putative European A2 (Werres & De Merlier, 2003) while 

some A1 isolates were reported in American nurseries (Hansen and others, 2003), 

therefore suggesting some possible crossing between both mating types. However, 

attempts to produce oospores in vitro with classical methods were difficult compared 

to other heterothallic species, therefore suggesting a weak functionality of the sexual 

system in this Phytophthora species (Brasier & Kirk, 2004). The objectives of this 

study were to evaluate the role of different parameters on the production of oospores 

in vitro, and to assess their viability. 

Methods and Materials 

The isolates used in this study are listed in Table 1. The Belgian isolates of P. 

ramorum were collected in the framework of the survey carried out by the Belgian 

Plant Protection Service. 

The production of gametangia by Phytophthora ramorum as well as by other 

Phytophthora species used as references was compared on media differing in nutrient 

source (carrot / tomato) and gelling agent (technical agar, bacto-agar, noble agar and 

agarose) using the method developed by Brasier and Kirk (2004). The amount of 

gametangia was scored on a scale of 0-4 based on the observation of the Petri dishes 

under the microscope (125x): 0=no gametangia, 1=less than 10 gametangia per 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2 Walloon Agricultural Research Centre (CRA-W), Dept. of Biological Control and Plant Genetic Resources, Rue de 

Liroux, 4, B-5030, Gembloux (Belgium). 

Corresponding author: chandelier@cra.wallonie.be.
GENERAL TECHNICAL REPORT PSW-GTR-221

258 

pairing, 2 = up to 5 gametangia per field, 3 = 5-25 gametangia per field and 4 = more 

than 25 gametangia per field. 

Oospores of P. ramorum and other Phytophthora used as references were extracted 

after 60 days. To this end, agar blocks containing oospores were comminuted, 

filtrated and incubated in a Glucanex solution during 24 h. The viability of oospores 

was evaluated by a staining method using tetrazolium bromide (MTT) developed by 

Sutherland and Cohen (1983). 

Table 1—Isolates of Phytophthora ramorum and other Phytophthora species 

used in this study. 

Results and Discussion 

Optimization of the production of oospores in vitro 

The quality of the gelling agent and the genotype of the strain were evaluated in 

pairings between mating partners. By comparing different agar sources on a carrot 

based medium, a delay or a failure in the production of oospores was observed in 

pairings carried out on media supplemented with technical agar. In contrast, oospores 

were produced on other agar types, the production on media supplemented with 

agarose being slightly higher (Table 2).These differences in oospores production 

were not observed with other heterothallic Phytophthora species used as reference 

material, except for P. cryptogea on Carrot Agar. 

Strain Nr Species Original Host Collection Mating Type Origin* 

2299 P. ramorum Viburnum sp. CBS1 101330 A1 EU 

3237 P. ramorum Viburnum bodnantense Belgium2 A2 EU 

2387 P. ramorum Rhododendron sp. Belgium2 A1 EU 

3206 P. ramorum Rhododendron sp. Belgium2 A1 EU 

3528 P. ramorum Quercus sp. USA3

 (014) A2 US 

3229 P. cambivora na BBA4 21/95-K2 A1 - 

3230 P. cambivora na BBA4

 20/95-2b3 A2 - 

3231 P. cinnamomi na BBA4

 69094 A1 - 

3232 P. cinnamomi na BBA4

 62660 A2 - 

3233 P. cryptogea na BBA4

 65909 A1 - 

3234 P. cryptogea na BBA4

 63651 A2 - 

2542 P. cactorum Fragaria ananassa Belgium2

 Homothallic - 

1: CBS Collection, Utrecht, The Netherlands 

2: Our collection 

3: Plant Protection service (PD) Geertjesweg 15, Wageningen, The Netherlands (Dr De Gruyter) 

4: BBA, Messeweg 11/12 , Braunschweig, Germany (Dr Werres) 

*origin of the Phytopthora ramorum isolate (EU or US) determined by PCR-RFLP according to Kroon et al

(2004). 

na: not available 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

259 

Table 2—Production of gametangia from four heterothallic and one 

homothallic Phytophthora species on different pairing media (CBA=Carrot 

Bacto-Agar, CA= Carrot Agar, CAN=Carrot Noble Agar, Caose= Carrot 

Agarose). Amount of gametangia according to a 10-4 scale. 

Pairing

A1 x A2 CBA CA CAN Caose

P. ramorum 2299 x 3528 4 2 4 4

P. ramorum 2299 x 3237 4 2 4 4

P. ramorum 2387 x 3528 2 0 2 3

P. ramorum 2387 x 3237 2 1 2 3

P. ramorum 3206 x 3528 2 1 2 2

P. ramorum 3206 x 3237 3 2 2 2

P. cambivora 3229 x 3230 4 4 4 4

P. cinnamomi 3231 x 3232 4 4 4 4

P. cryptogea 3233 x 3234 4 0 4 4

P. cactorum 542 (homothal 4 4 4 4

Species

Pairing media

Besides the gelling agent, the formation of gametangia was also influenced by the 

genotype of the strains involved in the pairing. A European A1 strain producing very 

few chlamydospores was found to be a better mating partner than other A1 strains. 

Moreover, media containing carrot nutrient were more suitable than media containing 

tomato (data not shown). 

Viability of the oospores 

Oospores from intraspecific pairings between European strains were extracted after 

60 days and coloured with the MTT method. As shown on figure 1, the distribution 

of oospores of P. ramorum in the different classes of coloration (purple, blue, black 

and colourless) was similar to that found in other Phytophthora species, i.e. a vast 

majority of oospores displayed a purple coloration corresponding to dormant 

structures. Blue coloration seemed to indicate an activated state as germinated 

asexual spores were stained in blue (data not shown). Unstained and black oospores 

were considered as non viable oospores. However, a high variation of color intensity 

was observed between oospores and the distinction between dark blue and black 

oospores was sometimes questionable, leading to potential misinterpretation. 

Germinated oospores were observed in several cases. Some of them died rapidly. The 

surviving progenies were subcultured in order to be characterised. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 260

0

20

40

60

80

100

Purple Blue Black Unstained

% oospores 

P. cactorum (197)

P. ramorum (91)

P. cryptogea (853)

P. cambivora (269)

Figure 1—Distribution of 60 day-old oospores in different color classes after 

incubation in MTT solution for P. ramorum and three other Phytophthora

species (in brackets= number of oospores) 

Literature Cited 

Brasier, C.M.; Kirk, S. 2004. Production of gametangia by Phytophthora ramorum in vitro. 

Mycological Research. 108: 823-827. 

Kroon, L.; Verstappen, E.; Kox, L.; FlierW.; Bonants, P. 2004. A rapid diagnostic test to 

distinguish between American and European Populations of Phytophthora ramorum. 

Phytopathology. 94: 613-620. 

Hansen, E.M.; Reeser, P.W.; Sutton, W.; Winton, L.M. 2003. First report of A1 mating 

type of Phytophthora ramorum in North America. Plant Disease. 87: 1267-1267. 

Sutherland, E.D.; Cohen, S.D. 1983. Evaluation of Tetrazolium Bromide as a vital stain for 

fungal oospores. Phytopathology. 73: 1532-1535. 

Werres, S.; De Merlier, D. 2003. First detection of Phytophthora ramorum mating type A2 

in Europe. Plant Disease. 87: 1266-1266. 

Werres, S.; Marwitz, R.; Veld, W.; De Cock, A.; Bonants P.; De Weerdt, M.; Themann, 

K.; Ilieva, E.; Baayen, R.P. 2001. Phytophthora ramorum sp nov., a new pathogen on 

Rhododendron and Viburnum. Mycological Research. 105: 1155-1165. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

261 

Phytophthora cinnamomi Populations on 

Quercus Forests From Spain and Portugal1

P. Caetano,2 A. Ávila,3 M.E. Sánchez,3 A. Trapero,3 A.C. Coelho4

Abstract 

Molecular characterization of P. cinnamomi isolates obtained from soil or root 

samples from declining holm and cork oak trees from different locations of the 

southwest region of the Iberian Peninsula (Spain and Portugal), was performed by 

AFLP (amplified fragment length polymorphism). Phytophthora cinnamomi

isolates from Quercus spp. structured in six populations according to their 

geographical origin, and were separated into two clusters based upon their AFLP 

genotypic profiles, showing only 20 percent of similarity between them. Cluster 1 

comprises the populations coming from Portugal and southwestern Spain from 

both Q. rotundifolia and Q. suber. Cluster 2 comprises populations from the 

eastern Spanish part of our area of study and also comprises isolates coming from 

Q. rotundifolia and Q. suber. Both populations of P. cinnamomi are in good 

agreement with previous results based upon morphology and temperature-growth 

relationships of the isolates. 

Introduction 

In recent years, oak forests have suffered several episodes of decline all around 

Europe. In the Iberian Peninsula, symptoms of declining trees are unspecific, 

including dieback of branches and part of the crown, and yellowing and wilting of 

leaves (Brasier 1996). In southern Spain and Portugal, a strong association was 

shown between P. cinnamomi root infection and decline and mortality of cork 

(Quercus suber) and holm oak (Quercus rotundifolia) (Sánchez and others 2002). 

Attempts to distinguish isolates of P. cinnamomi infecting Quercus spp. through 

molecular tools has not been performed in the past. Molecular analysis is important 

to understand the genetic diversity and structure of Phytophthora populations in order 

to help our understanding of disease development. The aim of the present study was 

to fingerprint P. cinnamomi isolates from Quercus spp., exploring the intraspecific 

variation to try to correlate the genetic profiles with their plant hosts, geographical 

location, mycelial morphology and temperature-growth relationships. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2

 FERN, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. 

3

 ETSIAM, Universidad de Córdoba, Campus de Rabanales. 14080 Córdoba, Spain. 4

 ESE, Universidade do Algarve, Campus da Penha, 8005-139 Faro, Portugal. 

Corresponding autor: ag1sahem@uco.es. 
GENERAL TECHNICAL REPORT PSW-GTR-221

262 

Materials and Methods 

Fungal isolates 

Isolates of P. cinnamomi (46) were obtained from soil or root samples taken from 

declining holm and cork oak trees from different locations of the southwest region of 

the Iberian Peninsula (Spain and Portugal) (Table 1), the geographical area more 

severely affected by Mediterranean oak decline (Sánchez and others 2002). 

Table 1—Characteristics of Phytophthora cinnamomi isolates from Quercus 

Geographic origin 

Group Clade Code Host Country Province Location Source 

PA001 Quercus suber Portugal Algarve S. Brás Alportel Root 

PA003 Quercus suber Portugal Algarve Loulé Soil 

PA006 Quercus suber Portugal Algarve Lagos Soil 

PA007 Quercus suber Portugal Algarve Lagos Root 

PA008 Quercus suber Portugal Alentejo Grândola Root 

PA011 Quercus suber Portugal Alentejo Sines Soil 

PA012 Quercus suber Portugal Alentejo Sines Root 

PA013 Quercus suber Portugal Algarve S. Brás Alportel Soil 

PA014 Quercus suber Portugal Algarve S. Brás Alportel Root 

PA019 Quercus suber Portugal Algarve S. Brás Alportel Soil 

PA020 Quercus suber Portugal Algarve S. Brás Alportel Soil 

PA021 Quercus suber Portugal Algarve S. Brás Alportel Root 

PA043 Quercus suber Portugal Alentejo Alcácer do Sal Soil 

PA044 Quercus suber Portugal Alentejo Grândola Soil 

PE008 Quercus rotundifolia Spain Huelva El Andévalo Root 

PE010 Quercus rotundifolia Spain Huelva El Andévalo Soil 

PE011 Quercus rotundifolia Spain Huelva El Andévalo Soil 

PE013 Quercus rotundifolia Spain Huelva El Andévalo Soil 

PE054 Quercus rotundifolia Spain Huelva Srra Aracena Root 

PE055 Quercus rotundifolia Spain Huelva Srra Aracena Soil 

PE056 Quercus rotundifolia Spain Huelva Srra Aracena Soil 

PE058 Quercus rotundifolia Spain Huelva El Andévalo Root 

PE059 Quercus rotundifolia Spain Huelva El Andévalo Soil 

PE060 Quercus rotundifolia Spain Huelva El Andévalo Soil 

PE061 Quercus rotundifolia Spain Huelva El Andévalo Soil 

PE062 Quercus rotundifolia Spain Huelva El Andévalo Soil 

PE085 Quercus rotundifolia Spain Huelva Calañas Soil 

PE086 Quercus rotundifolia Spain Huelva El Andévalo Soil 

PE095 Quercus rotundifolia Portugal Algarve S. Brás Alportel Root 

A 1 

PE097 Quercus rotundifolia Portugal Algarve Tavira Root 

2 PA025 Quercus suber Spain Cádiz Los Alcornocales Soil 

2 PA030 Quercus suber Spain Cádiz Los Alcornocales Soil 

2 PA031 Quercus suber Spain Cádiz Los Alcornocales Soil 

2 PA032 Quercus suber Spain Cádiz Los Alcornocales Soil 

2 PA033 Quercus suber Spain Cádiz Los Alcornocales Soil 

2 PA035 Quercus suber Spain Cádiz Los Alcornocales Soil 

2 PA037 Quercus suber Spain Cádiz Los Alcornocales Root 

1 PA040 Quercus suber Portugal Ribatejo Ponte de Sôr Root 

1 PA041 Quercus suber Portugal Ribatejo Ponte de Sôr Soil 

1 PA042 Quercus suber Portugal Ribatejo Ponte de Sôr Root 

2 PA118 Quercus suber Spain Cádiz Los Alcornocales Soil 

2 PE078 Quercus rotundifolia Spain Cáceres Herreruela Soil 

2 PE079 Quercus rotundifolia Spain Cáceres Herreruela Soil 

2 PE080 Quercus rotundifolia Spain Cáceres Herreruela Root 

2 PE082 Quercus rotundifolia Spain Badajoz Azagala Root 

B 

2 PE094 Quercus rotundifolia Spain Córdoba Pozoblanco Soil 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

263 

In molecular analysis, some additional isolates were used as controls: two isolates of 

P. cinnamomi from Castanea sativa, two P. megasperma and two P. inundata

isolates from Olea europaea, and two P. drechsleri and two P. cryptogea isolates 

from Q. rotundifolia seedlings (Sánchez and others 2001, 2005). 

Hyphal morphology and growth-rate 

To determine mycelial growth, CA plugs of 7-mm diameter from edges of P. 

cinnamomi colonies in active growth were placed in the centre of CA Petri dishes (9-

cm diameter, 20-ml media/plate) and incubated at different temperatures in darkness. 

Temperatures tested were 5, 10, 15, 20, 25, 30 and 35 °C. Three replicates per isolate 

and temperature were prepared. Two colony diameters were measured after 2, 4, 6, 

10 and 15 days. 

Growth-rates per day were calculated for the different temperatures and average data 

of the three replicates were adjusted to a regression curve. Adjustment was made 

using Statistix (Statistix for Windows. Analytical Software, Tallahassee, Florida, 

2000). The best polynomial model for all the isolates was chosen from several 

combinations of terms, based on the significance of the estimated parameters 

(P0.05): coefficients of determination (R2), coefficients of determination adjusted 

for degrees of freedom (Ra2), and pattern of residuals (Draper and Smith, 1981). The 

optimum growth temperature was estimated in the adjusted model. 

Colony morphology was assessed immediately after measurement at both 4 and 6 

days of growth at 20o C. Production of hyphal swellings and chlamydospores was 

assessed after removal of small portions of mycelium to a glass microscope slide and 

staining with acid fuchsin in lactophenol. 

Molecular analysis 

Petri dishes containing 20 ml of carrot broth and one agar plug cut from the growing 

margin of fresh cultures of P. cinnamomi and other Phytophthoras growing on 

clarified V8 juice agar were prepared. After 5 days of incubation at 25 °C, the 

resulting mycelium was washed in sterile distilled water. Clean mycelia were 

separately frozen in liquid nitrogen and ground into a fine powder with a mortar and 

pestle. DNA extraction was performed with the DNeasy Plant Mini kit following 

manufacturer instructions. 

AFLP analysis was performed using the AFLP Analysis System (Gibco BRL, Grand 

Island, NY) with some modifications of the manufacturer’s recommended protocol 

(Vos and others 1995). 

Oomycete genomic DNA (300 ng) was digested for 2 h at 37 °C with EcoRI (2.5 U) 

and MseI (2.5 U) restriction enzymes. Two oligonucleotide adaptors, complementary 

to the restricted sites of the DNA fragments, were ligated. A pre-amplification 

reaction was performed with primers annealing to the EcoRI and MseI adaptors with 

an additional selective 3’ nucleotide (A or T). Selective PCR reactions were 

performed using five primer combinations. The EcoRI primer was radioactively 
GENERAL TECHNICAL REPORT PSW-GTR-221

 264

labelled and the amplification products were separated on 6 percent polyacrylamide 

gels. The DNA fragments were visualised after exposition of the gel to X-ray film. 

The reproducible bands were scored manually as present (1) or absent (0) for each 

DNA sample. One hundred and forty nine bands were scored, resulting in a binary 

matrix of 56 × 149 bands. 

A similarity matrix was constructed based upon the Jaccard coefficient using the 

SIMQUAL program in the NTSYS-pc software package (Rohlf, 1998). Cluster 

analysis of the matrix values was performed using the SAHN program in NTSYS-pc 

2.0 and a dendrogram was built. 

Populations were defined according to their geographic origin and plant host, and the 

binary matrix was analysed using the POPGENE 32 software. The corresponding 

dendrogram was constructed based on a matrix of genetic distances using unweighted 

pair-group mean analysis (UPGMA) through the same software. 

The assessment of the genetic diversity within P. cinnamomi species and population 

genetic structure was evaluated using AFLP-SURV 1.0 (Vekemans, 2002). 

Analysis of molecular variance (AMOVA; Excoffier and others 1992), based upon 

Euclidian distances between AFLP multilocus phenotypes, was conducted with 

ARLEQUIN 3.01. 

Results 

Hyphal morphology and growth rates 

Colony morphology assessment at 20 oC in CA medium did not clearly let us to 

separate the isolates into groups. Each isolate produced flat to slight petaloid colonies 

after 4 days growth, with abundant, cottony aerial mycelium after 6 days of growth. 

However, two different groups could be well distinguished based on hyphal 

morphology: Phytophthora cinnamomi Group A produces abundant, subspherical 

hyphal swellings, mainly in terminal clusters. In contrast, Group B isolates show 

hyphal swellings irregularly shaped, in a botryose branching pattern. 

After the evaluation of different models, the growth rate data were adjusted to a third 

degree polynomial model: y = a*t + b*t2 + c*t3, in which y = the growth rate 

(mm/day), t = temperature, and a, b, c are the regression constants. Analysis of 

variance was performed and Fisher's test showed significant differences in average 

growth rate at the different temperatures tested. Two different growth patterns were 

detected in good agreement with the morphological groups A and B (fig. 1). 

According to the model selected, the estimated optimal temperature for growth was 

30.1 °C for Group A and 26.9 °C for Group B. Average maximum growth rates 

recorded at the temperatures tested are in table 2. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

265 

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40

Temperatura (oC)

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40

Temperatura (oC)

y = - 0,00337 t3 + 0,14872 t2 - 0,68682 t

R2 = 0,983 

y = - 0,00200 t3 + 0,09296 t2 - 0,15988 t

R2 = 0,980 

)

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40

Temperatura (oC)

0

5

10

15

20

25

30

0 5 10 15 20 25 30 35 40

Temperatura (oC)

y = - 0,00337 t3 + 0,14872 t2 - 0,68682 t

R2 = 0,983 

y = - 0,00200 t3 + 0,09296 t2 - 0,15988 t

R2 = 0,980 

)

Growth Rate (mm/day) Growth Rate (mm/day)

Temperature (oC)

Temperature (oC)

Figure 1. Growth patterns of P. cinnamomi morphological Groups A and B. Arrows indicate the 

estimated optimum growth temperature (30.1 and 26.9o C respectively).

Group A

Group B

Table 2—Growth rate, minimum and optimal temperatures for growth in CA 

medium of the morphological Groups of P. cinnamomi

Group Growth model R2

Minimum 

Temperature 

Estimated 

(C) 

Optimal 

Temperature 

Estimated 

(C) 

Average 

Maximum 

Growth Rate 

(mm/day) 

A y = -0.002t3

 + 0.09296t2

- 0.15988t 0.980 1.8 (0-4.4) 30.1 (28.3-32.4) 

24.9 (19.4-

29.8) 

B y = -0.00337t3

 + 

0.14872t2

 + 0.68682t 0.983 5.2 (3.9-6.8) 26.9 (25.2-28.0) 

23.5 (22.6-

24.6) 

Molecular analysis 

Isolates of P. cinnamomi obtained from Q. suber and Q. rotundifolia in Portugal 

(Algarve, Alentejo) and Spain (Cádiz, Badajoz, Cáceres, Córdoba, Huelva) were 

analysed by AFLP generating a total of 149 DNA fragments. One hundred and one 

(67.8 percent) out of 149 amplified fragments were polymorphic within or among 

populations. In single populations the percentage of polymorphic fragments ranged 

from 41.6 percent to 54.4 percent. All the individuals tested displayed unique AFLP 

band patterns, suggesting a high level of genetic variability. The results of the 

analysis of genetic diversity were 0.2267 for Ht (total diversity); 0.2070 for Hw 

(average diversity within populations); 0.0198 for Hb (average diversity among 

populations), and 0.0868 for Fst (differentiation between populations). High levels of 

gene diversity within populations (Hw = 0.2070) were observed. AMOVA revealed 

that 88.59 percent of the total genetic variation was attributed to differences among 

isolates within populations, while 11.41 percent was attributable to differences 
GENERAL TECHNICAL REPORT PSW-GTR-221

266 

among populations. Of the total variance only 5.09 could be attributed to geographic 

structure. 

Phytophthora cinnamomi isolates from Quercus spp. structured in six populations 

according to their geographical origin and host provenance and were separated into 

two groups based upon the AFLP genotipic profiles, showing only 20 percent of 

similarity between them. The dendrogram calculated from Nei ́s unbiased genetic 

distances among populations by the UPGMA method, showed two distinct clusters 

(fig 2). Cluster 1 comprises the populations coming from Portugal and Huelva 

(southwestern Spain) from Q. rotundifolia or Q. suber. Cluster 2 comprises 

populations from the eastern Spanish part of our area of study (province of, Badajoz 

–single isolates from Córdoba and Cáceres included- and Cádiz), and also comprises 

isolates coming from Q. rotundifolia and Q. suber. 

Figure 2—Unweighted pair group mean analysis dendrogram illustrating 

Nei ́s genetic distance based on amplified fragment length polymorphism 

from geographical origin and host populations of Phytophthora cinnamomi

from Quercus spp. 

Both Clusters 1 and 2 showed a good correspondence with the morphological Groups 

A and B respectively (Table 1). Only three isolates (PA040, PA041 and PA042) 

coming from Q. suber Ribatejo (Portugal) belong to the morphological Group B but 

their AFLP profiles are in better agreement with the rest of isolates on Group A, 

belonging to the Cluster 1 (Table 1). 

Algarve-Q. suber

Huelva-Q. rotundifolia

Riba-Alentejo-Q. 

Algarve-Q. rotundifolia

Badajoz-Q. 

Cádiz-Q. suber

0.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

267 

Figure 3-Geographical origin of P. cinnamomi isolates belonging to the 

morphological Group A and B. Isolates coming from Ribatejo (Portugal) 

(encircled) belong to the Group B but their AFLP profile correspond to the 

Cluster 1. 

Discussion 

Two different populations have been detected among P. cinnamomi isolates infecting 

Mediterranean Quercus species in southern Spain and Portugal by using molecular 

tools as AFLP fingerprintings. In previous studies, dealing with the morphological 

characterisation of P. cinnamomi isolates coming from Quercus roots located in this 

geographical area (southwest of the Iberian Peninsula), two different hyphal patterns 

were detected (Sánchez and others 2003a). A first group, coming from the province 

of Cádiz (eastern part of our area of study) showed straight hyphae, poorly branched 

and with abundant spherical hyphal swellings. The second group, coming from 

Quercus roots located in the west area of southern Spain, was characterised by the 

presence of a botryose-coralloid mycelium clearly different from the first one 

(Sánchez and others 2003a). As the first group of isolates came from Q. suber 

samples and the second one came from Q. rotundifolia, it was hypothesized that these 

differences could be related with the host origin (Sánchez and others 2003a, 2006). 

Analysing a higher number of isolates in a wider range of locations, it seems that 

these morphological differences former detected were better associated with the 

geographical origin of the isolates. The differences among both morphological 

groups of P. cinnamomi were also noticed when their cardinal temperatures of 

growth were studied. Isolates coming from the western part of southern Iberia (Group 

A) showed a higher optimal growth temperature (30.1o C) in CA medium than the 

isolates coming from the east (Group B, optimum temperature 26.9o C). In contrast, 

Group A

Group B

PORTUGAL

SPAIN

Alentejo

Algarve

Huelva

Badajoz

Córdoba

Cádiz

Cáceres

Ribatejo
GENERAL TECHNICAL REPORT PSW-GTR-221

268 

minimal temperatures for growth were lower in Group A than in Group B, suggesting 

a higher adaptability of Group A to survive in a wider range of temperatures. 

Previous studies carried out in the region of Andalucía (southern Spain) showed that 

the oak decline was strongly associated with P. cinnamomi root rot in the province of 

Huelva, where found the Cluster 1-GroupA of the pathogen (Sánchez and others 

2002, 2003a, 2006, Navarro and others 2004). In this province, P. cinnamomi appear 

near the only factor associated whith decline, as also noticed in south Portugal 

(Moreira and Martins, 2005). In contrast, in the oaklands affected by decline in the 

province of Córdoba, the root rot caused by P. cinnamomi is the main biotic factor 

involved, but not the only one (Sánchez and others 2003a, 2006, Navarro and others 

2004). In the south (province of Cádiz), the main disease associated with oak decline 

is the trunk canker caused by Botryosphaeria corticola (Sánchez and others 2003a; 

2003b, Navarro and others 2004), remaining the Phytophthora root rot as secondary 

importance (Sánchez and others 2003a, Navarro and others 2004). In both provinces 

P. cinnamomi population belong to the Cluster 2-Group B. Nevertheless, despite the 

fact that both populations seem to play different roles in oak decline development in 

southern Iberia, differences on pathological ability between them seem to be 

inexistent when tested by artificial inoculations (data not published). 

The present results suggest that there are two different populations of P. cinnamomi

causing root rot on Quercus species in southern Iberia, one very aggressive, coming 

from Portugal and also colonizing the southwestern part of Spain (Huelva) causing 

root disease, and a second Spanish population of P. cinnamomi acting as decline 

factor in the eastern part of the geographical area affected by decline. 

Literature Cited 

Brasier, C.M. 1996. Phytophthora cinnamomi and oak decline in southern Europe. 

Environmental constraints including climate change. Ann. Sci. For. 53: 347-358. 

Draper, N.R.; Smith H. 1981. Applied Regression Analysis. John Wiley and Sons, New 

York. 

Excoffier, L.; Smouse, P.E.; Quattro, J.M. 1992. Analysis of molecular variance inferred 

from metric distances among DNA haplotypes: application to human mitochondrial DNA 

restriction data. Genetics 131: 479-491. 

Moreira, A.C.; Martins, J.M.S. 2005. Influence of site factors on the impact of 

Phytophthora cinnamomi in cork oak stands in Portugal. For. Path. 35: 145-162. 

Navarro, R.M.; Fernández, P.; Trapero, A.; Caetano, P.; Romero, M.A.; Sánchez, M.E.; 

Fernández, A.; Sánchez, I., López, G. 2004. Los procesos de decaimiento de encinas y 

alcornoques. Dirección Gral. de Gestión del Medio Natural. Consejería de Medio Ambiente, 

Junta de Andalucía, Sevilla. 

Rohlf, F.J. 1998. NTSYSpc. Numerical taxonomy and multivariate analysis system. Version 

2. User guide. Applied Biostatistics, Inc., Setauket, NY 

Sánchez, M.E.; Muñoz, M.; Brasier, C.M.; Trapero, A. 2001. Identity and pathogenicity 

of two Phytophthora taxa associated with a new root disease of olive trees. Plant Dis. 85: 411-

416. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

269 

Sánchez, M.E.; Caetano, P.; Ferraz, J.; Trapero, A. 2002. Phytophthora disease of 

Quercus ilex in southwestern Spain. For. Path. 32: 5-18. 

Sánchez, M.E.; Sánchez, J.E.; Navarro, R.M.; Fernández, P.; Trapero, A. 2003b. 

Incidencia de la podredumbre radical causada por Phytophthora cinnamomi en masas de 

Quercus en Andalucía. Bol. San. Veg. Plagas 29: 87-108 

Sánchez, M.E.; Venegas, J.; Romero, M.A.; Phillips, A.J.L.; Trapero, A. 2003b. 

Botryosphaeria and related taxa causing oak canker in southwestern Spain. Plant Dis. 87: 

1515-1521. 

Sánchez, M.E.; Andicoberry, S.; Trapero, A. 2005. Pathogenicity of three Phytophthora

spp. causing late seedling rot of Quercus ilex ssp. ballota. For. Path. 35: 115-125. 

Sánchez, M.E.; Caetano, P.; Romero, M.A.; Navarro, R.M.; Trapero, A. 2006. 

Phytophthora root rot as the main factor of oak decline in southern Spain. IUFRO Forest 

Phytophthora Research Workshop. Proceedings of the Third International Meeting on 

Phytophthora in Forests and Natural Ecosystems. Freising, Germany. 

Vekemans X. 2001 AFLPsurv V.1.0 a software for genetic diversity analysis with AFLP 

population data, distributed by the author, xvekema@ualb.ac.be, Université Libre de 

Bruxelles. 

Vos, P.; Hogers, R.; Bleeker, M.; Reijans, M.; van de Lee, T.; Hornes, M.; Frijters, A.; 

Pot, J.; Peleman, J.; Kuiper, M.;Zabeau, M. 1995: AFLP: a new technique for DNA 

fingerprinting. Nucleic Acids Res. 23: 4407-4414. 
GENERAL TECHNICAL REPORT PSW-GTR-221

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Genetic Transformation of Phytophthora 

ramorum With the Jellyfish GFP Gene1

Gautier Calmin,

2

 Marko Riedel,3 Lassaad Belbahri,2 Stefan 

Wagner,3

 Sabine Werres,3 and François Lefort2 

Abstract 

The important quarantine organism Phytophthora ramorum has been dramatically 

increasing its host range in the past years and most of the studies concerning P. 

ramorum focus on these issues. Very little is known about the latency period. For 

sampling and analyzing potentially infected plant material, detailed information on the 

latency of the disease is of high importance. In order to provide such a tool for the 

infection process follow-up, we established a reliable method for the stable genetic 

transformation of P. ramorum isolates. The transferred genes were the marker gene 

nptII for resistance to geniticin and the target gene GFP. The first and most important 

step in this protocol was to develop a stable system to produce protoplasts from P. 

ramorum tissue. The next step was the transformation of protoplasts with plasmids 

containing marker gene and target gene, following an improved polyethylene glycol 

(PEG)-mediated protocol of protoplasts transformation. After transformation, 

protoplasts were cultivated on a selective medium and allowed to regenerate 

mycelium. The selected transformants were checked for integration and expression of 

transferred genes by PCR amplification and the use of anti-GFP antibodies. 

About forty-three different transformed isolates were produced and then tested for GFP 

fluorescence, GFP expression and GFP gene integration. They were further tested for 

their infection potential on Rhododendron plants. 

Introduction 

The oomycete Phytophthora ramorum Werres, De Cock & Man in’t Veld sp. nov. is 

a ubiquitous plant pathogen with a wide host range. One of the major diseases caused 

by P. ramorum is sudden oak death (SOD), a serious threat to native American oaks 

and European relatives where it has been discovered in numerous ornamental plant 

nurseries. 

We are interested in the molecular, cellular and physiological processes underlying 

infection pathways and tissue colonization. Using visible genetic markers has been 

proved to be an useful tool for the analysis of plant-pathogen interactions. 

Here, we analyse the suitability of the Green Fluorescent Protein (GFP) as a reporter 

gene in P.ramorum. A CaCl2 and polyethylene-glycol-based DNA transformation 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Laboratory of Applied Genetics and Plant Health, Institute Earth Nature and Landscape, University of Applied 

Sciences of Western Switzerland, 150 Route de Presinge 1254 Jussy, Switzerland. 

3 Institute for Plant Protection in Horticulture, Federal Biological Research Centre for Agriculture and Forestry 

(BBA), Messeweg 11/12, D-38104 Braunschweig, Germany. 

Corresponding author: gautier.calmin@hesge.ch. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

271 

protocol was developed and transformants expressing GFP were evaluated for 

usefulness in future studies. 

Material and Methods 

Transformation 

The transformation vector used in the present study was p34GFN (Si-Ammour and 

others 2003) which contained the selectable marker nptII and the visible marker gfp 

expressed under the control of the ham34 promoter and terminator sequences. Four 

isolates of P. ramorum (BBA 9/95, BBA 26/02, BBA MSOD03-002 and PR01) were 

used as recipients for transformation according to the protocol of Judelson and others 

(1991) with some modifications. 

Immunocytochemistry 

The GFP expression was verified by immunocytochemical detection with Alexa 

Flour 488-labeled anti-GFP antibodies (Molecular Probes). 

Microscopical analysis 

For microscopical observation the Leica TCS SP2 confocal laser scanning 

microscope (CLSM) was used. GFP and anti-GFP antibody excitation was 

done with a 488 nm Argon Krypton laser at power levels from 10 to 50 

percent. GFP expression of transgenic P. ramorum structures was measured 

by generating emission spectra profiles (lambda scan) from 500 to 580nm. 

Images and lambda scans were analysed with the Leica LCS software. 

Results 

About forty-three different transformed isolates were produced and then tested for GFP gene 

integration and GFP expression. Integration of GFP genes was approved with Real Time PCR 

and sequencing. GFP expression could be observed in all P. ramorum propagules obtainable 

from in vitro cultures like: germinating cysts, hyphae, zoosporangia and chlamydospores (Fig. 

1). 
GENERAL TECHNICAL REPORT PSW-GTR-221

272 

10 μm 10 μm

16 μm 16 μm

16 μm 16 μm 16 μm 16 μm

20 μm 20 μm 16 μm

A B

C D

E F

Figure 1-CLSM micrographs indicating GFP expression of transgenic P. ramorum

strains BBA26/02_4 (A and C) and BBA9/95_6G (B, D, E and F). A: germinating 

cyst, B: hyphae grown in vitro, C, D and E: zoosporangia releasing zoospores and 

germinating respectively, F: chlamydospores grown in vitro. Each left image:

transmission images. Each right image: CLSM signals with green signal indicating 

GFP expression in protoplasm and yellow signals showing natural P. ramorum 

autofluorescence in wall and papilla of zoosporangia and wall of chlamydospores. 

Acknowledgements: 

We would like to thank USDA-Forest Service, Pacific Southwest Research Station 

for funding the project. 

Literature Cited 

Judelson, H.S.; Tyler, B.M. and Michelmore, R.W. 1991. Transformation of the Oomycete 

Pathogen Phytophthora infestans. Molecular Plant-Microbe Interactions. 4: 602-607. 

Si-Ammou, A.; Mauch-Mani, B.; Mauch, F. 2003. Quantification of induced resistance 

against Phytophthora species expressing GFP as a vital marker: beta-aminobutyric acid but 

not BTH protects potato and Arabidopsis from infection. Molecular Plant Pathology. 4: 237-

248. 

Posters 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

273 

Phytophthora pseudosyringae Found 

on European Beech and Hornbeam 

Trees in the United Kingdom1

S. Denman,2 J. Rose,2 and B. Slippers3 

There is major concern about the spread and impact of Phytophthora ramorum and P. 

kernoviae on trees and plants in the U.K. Annual national surveys monitoring their presence 

on Rhododendron ponticum are carried out but intensive spot surveys are also done on sites of 

particular concern. At two such sites in Wales further inspections identified a few trees with 

bleeding cankers potentially caused by P. ramorum. Three very mature trees (>150 yr-old) 

were affected: two European beech (Fagus sylvatica) and a hornbeam (Carpinus betulus), all 

were in close proximity to rhododendrons infected with P. ramorum. 

Two bark panels were excised from each tree and isolations made from the dead live junctions 

of necrotic inner bark tissue onto a Phytophthora selective medium (SMA). Tissue from the 

hornbeam was also washed several times before Phytophthora could be isolated. Colonies did 

not show typical morphological characteristics of P. ramorum, so they were sub-cultured on 

fresh CA and sexual structures were examined after 10 d. Plugs taken from the margins of 

actively growing colonies were also placed in pond water and incubated at 20°C on the 

laboratory bench to stimulate sporangial formation. 

Detailed examination of the cultures and sporangia revealed a homothallic Phytophthora

species with semi-papillate, caducous sporangia and catenulate hyphal swellings formed in 

liquid culture. The morphological features correspond to those described for P. 

pseudosyringae. Sequences of the ITS1 and ITS2 rDNA matched those of P. pseudosyringae

in GenBank (>99 percent). 

Pathogenicity tests conducted on logs 1.2m long, ca. 40cm diam. of beech, included a 

reference isolate of P. pseudosyringae obtained from infected oak trees in Bavaria. P. 

pseudosyringae caused lesions on the beech logs, but isolates originally obtained from beech 

caused significantly larger lesions (1520 cm2

) than isolates from either hornbeam or oak (2 

cm2

). All isolates used in inoculations were re-isolated. 

This is the first time that P. pseudosyringae has been reported from the U.K. and also the first 

record of this pathogen on hornbeam. The infected trees at both sites were very close to 

pathways running through the woodlands with a source of water close by. In Italy, this 

pathogen has also been recorded from mature beech with bleeding cankers, situated close to 

pathways. This study raises questions about host specificity and aggressiveness of isolates of 

P. pseudosyringae and the possibility of anthropogenic introduction to the U.K. through 

recreational activities. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2 Forest Research, Alice Holt Lodge, Farnham, Surrey, GU10 4LH, U.K. 

3 Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, 

South Africa. 

Corresponding author: sandra.denman@forestry.gsi.gov.uk. 
GENERAL TECHNICAL REPORT PSW-GTR-221

274 

Introduction 

There is major concern about the spread and impact of Phytophthora ramorum and P. 

kernoviae on trees and plants in the U.K. With Rhododendron ponticum being the 

main foliar host on which inoculum is produced in Britain, annual national surveys 

monitoring the presence of these two pathogens on wild rhododendron are carried out 

but intensive spot surveys are also done on sites of particular concern. At two such 

sites in Wales further inspections identified two very mature European beech (Fagus 

sylvatica) and a hornbeam (Carpinus betulus) with bleeding cankers potentially 

caused by P. ramorum. These trees were investigated to determine the cause of the 

bleeding lesions. 

Symptoms and tree characteristics 

On the beech trees bleeding lesions were present on 

root flares extending up the trunk to between 1.5m 

and 2.5m respectively. On one of the two trees the 

lesions extended from the soil-surface interface 

upwards. The bleeds were a purplish-brown colour 

and towards the end of the growing season when the 

bleeding had slowed down, dark blobs of ooze 

congealed on some of the necrotic lesions (fig. 1). 

Underneath the outer bark the inner bark was stained 

a pale smoked-salmon orange colour. Numerous 

additional stresses including damage to bark (graffiti 

engraving, lightning, mammal damage), loss of 

limbs and colonisation by wood rot fungi and 

Armillaria further compromised tree health. The 

canopy of one of the beech trees was very thin, the 

other was less affected. 

The hornbeam had similar situation to the beech 

trees. It was also a very mature tree, coppiced with 

six stems. Bleeds were present on most stems but 

extended up to 6m. Overall health of the tree was 

also compromised evidenced by a broken out limb, 

decay fungi present, and Armillaria on the roots. 

The crown however, was in good condition. 

All trees were mature with ages estimated between 150−180 years-old. 

Environment 

Both sites are managed woodland-parkland areas that have networks of foot and 

cycle paths lacing the area and are highly visited by the public for recreational 

purposes. All the trees were close to water sources (10-15 m) and pathways (2−3 m) 

about midway up a slope. Both sites were at a low elevation (70-75 m above sea 

level). At the site with the infected beech the soil was brown earth but was stony 

with a pH of 6.5. The two infected trees were about 20 m apart and the setting was 

similar to parkland with an open undercanopy and rhododendron along the 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

275 

watercourse. The site with the hornbeam by comparison, was more akin to woodland 

having brown earth that had a gleyed layer and a pH of 6.0. The hornbeam site was 

generally wetter than the beech site. 

Methods 

Isolation and identification 

Panels of diseased bark (3x5 cm2) were excised from the trees over a dead-live 

junction, so that necrotic inner bark and a fresh margin of active lesion were 

obtained. Samples were washed under running tap water for 1 to 24 hours and pieces 

of tissue from the dead-live margins of the inner bark plated on Phytophthora

selective medium (SMA) (Brasier and Jung 2001). Isolates that developed on SMA 

were sub-cultured on carrot agar (CA) (Brasier 1969) for morphological studies. 

Pieces of the colonies were cut from the actively growing margins, placed in to pond 

water and exposed to natural light for 48 h (Jung and others 2003) to induce 

sporangia formation; oospores were formed on CA after 10 days. Morphological 

features were examined using the microscope (x40 magnification). Measurements of 

10 sporangia, antheridia and oogonia per isolate were made and compared with those 

described by Jung and others (2003). Daily radial growth rate on CA at 20oC were 

also compared with the description given in Jung and others (2003). Molecular 

diagnosis was used to confirm identification. DNA was extracted using and the ITS 

regions amplified using primers ITS1 and ITS4 (White and others 1990). Sequences 

were compared with those in GenBank using BLAST. 

Inoculations 

Log inoculation tests were carried out using 1 cm diameter mycelial plugs on 

wounded beech logs (ca. 20 cm diam.) according to the method described by Brasier 

and Jung (2001). Four isolates of P. pseudosyringae were tested on beech and two 

control Phytophthora species - P. cambivora as an aggressive positive control, and P. 

alni as a non-pathogenic negative control were included (Table 1). 
GENERAL TECHNICAL REPORT PSW-GTR-221

276 

Table 1—Phytophthora species and isolates used in pathogenicity tests and 

details of original host and country isolates were obtained from 

Species Isolate number Host of origin Country of origin 

P. alni subsp alni P772 Alnus glutinosa England, U.K. 

P. cambivora P1832 Fagus sylvatica England, U.K. 

P. pseudosyringae PSEU 6* Quercus robur Germany 

P. pseudosyringae TLB 14 Fagus sylvatica Wales, U.K. 

P. pseudosyringae TLB 13 Fagus sylvatica Wales, U.K. 

P. pseudosyringae TLB 105 Carpinus betula Wales, U.K. 

* Isolate kindly supplied by T. Jung.

Inoculated logs were incubated at 20°C for 5 wk (Brasier and Jung 2001). Paring 

away the outer bark exposed lesions (fig. 2) which were traced, the tracings cut out 

and weighed and disease area determined (Brasier and Jung 2001). 

Results 

Cultural and morphological features were in close agreement with those described for 

P. pseudosyringae (Jung and others 2003) (fig. 3) and the ITS BLAST searches 

showed >99 percent homology to sequences of P. pseudosyringae, confirming the 

morphological identification. 

Figure 2—Exposed, inner bark lesions after 5 wk on wound inoculated European beech, 

caused by P. cambivora (left) and P. pseudosyringae (TLB 14A) (right).
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

277 

Figure 3—Morphological features of U.K. isolates of P. pseudosyringae – (a) Colony 

morphology similar to German isolate: (top, left to right) PSEU 6; TLB 105C; TLB 105D 

(bottom, left to right) TLB 13A; MAR 13A; (b) Sporangium: Attached (although some 

caducous in colony), terminal, semi-papillate; (c) Oogonium with attached single celled, 

paragynous antheridium, and golden oospore; (d) Catenulate hypal swellings. 

The inoculation studies confirmed that P. pseudosyringae was pathogenic on beech 

but there was much variation in aggressiveness amongst the isolates. One isolate was 

highly aggressive, one moderately so and two were benign (fig. 4), the more 

aggressive two originating from beech while the two benign isolates originated from 

other hosts (oak and hornbeam, fig. 4; Table 1). 

P. cambivora was the most aggressive Phytophthora species tested while P. alni

subsp. alni was non-pathogenic. 

A 

B 

C 

D 
GENERAL TECHNICAL REPORT PSW-GTR-221

278 

Discussion 

This is a first record of P. pseudosyringae in the U.K. and hornbeam is a new 

host for this pathogen. Although our isolates closely matched previous 

descriptions (Jung and others 2003), since P. pseudosyringae is a recently 

described species and very similar morphologically to P. syringae, it is 

possible that it has been misidentified previously in the U.K. (Jung and others 

2003). Koch’s postulates need to be carried out on hornbeam to confirm 

pathogenicity on this host. 

Initial pathogenicity tests on beech indicate that P. pseudosyringae is much 

less aggressive than P. cambivora corroborating previous work (Brasier and 

Kirk 2001, Fleischmann and others 2004, Jung and others 2003), but the large 

range in aggressiveness of isolates suggests that there may be a host 

specificity isolate aggressiveness relationship which has also been mentioned 

by others (Brasier and Kirk 2001, Jung and others 2003, Martin and Tooley 

2003). Cross-pathogenicity tests need to confirm this hypothesis. Fleishmann 

and others (2004) show that P. pseudosyringae has a different pathogenic 

effect on beech seedlings than some of the more aggressive Phytophthora

species such as P. cambivora or P. citricola.

Much remains unknown about the ecological role and behaviour of P. 

pseudosyringae. In Europe it is regarded as a pathogen causing fine root rot 

and bleeding stem cankers of native tree species. However, in the USA it is 

isolated from necrotic leaves and twig cankers in tree canopies specifically 

Figure 4—Relative aggressiveness of Phytophthora pseudopsyringae to inner bark of Fagus 

sylvatica using wound inoculation tests. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

279 

bay laurel (Umbellularia californica), with only a few records of it being 

associated with oak bleeding stem cankers (Murphy and Rizzo 2005, 

Wickland and Rizzo 2005). Another interesting ecological aspect of P. 

pseudosyringae is that other Phytophthora species are frequently isolated with 

it whether from infected roots and stems (Diana and others 2006, Fleishmann 

and others 2004, Jung and others 2003, and also in this study) or from leaves 

where it is reported to co-exist with other Phytophthora species in tree 

canopies (Murphy and Rizzo 2005, Wickland and Rizzo 2005). 

The site details reveal that the diseased trees in Wales were close to pathways 

and a source of water. First reports of this pathogen in Italy also emphasise 

this observation (Diana and others 2006). This could suggest disease 

development either through introduction of a foreign pathogen connected with 

human recreational activity; or to stress imposed on the tree by compaction 

and altered drainage caused by the pathways as well as activity around its 

roots thus altering their susceptibility to this particular Phytophthora. 

Acknowledgement 

We thank Dai Evans, FC Technical Support Unit, Talybont, Wales for field 

assistance. 

Literature Cited 

Brasier, C.M. 1969. The effect of light and temperature on reproduction in vitro in two 

tropical species of Phytophthora. Transactions of the British Mycological Society 52, 105–

13. 

Brasier, C.M.; Kirk, S.A. 2001. Comparative aggressiveness of standard and variant hybrid 

alder phytophthoras, Phytophthora cambivora and other Phytophthora species on bark of 

Alnus, Quercus and other woody hosts. Plant Pathology 50, 218–229. 

Brasier, C.M.; Jung, T. 2001. Progress in understanding Phytophthora diseases of trees in 

Europe. ‘Phytophthora in Forests and Natural Ecosystems’. 2nd International IUFRO 

Working Party 7.02.09 Meeting, Albany, W. Australia 30th Sept – 5 Oct 2001. Eds. McComb 

JA, Hardy GE StJ and Tommerup IC (Murdoch University Print) pp 4–18. 

Diana, G.; Pane, A.; Raudion, F.; Cooke, D.E.L.; Cacciola, S.O.; Magnano di San Lio, 

G. 2006. A decline of beech trees caused by Phytophthora pseudosyringae in central Italy. 

Progress in Research on Phytophthora Diseases of Forest Trees, Chapter 33, 142–144. 

Fleischmann, F.; Göttlein, A.; Rodenkirchen, H.; Lütz, C.; Oßwald, W. 2004. Biomass, 

nutrient and pigment content of beech (Fagus sylvatica) saplings infected with Phytophthora 

citricola, P. cambivora, P. pseudosyringae and P. undulata. Forest Pathology 34, 79–92. 

Jung, T.; Nechwatal, J.; Cooke, D.E.L.; Hartmann, G.; Blaschke, M.; Oßwald, W.F.; 

Duncan, J.M.; Delatour, C. 2003. Phytophthora pseudosyringae sp. nov., a new species 

causing root and collar rot of deciduous tree species in Europe. Mycological Research 107, 

772–789. 
GENERAL TECHNICAL REPORT PSW-GTR-221

280 

Linze, R.; Garbelotto, M. 2005. AFLP Analysis of Phytophthora nemorosa and P. 

pseudosyringae Genetic Structure in North America. In: Frankel, S.J.; Shea, P.J.; Haverty, 

M.I., tech. coords. Proceedings, sudden oak death second science symposium: the state of our 

knowledge. 2005 January 18-21; Monterey, CA. Gen. Tech. Rep. PSW-GTR-196. Alabany, 

CA; U.S. Department of Agriculture, Forest Service, Pacific Southwest Research Station: 

January 18-21, 2005. 

Martin, F.N.; Tooley, P.W. 2003. Phylogenetic relationships of Phytophthora ramorum, P. 

nemorosa, and P. pseudosyringae, three species recovered from areas in California with 

sudden oak death. Mycological Research 107 (12) 1379–1391. 

Murphy, S.; Rizzo, D.M. 2005. Incidence of Phytophthora ramorum, P. nemorosa and 

Phytophthora pseudosyringae in Three Coastal Californian Forest Communities. In: Frankel, 

S.J.; Shea, P.J.; Haverty, M.I., tech. coords. Proceedings, sudden oak death second science 

symposium: the state of our knowledge. 2005 January 18-21; Monterey, CA. Gen. Tech. Rep. 

PSW-GTR-196. Alabany, CA; U.S. Department of Agriculture, Forest Service, Pacific 

Southwest Research Station: January 18-21, 2005. 

White, T.J., and others. 1990 Amplification and direct sequencing of fungal ribosomal RNA 

genes for phylogenetics. In PCR Protocols: a guide to methods and applications (M.A. Innis, 

D.H. Gelfand, J.J. Sninsky and T.J. White, eds): 315-322. Academic Press, Inc., New York. 

Wickland, A.C.; Rizzo, D.M. 2005. Ecology of Phytophthora nemorosa and Phytophthora 

pseudosyringae in Mixed Evergreen Forests. In: Frankel, S.J.; Shea, P.J.; Haverty, M.I., tech. 

coords. Proceedings, sudden oak death second science symposium: the state of our 

knowledge. 2005 January 18-21; Monterey, CA. Gen. Tech. Rep. PSW-GTR-196. Alabany, 

CA; U.S. Department of Agriculture, Forest Service, Pacific Southwest Research Station: 

January 18-21, 2005.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

281 

Root Associations of Phytophthora 

ramorum and Phytophthora 

kernoviae in U.K. Woodlands1

Elizabeth J. Fichtner,2 David M. Rizzo,2 Susan A. Kirk,3 

Alistair Whybrow,3 and Joan Webber3

Abstract 

Phytophthora kernoviae and Phytophthora ramorum, two pathogens recently introduced to 

the U.K., incite foliar lesions, shoot necrosis, and death of Rhododendron ponticum, an 

invasive weed pervading U.K. woodlands. In infested woodlands, R. ponticum serves as an 

epidemiologically important host, supporting sporulation of both pathogens. Bleeding cankers 

on trunks of European beech (Fagus sylvatica) caused by either P. ramorum or P. kernoviae 

are often associated with neighboring infected R. ponticum. 

Rhododendron ponticum has been removed from several woodlands as an inoculum 

management strategy, but the long-term efficacy of plant removal is unknown, in part due to 

lack of knowledge of pathogen persistence in roots and in emerging seedlings. 

The potential for P. ramorum and P. kernoviae to infect roots of R. ponticum in U.K. 

woodlands is unknown. To assess pathogen association with rhododendron roots, roots 

initiated from natural layering were excavated from two sites infested with P. kernoviae and 

one site infested with P. ramorum. At each site, four sets of layered roots were sampled, in 

addition to the associated leaf litter, rhizosphere soil, and symptomatic leaves. In the 

laboratory, soil and leaf litter were individually baited using leaf disks of Rhododendron 

catawbiense ‘Cunninghams White.’ Tissue from symptomatic leaves was embedded in SMA 

agar for isolation of Phytophthora spp. Neither pathogen was baited from rhizosphere soil, but 

both were routinely recovered from leaf litter. Phytophthora ramorum was baited from one 

set of layered roots; P. kernoviae was baited from three sets of roots at one site and from two 

sets at another site. 

A second objective focused on investigating the potential for infection of R. ponticum 

seedlings in a woodland cleared of R. ponticum in 2005 for management of P. kernoviae. 

Nineteen seedlings were excavated from the woodland and all foliar lesions were sampled for 

pathogen isolation. Rhizosphere soil and roots were independently baited with rhododendron 

leaf disks. Phytophthora kernoviae was recovered from foliar tissue on 2 seedlings, from 

roots of 5 seedlings, and from two samples of rhizosphere soil. 

The results suggest that both P. ramorum and P. kernoviae are associated with R. ponticum 

roots in infested U.K. woodlands. Furthermore, the presence of inoculum in litter but rarely 

in soil suggests that the pathogens may infect the roots, rather than simply persist on the 

rhizoplane. Further research is needed to assess the frequency of root associations and to 

histologically visualize root infections of thesetwo pathogens. These preliminary data suggest 

that the potential persistence of these pathogens in roots and litter should be considered when 

managing the diseases in infested woodlands. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2 Department of Plant Pathology, Univ. of California, One Shields Ave., Davis, CA 95616, USA. 3 Forest Research, Alice Holt Lodge, Farhnam, Surrey, GU10 4LH, U.K. 

Corresponding author: ejfichtner@ucdavis.edu.
GENERAL TECHNICAL REPORT PSW-GTR-221

282 

“Mal del Ciprés” Disease: Analysis 

 of the Association Between Aerial 

Symptoms and Vitality of Trees1

María Pía Floria2 and Alina G. Greslebin3

Abstract 

Austrocedrus chilensis is an indigenous Cupressaceae of the Patagonian Andes forests in 

southern Argentina and Chile that suffers a disease called “mal del ciprés”. Phytophthora 

austrocedrae, a new Phytophthora species, was recently shown to be the main cause of the 

disease. The progressive withering and subsequent defoliation of the tree, which finally dies, 

has been traditionally considered the main symptom. Other symptoms are basal resinous 

exudates and red-brown necrotic lesions in the inner bark extending up the bole from killed 

roots. Brown cubic rots in roots and sapwood are many times associated with dead or dying 

trees. 

Although the symptomatology of the disease has been described as a slow process of 

defoliation that culminates with the death of the tree, dying and recently dead trees with 

abundant foliage are frequently observed. This shows that sudden death with little or no 

previous defoliation also happens. On the other hand, trees with crowns looking almost 

healthy but with many big lesions at the root collar, and defoliated trees with few or no 

lesions at the root collar have been observed several times. 

Traditionally, it was thought that crown transparency is a suitable measure of the vitality of a 

tree. Nevertheless, the contradictory observations previously mentioned make such a 

supposition doubtful and make it difficult to assess the extent of disease in a tree or a stand. 

Since an accurate method for evaluating the vitality of affected trees is essential for 

establishing management and conservation strategies in affected forests, an evaluation of 

symptomatology and its association with tree vitality was done. The aims of this work were 

to analyze the association between aerial symptoms and vitality and to adjust the method used 

for determining vitality of trees affected by “mal del ciprés”. 

Sixty trees, in three different vitality classes, of a stand affected by “mal del ciprés” were 

evaluated. Percentage of defoliation was estimated visually, with an accuracy of 5 percent, 

using two different methods. The pattern of defoliation, the presence of yellow or red leaves 

in the crown, cankers and resinous exudates in the stem, and other features that could be 

related to vitality were also recorded. Bark was removed to expose inner bark and the vitality 

at the collar root was determined as the percentage of the perimeter with dead tissues. Then 

the root system was excavated to expose roots in a perimeter of 1m from the base of the tree. 

Bark of main roots was removed and vitality of each root was evaluated as percentage of 

tissues dead/affected. Vitality of root system was estimated as the total percentage of affected 

tissues of main roots. The association between aerial symptoms and tissue vitality was 

evaluated through a correlation analysis between each of the three variables (defoliation, 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2 Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia, Ruta 259 Km 4, 9200, Esquel, Chubut, 

Argentina.

3 Área de Protección Forestal, Centro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP), CC 14, 

9200, Esquel, Chubut, Argentina. 

Corresponding author: agreslebin@ciefap.org.ar.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

283 

vitality at root collar and vitality of roots). The association between root collar and root 

vitality and the other recorded symptoms was also evaluated through a correlation analysis. 

This work presents the results of the study and a discussion on the reliability of aerial 

symptoms for estimating vitality of trees affected by the “mal del ciprés”. 
GENERAL TECHNICAL REPORT PSW-GTR-221

284 

 The Phytophthora Species 

 Known as “Pg chlamydo”1

Everett Hansen,2 Paul Reeser,2 and Wendy Sutton2

Abstract 

Phytophthora taxon Pg chlamydo is perhaps the second most abundant Phytophthora species 

in the world, after P. gonapodyides, although it is commonly misidentified. Pg chlamydo is 

frequently encountered in streams and rivers in western North America, Argentina, China, and 

Europe. It has occasionally been recovered from forest soil and was once isolated from a bole 

canker on a tanoak tree; it was pathogenic to tanoak in artificial inoculation. 

Pg chlamydo resembles P. gonapodyides in culture, and is related to that species. It is 

apparently sterile, not itself forming sexual structures even when paired with tester isolates of 

known mating type. Sporangia are non-descript nonpapillate, similar to other species in the P. 

megasperma/P. gonapodyides ITS clade. It is distinguished from P. gonapodyides by the 

formation of chlamydospores in culture. It has also been misidentified as P. lateralis, P. 

drechsleri or P. cryptogea, although the latter species are heterothallic, and readily 

distinguished with a mating test. 

ITS-DNA sequences of isolates from Oregon, California, Argentina, and France were 

identical, but at least three mitochondrial genotypes were distinguished. 

Introduction 

Phytophthora taxon “Pg chlamydo” is perhaps the second most abundant 

Phytophthora species in the world, after P. gonapodyides, although it is commonly 

misidentified. It resembles P. gonapodyides in culture, but is distinguished by the 

formation of chlamydospores and chains of hyphal swellings. Like P. gonapodyides, 

it is a sterile species, but has also been misidentified as P. lateralis, P. drechsleri or 

P. cryptogea. “Pg chlamydo” is frequently encountered in streams and rivers in 

western North America, Argentina, and Europe. It has occasionally been recovered 

from forest soil and was once isolated from a bole canker on a tanoak tree; it is 

occasionally encountered as a foliar pathogen of woody plants in ornamental 

nurseries (Blomquist unpublished). It was associated with root rot of Port-Orfordcedar in German nurseries, where it was initially misidentified as P. lateralis (Hansen 

and others 1999), and with root rot and stem cankers of Abies species in nurseries and 

Christmas tree plantations, where it was misidentified as P. drechsleri (Brasier and 

others 1993). 

Objectives 

1. To raise general awareness of this common but poorly understood Phytophthora. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2

 Department of Botany and Plant Pathology, Oregon State University, Corvallis OR 97331, USA. 

Corresponding author: hansene@science.oregonstste.edu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

285 

2. To explore the population genetics of this sexually sterile and very widespread 

species. 

Methods 

Isolates recovered from water, soil and plant tissues originating from Europe (France 

and Germany), Argentina, and western United States (California and Oregon) were 

compared. All isolates from Argentina were baited from different locations on a 

single stream near Corcovado. Water isolates from western USA were all from 

different streams in SW Oregon and northern California. 

Isolates were grown up on CMA and DNA was extracted from colonized agar plugs 

using a CTAB buffer protocol. DNA was PCR amplified with each of the five primer 

sets listed below (Table 1) and sequenced. These sequences were then edited and 

aligned. 

Table 1—DNA regions analyzed. 

Gene or DNA 

Region Primers Length Amplified Citation 

ITS rDNA ITS 4 and ITS5 ~820bp White,T.J,.and others 1990 

Mitochondrial 

COX spacer FMPH8 and FMPH10 ~340bp Martin,F.N. and Tooley,P.W. 2003

60S ribosomal 

protein L10 60SL10F and 60SL10R ~420bp http://www.phytophthoradb.org/ 

B tubuln BTubF1 and BTubR1 ~990bp Kroon, L.P.N.M., and others 2004 

Elongation 

Factor EF1AF and EF1AR ~865bp Kroon, L.P.N.M., and others 2004 

Morphology 

Morphology and growth were similar for all isolates examined regardless of source. 

 A B C D 

Figure 1—Sporangia of “Pg chlamydo” are non-papillate and proliferate both internally 

and externally (A and B). Chlamydospores (C) and hyphal swellings (D) characteristic 

of “Pg chlamydo”. Photos C. Delatour 
GENERAL TECHNICAL REPORT PSW-GTR-221

286 

Population Structure 

Although isolates identified as “Pg chlamydo” are monophyletic within ITS clade 6, 

they exhibit remarkable intra-specific molecular variation in all genetic regions 

examined. “Double peaks,” commonly interpreted as evidence for past hybridization 

events, are unusually common in the DNA sequences. Only the mitochondrial COX 

spacer region lacked double peaks. 

All isolates shared a basic ITS DNA sequence, but four ITS groups were 

distinguished based on the presence or absence of double peaks at four loci. 

The mitochondrial COX spacer DNA region also had four genotypes among the 24 

isolates fully characterized, although two genotypes were represented by single 

isolates. The 60S L 10 gene (RL10) encodes a ribosomal protein. Most isolates 

exhibited double peaks at 4-5 loci. Four related genotypes were identified among the 

isolates. 

Elongation factor and β-tubulin gene sequences have not yet been not fully aligned, 

but many isolates exhibit double peaks at multiple loci. 

Multilocus DNA Phenotype 

With four genotypes recognized within each of the three DNA sequences fully 

analyzed, a total of 64 multi-locus genotypes are possible. Only five of these were 

identified in the 24 isolates fully characterized (Table 2). 

Five isolates formed multi-locus Group A. There were no double peaks scored for 

Group A in any of the three DNA regions. These isolates were from streams and soil 

in Oregon, and water in France. 

All of the isolates from Argentina were identical, and formed multi-locus Group B, 

distinguished by double peaks at four RL 10 loci, and a single ITS locus. 

Multi-locus Group E had a distinctive COX spacer sequence and unique patterns of 

double peaks in the RL10 and ITS sequences. The ITS variation within Group E 

resulted from differences in relative peak strengths at the same double peak loci. 

The remaining multi-locus Groups, Group C and Group D, were represented by 

single isolates. 

Conclusions 

Phytophthora isolates identified as P.sp.”Pg chlamydo” from Europe, North America, 

and Argentina are indistinguishable morphologically and closely related 

phylogenetically. 

Isolates could be grouped in three main multi-locus DNA clades. Isolates from 

Oregon, California, and Europe were mixed in two clades, and all isolates from 

Argentina formed the third main clade. 

Isolates in clade A (Oregon and France) exhibited no double peaks in the sequences 

analyzed. The other clades were distinguished by patterns of multiple double peaks. 

P.sp.”Pg chlamydo” evidently has a complex evolutionary history. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

287 

Table 2—Groupings according to DNA alignments of three DNA regions. 

Isolate Source Substrate Collector FMPH GROUP 60SL10 GROUP ITS GROUP MULTI-LOCUS GROUP

9364 Oregon Tanoak canker Hansen lab I c 2 A

33-2-2-0603 Oregon Soil Hansen lab I c 2 A

Haye3b Europe Water Hansen & Delatour I c 2 A

WA42.2-062204 Oregon Water Hansen lab I c 2 A

WA49-031907 Oregon Water Hansen lab I c 2 A

AG27 Argentina Water Greslebin I d 4 B

AG29 Argentina Water Greslebin I d 4 B

AG30 Argentina Water Greslebin I d 4 B

AG48 Argentina Water Greslebin I d 4 B

AG50 Argentina Water Greslebin I d 4 B

WCK2.7.21 California Water Rizzo lab II e 1 C

WA46.3-100404 Oregon Water Hansen lab III d 2 D

107 Europe POC soil Hansen IV a 3.1 E1

62689 Europe POC soil Hansen IV a 3.1 E1

Haye3.1 Europe Water Hansen & Delatour IV a 3.1 E1

RIZZO-BUL 1-2.7.8 California Water Rizzo lab IV a 3.1 E1

WA20.1-041904 Oregon Water Hansen lab IV a 3.1 E1

WA38.1-083104 Oregon Water Hansen lab IV a 3.1 E1

WA39-100404 Oregon Water Hansen lab IV a 3.1 E1

WA6-011304 Oregon Water Hansen lab IV a 3.1 E1

H1 Europe Water Hansen & Delatour IV a 3.2 E2

WA48.2-070404 Oregon Water Hansen lab IV a 3.2 E2

RIZZO-RCK1.9.2 California Water Rizzo lab IV a 3.3 E3

WA5.1-072003 Oregon Water Hansen lab IV a 3.3 E3

Literature Cited 

Hansen, E.M.; Streito, J.C.; Delatour, C. 1999. First confirmation of Phytophthora lateralis

in Europe. Plant Disease 83:587. 

Brasier, C.M.,Cooke, D.E.L.; Duncan, J.M.; Hansen, E.M. 2003. Multiple new 

phenotypic taxa from trees and riparian ecosystems in Phytophthora gonapodyides-P. 

megasperma ITS Clade 6, which tend to be high-temperature tolerant and either inbreeding or 

sterile. Mycol. Res. 107(3): 277-290. 

Brasier, C.M., Hamm, P.B.; Hansen, E.M.. 1993. Cultural characteristics, protein patterns 

and unusual mating behavior of Phytophthora gonopodyides isolates from Britain and North 

America. Mycol. Res 97:1287-1298. 

Delatour, C. Atlas illustre de quelques Phytophthoras. Institut National de la Recherche 

Agronomique. 

Kroon, L.P.N.M.; Bakker F.T.; van den Bosch, G.B.M.; Bonants, P.J.M.; Flier, W.G. 

2004. Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear 

DNA sequences. Fungal Genetics and Biology 41:766-782 

Martin, F.N. and Tooley, P.W. 2003. Phylogenetic relationships among Phytophthora

species genes. Mycologia 95: 269-284. 

White, T.J.; Bruns, T.; Lee, S.; Taylor, J. 1990 Amplification and direct sequencing of 

fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: a guide to methods and 

applications (M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White, eds): 315-322. Academic 

Press, Inc., New York. 
GENERAL TECHNICAL REPORT PSW-GTR-221

288 

Virulence Behaviour and Gene Expression 

Patterns of a Phytophthora cinnamomiCinnamomin Silenced Transformant1

Marília Horta,2 Paula Caetano,2 Ana C. Coelho,3 Dina Neves,4

Nelson Sousa,2 and Alfredo Cravador2

Abstract 

Phytophthora cinnamomi is an oomycete parasite of Quercus suber roots. This pathogen 

secretes abundantly cinnamomins, proteins from the elicitin family. It was demonstrated that 

these proteins interact with several lipidic molecules, namely sterols but their biological role 

remains unknown. 

The silencing of the gene coding for β-cinnamomin was induced by genetic transformation of 

protoplasts by chemical methods (liposomes and CaCl2/PEG) with an antisense sequence of 

the β-cin gene (our recent work). Selection of transformants was achieved through cotransformation with a gene conferring resistance to hygromicin B. The presence of the 

transgenes was certified by PCR. The absence of the protein in the culture media was 

confirmed through western blotting using monoclonal antibodies anti-β-cinnamomin, and the 

absence of the coding mRNA was proven by means of real time RT-PCR quantification. The 

geneticexpression of the genes coding to the other elicitins was shown to decrease as well. 

Greenhouse pathogenicity tests were now carried out in parallel in Q. suber plants with the 

only stable cotransformant obtained (antibiotic resistant and with the β-cin gene silenced), 

with a simple transformant (antibiotic resistant) and with the wild type isolates. The results 

revealed that the β-cin silenced isolate has a decreased virulence; and that this impaired 

virulence could not be caused solely by the presence of the resistance gene. 

Genetic expression profiles obtained by cDNA-AFLP disclosed marked differences between 

the cotransformant and the wild isolate. Sequencing of some fragments differentially 

expressed showed that these are potentially associated with the expression of the β-cin gene; 

among them, a gene associated with the fatty acids metabolism is particularly important. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2 Faculdade de Engenharia dos Recursos Naturais, Universidade do Algarve Campus de Gambelas, 8005-139 FARO, 

Portugal.

3 Escola Superior de Educaçáo, Univ. do Algarve Campus da Penha, 8005-139 FARO, Portugal. 4 Instituto Transfronteiriço Universitário para a Ciência, a Cultura e o Ambiente, Universidade do Algarve Campus 

de Gambelas, 8005-139 FARO, Portugal. 

Corresponding author: mhorta@ualg.pt.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

289 

The Spatial and Temporal Dynamics of 

Phytophthora lateralis-Related Mortality on 

Chamaecyparis lawsoniana After Ten-Plus 

Years on Two Field Sites1

Scott E. Kolpak,2 Everett M. Hansen,3 Richard A. Sniezko,2 and 

Wendy Sutton3

Abstract 

Chamaecyparis lawsoniana (Port-Orford cedar, POC) is distributed throughout southwest 

Oregon and Northern California, located predominantly along river drainages. A non-native 

root pathogen, Phytophthora lateralis, was first detected in a Seattle Nursery in 1923 and is 

responsible for extensive mortality across the range of POC. The spatial structure of infection 

was examined in the Page Mountain area (Siskiyou Mountains, Oregon/California, USA) 

using dendrochronology methods and the Smith River National Recreation Area (California, 

USA) using GIS methods. There is no published data on the effects of site and micro-site 

variation on the patterns of P. lateralis infection and its relative virulence using a uniform 

population background. 

Two replicated field trials (Quosatana and Flannigan sites) were established in 1993 in the 

Siskiyou National Forest planted with seedlings from 28 open-pollinated families from a 

subset of 200 phenotypic resistant selections chosen in the late 80s. A single seedling from 28 

open-pollinated families was planted in each plot. The trees were planted in a circular plot 

consisting of an outer ring and an inner ring centered around a dead POC to provide a 

consistent and uniform inoculum hazard. A group of four to eight plots were planted within 

five meters of a neighboring plot and groups were separated by 35 to 65m. The trees were 

monitored for mortality (P. lateralis and other factors) over ten-plus years; P. lateralis 

isolation/confirmation was attempted in the first two years after planting establishment and 

again ten years later. 

There are significant differences among families in the level of mortality over 10 years, with 

family 510015 showing greater survivorship than the most susceptible families. However the 

survivorship functions are not different among families because survival curves have a similar 

shape but plateau at different mortality levels. Site by family interactions and site effects are 

not significant but the group (clusters of plots) effects are significant in the ANOVA. 

Survivorship analysis found significant difference across sites, groups, and plots. The 

Quosatana site has higher mortality (88 percent) than Flannigan site (74 percent) after 10 

years and the survivorship functions are different. The absolute level of mortality is different 

between groups and plots with plot mortality ranging between zero and 100 percent. The data 

suggests that the density P. lateralis in the early years of the planting was not uniform within 

or between sites reflected by the differences in mortality. There are some weak associations of 

group and plot mortality with the surrounding vegetation composition. Phytophthora lateralis 

was isolated from most plots in the first couple years after planting and ten years later the 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 USDA Forest Service, Dorena Genetic Resource Center, 34963 Shoreview Road, Cottage Grove, OR 97424, USA. 3

 Department of Botany and Plant Pathology, Oregon State University, 2082 Cordley Hall, Corvallis, OR, 97331, 

USA. 

Corresponding author: skolpak@fs.fed.us. 
GENERAL TECHNICAL REPORT PSW-GTR-221

290 

pathogen is detectable in less the half of the plots indicating the density of P. lateralis spores 

are significantly reduced or now absent in some areas of the plantings. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

291 

Adaptability and Susceptibility of Oaks to 

Phytophthora quercina Under Drought and 

Nitrogen Stress1

Sindy Leonhard,2 Jörg Schumacher,2 and Alfred Wulf2

Abstract 

The effects of global climate change are nowadays a frequently discussed problem. Among 

them especially temperature increase and drought stress during the growing season can 

predispose plants to diseases. Therefore a project of the Deutsche Bundesstiftung Umwelt 

(DBU) was started in 2005 to investigate the influence of changing environmental factors to 

autochthonic tree species as well as their adapted and potential pathogens. The purpose of the 

present study is to examine the effects of controlled stress factors, particularly drought and 

nitrogen stress, on the susceptibility and adaptability of two to three years old Quercus 

petraea plants to infection by Phytophthora quercina. The host-pathogen interactions of two 

different German proveniences of Q. petraea under controlled greenhouse conditions have 

been studying since 2005. First results about the differences in the response of the species to 

drought and nitrogen stress are compared and discussed. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Protection in Forests 

Braunschweig Messeweg 11/12, 38104 Braunschweig, Germany. 

Corresponding author: s.leonhard@bba.de. 
GENERAL TECHNICAL REPORT PSW-GTR-221

292 

Prevalence and Development of Disease on 

Coast Redwood Seedlings Caused by 

Phytophthora ramorum1

Sunny Lucas,2 Jennifer Parke,2 and Yana Valachovic3

Abstract 

Coast redwood (Sequoia sempervirens) is a host for Phytophthora ramorum but it is unclear if 

the pathogen represents a significant disease risk to this tree species. In an on-going field 

experiment, we are examining the prevalence of infection and the development of symptoms 

on coast redwood seedlings in naturally infested sites in southern Humboldt Co., California. 

In November 2006, healthy redwood seedlings were placed amid tanoak and bay trees 

naturally infected with P. ramorum. Disease incidence and symptom development are being 

observed monthly, and every two months, a subset of redwood seedlings is destructively 

sampled to investigate the location and extent of tissue colonization by P. ramorum. In order 

to correlate inoculum levels across field sites with disease development, rainwater is being 

collected and baited with rhododendron leaves to determine presence and frequency of 

positive P. ramorum rain traps. Results of this study will help evaluate the risk of P. 

ramorum to coast redwood seedlings and inform land managers of the potential for 

reforestation of infested sites with this species. 

Introduction 

Coast redwood (Sequoia sempervirens) is a host for Phytophthora ramorum but it is 

unclear if the pathogen represents a significant disease risk to this species. P. 

ramorum was first isolated from saplings in Sonoma and Santa Cruz Counties, 

California in 2002 and Koch’s Postulates were completed (Maloney and others 

2002). Symptoms include needle lesions, cankers on small branches, and dieback of 

epicormic sprouts (Davidson and others 2003). Researchers also observed 

discoloration in sapling xylem up to 90 cm from stem inoculation (Maloney and 

others 2002). Detection from mature redwood tissue has been reported, but 

conclusive disease causality was not determined (Garbelotto and others 2003). In a 

comparison of artificial inoculation methods, whole plant dips led to symptoms most 

similar to those found in nature (Hansen and others 2005). 

The objectives of this study were to evaluate the risk of disease to coast redwood 

seedlings caused by P. ramorum. To do this, we attempted to determine disease 

incidence in relation to time of year, weather and inoculum. We also wanted to 

examine disease development on redwood seedlings in association with a specific 

overstory species, California bay (Umbellularia californica). 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Department of Botany & Plant Pathology, Oregon State University, Corvallis, OR 97331 USA. 3 University of California Cooperative Extension, Humboldt & Del Norte Counties, Eureka, CA USA. 

Corresponding author: lucass@science.oregonstate.edu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

293 

Methods 

Experimental Sites 

Experimental sites are located in southern Humboldt County, the site of the 

northernmost California SOD infestation (Table 1). Conditions in this area are 

potentially conducive for sporulation because of mild temperatures, heavy 

precipitation, and presence of hosts. Infestation around the Avenue of the Giants is 

unsettling due to the valuable ecosystem and the amount of human traffic in this area. 

All sites are located along the Eel River on alluvial flats. The overstory is dominated 

by coast redwood. Symptomatic tanoak (Lithocarpus densiflorus) is present at all 

sites. California bay is present at two of the sites (Table 1). 

Table 1—Location of experimental sites. 

Site Location 

Prevalence of 

disease symptoms 

on tanoak 

Presence of 

California bay

O’Meara Grove 

Redway, CA 

Humboldt Redwood State 

Park (HRSP) 

High No 

Tooby Park Garberville, CA 

Garberville Community Park High Yes 

Chandler Grove 

8 km north of Meyers Flat, 

CA 

HRSP 

Low No 

Chandler Grove 8 km north of Meyers Flat, 

CA 

HRSP 

Low Yes 

Seasonality of Disease Incidence 

Using a complete random design, 20 healthy, potted seedlings were placed at Tooby 

Park for a two-month period beginning in December, 2006. A new group of seedlings 

was placed at Tooby Park every two months through June, 2007. 

Presence of P. ramorum was determined by isolation and PCR from selected stems, 

needles, and buds of each seedling. No seedlings were found positive for P. ramorum

from any time period. Therefore, we were unable to determine when redwood 

seedlings are most susceptible to infection. 
GENERAL TECHNICAL REPORT PSW-GTR-221 

 294

Symptom Development 

Thirty healthy seedlings were planted at each of the four research sites in December, 

2006. Symptom development was monitored monthly through June, 2007. A count 

of necrotic needles, cankers and length of stem dieback per seedling was measured 

monthly. Growth of seedlings over time was used as an indicator of overall vigor. 

No symptoms were detected on seedlings at any of the sites. Therefore, no 

relationship between symptom development and inoculum levels or presence of 

California bay could be determined. 

Detecting Inoculum 

Rainwater was collected and attempts made to quantify inoculum (fig 1). Initially 

fifteen raintraps were established at each of the sites. The first method used, from 

December, 2006 through April, 2007, followed the protocol set forth by the Rizzo lab 

at UC Davis (Davidson and others 2005). Water was collected, and then filtered. The 

filter was placed onto pimaricin-ampicillin-rifampicin selective media (PAR), and the 

plates examined for colony forming units (CFU) at OSU. 

Due to erratic detection, methodology was altered at the end of April. The second 

method followed a detection protocol similar to that used in Curry County, Oregon. 

Rhododendron leaf baits were placed in each of 60 raintraps, collected semi-monthly, 

and plated onto PAR to determine presence or absence of inoculum. 

Daily Rainfall vs. Number of Raintraps Positive for P. ramorum 

0

1

2

3

4

5

6

7

8

December

January

February

March

April

May

Rainfall (cm)

0

1

2

3

4

5

6

7

8

9

10

Number of Positive Raintraps

Inches of Rain

Rain Collection Method I

Rain Collection Method II

Figure 1-Rainfall was measured at the Eel River Conservation 

Camp in Redway, CA (approx. 8 km from the O’Meara Grove field 

site). Rain collection method I (♦) was discontinued at the end of 

April. Rain collection method II () was begun at this time. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

295 

Discussion 

P. ramorum was rarely detected from rainwater in Humboldt County during the first 

half of 2007. In contrast, Curry County, Oregon had consistent detections from 

rainwater during this same period, but also received considerably more rainfall (179.7 

cm vs. 76.5 cm). Lack of symptoms on redwood seedlings may be due to lower than 

average rainfall (76.5 cm from Dec.-May vs. 132.6 cm average). An additional 

season is necessary to assess the potential threat to coast redwood by P. ramorum in 

the field. In future studies on incidence and symptom development, a susceptible 

host, such as rhododendron seedlings, should be included as a positive control. 

Controlled laboratory experiments are currently underway to determine the 

relationship between inoculum dose and disease incidence (or symptom 

development), examine distance of spread and tissues colonized within inoculated 

seedlings, and assess the level of sporulation on redwood seedlings. 

Acknowledgements 

California Department of Parks & Recreation, North Coast Redwoods District 

Tooby Park Board of Directors 

Plum Creek Nursery 

Literature Cited 

Davidson, J.M.; Wickland, A.C.; Patterson, H.A.; Falk, K.R.; Rizzo, D.M. 2005. 

Transmission of Phytophthora ramorum in mixed-evergreen forest in California. 

Phytopathology. 95: 587-596. 

Davidson, J.M.; Werres, S.; Garbelotto, M.; Hansen, E.M.; Rizzo, D.M. 2003. Sudden 

oak death and associated diseases caused by Phytophthora ramorum. Plant Health Progress. 

Doi:10.1094/PHP-2003-0707-01-DG. (1 December 2005). 

Garbelotto, M.; Davidson, J.M.; Ivors, K.; Maloney, P.E.; Huberli, D.; Koike, S.T.; 

Rizzo, D.M. 2003. Non-oak native plants are main hosts for sudden oak death pathogen in 

California. California Agriculture. 57: 18-23. 

Hansen, E.M.; Parke, J.L.; Sutton, W. 2005. Susceptibility of Oregon forest trees and 

shrubs to Phytophthora ramorum: a comparison of artificial inoculation and natural infection. 

Plant Disease. 89: 63-70. 

Maloney, P.E.; Rizzo, D.M.; Koike, S.T.; Harnik, T.Y.; Garbelotto, M. 2002. First report 

of Phytophthora ramorum on coast redwood in California. APS online. 

www.apsnet.org/online/SOD/Papers/APS/PDnote_86-11a.html.0020. (1 April 2006). 
GENERAL TECHNICAL REPORT PSW-GTR-221

 296

Vegetation Dynamics and Impacts by 

Phytophthora ramorum in Redwood-Tanoak 

Forests in California1

Shannon C. Lynch,2

 Richard C. Cobb,2,3 and David M. Rizzo2

Introduction 

Pathogen epidemiology, community ecology of host species, and land use history all 

play major roles in the current distribution of Phytophthora ramorum and the extent 

of damage this pathogen has caused in California forests (Rizzo and others 2005, 

Meentemeyer and others 2008b). Research on the community ecology and the 

interaction of community composition and disease intensity will help enable 

synthesis of management actions at the stand level and reduce rates of infection, 

mortality, and fuel accumulation. Disease and mortality of tanoak (Lithocarpus 

densiflorus) are patchy across California landscapes, and causative mechanisms for 

these patterns likely operate at different spatial and temporal scales. Theoretical and 

empirical models that reflect the interdependences of vegetation and pathogen 

ecology are important to identify high-risk stands and developing management 

practices to reduce disease impacts. 

 

In this study, we assess the ecological characteristics of redwood forests to 

understand impacts by P. ramorum on redwood-tanoak forests in California. Since 

2002, we have monitored the survival of 5769 trees in the central and southern parts 

of the distribution of redwood forests (Maloney and others 2005). Using this dataset, 

we have asked the following questions: How does variation of host community 

structure affect the frequency of infection and mortality due to P. ramorum? What 

are the ecological relationships which structure these vegetation types? We address 

these questions by examining relationships between species biomass and edaphic 

factors. We then use preexisting host community structure to explore extent of P. 

ramorum infection and resulting mortality across the main area of infestation. 

Methods 

The study was conducted within a network of 120 500m2 circular plots covering 

redwood forests within Sonoma, Marin, Santa Cruz, and Monterey counties 

(Maloney and others 2005). A total of 5,769 trees ≥ 1cm DBH were surveyed in 2002 

and 2007. Every tree species within a plot was measured for diameter at breast height 

and given a crown vigor rating. Basal area for each species was calculated at the plot 

level and reported in square meters per 1/20 ha. Susceptible host trees included coast 

redwood (Sequoia sempervirens), tanoak, California bay laurel (Umbellularia 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, 

Phytophthoras in Forests and Natural Ecosystems, August 26–31, 2007, Monterey, California. 

2

 University of California Davis, Department of Plant Pathology One Shields Ave. Davis, CA 95616 USA. 

3 Graduate Group in Ecology, University of California at Davis, One Shields Ave. Davis, CA 95616. 

Corresponding author: sclynch@ucdavis.edu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 297 

californica), Douglas-fir (Pseudotsuga menziesii), big leaf maple (Acer 

macrophyllum), Pacific madrone (Arbutus menziesii), coast live oak (Quercus 

agrifolia), and California black oak (Quercus kelloggii). Tissue symptomatic of P.

ramorum infection was collected from each tree; in the lab, pieces of collected leaf 

and bark tissue were placed in Petri dishes containing pimaricin-ampicillinrifampicin-Pentachloronitrobenzene agar (PARP), a selective medium for 

Phytophthora spp. (Davidson and others 2003). In 2003, one soil sample was 

collected within each plot to evaluate soil texture and chemical properties. Soil 

analysis was conducted with standard methods for soil analysis at the University of 

California Davis ANR Analytical Laboratory. Plot location was recorded using a 

global positioning system (GPS) device (GARMIN, Olathe, KS, USA); plot locations 

were used to derive edaphic variables including topographic moisture index (TMI), 

elevation, and percent maximum solar radiation input. Principal component analysis 

(PCA) was conducted to assess multivariate correlation patterns of tree distributions 

with environmental variables. 

Sites were divided into central (Sonoma and Marin counties) and southern (Santa 

Cruz and Monterey counties) redwood forest sub regions (Sawyer and others 2000). 

Principle components for vegetation were calculated using correlations of plot level 

basal area. Three tree species including redwood, tanoak, and bay laurel accounted 

for 90 percent (±14 percent standard deviation) of the total basal area within the plot 

network. Host community structure was analyzed by including estimated basal area 

of tanoak, bay laurel, and redwood with all other species collapsed into a general 

category of other species (including black oak, coast live oak, Douglas-fir, maple, 

and madrone). These categories reflect a general continuum of susceptibility 

weighted by the biomass of individual species. 

 

Two PCA analyses were conducted. The first used only vegetation structure (four 

categories) to examine multivariate associations in host community structure. A 

second PCA analysis included edaphic variables and vegetation structure in order to 

elucidate environmental controls on host community composition. Eigenvalues for 

the first five principal components were used to examine associations between 

vegetation and edaphic factors. Effects of host community composition on intensity 

of sudden oak death was examined with multiple linear regression (MLR) using 

vegetation structure as independent variables and amount of tanoak and bay laurel, 

and tanoak mortality as dependent variables (Maloney and others 2005). Independent 

and dependent variables were square root transformed prior to analysis. 

Results 

Community composition and disease impacts 

Results from PCA analysis indicate strong negative correlations among bay laurel, 

tanoak, and redwood when considered at the redwood forest community level (fig 1). 

The first and second principle components accounted for 62 percent of the variation 

in vegetation structure and suggest segregation of host species within redwood forest 

types. Redwood and bay laurel basal area were negatively correlated and both species 

showed less pronounced negative correlation with tanoak. All other species were 

positively correlated with tanoak basal area suggesting that stands with greater tanoak 

dominance have higher overstory tree diversity compared to redwood stands 
GENERAL TECHNICAL REPORT PSW-GTR-221

 298

dominated by redwood or bay laurel at the plot level. The large amount of spread in 

the data and lack of obvious plot clustering suggests that segregation of host species 

occurs along gradients within redwood forests. 

Figure 1—Score (left) and loadings (right) from a PCA analysis on 

correlations among tanoak (LIDE), bay laurel (UMCA), redwood (SESE), and 

all other species (Other) basal area from 120 plots in redwood forests. This 

analysis illustrates variation in host community structure and suggests that 

edaphicand/or competitive interactions are important in structure host 

populations. 

Results from our second PCA analysis suggest that stand level vegetation structure in 

redwood forests is related to edaphic factors but the relative importance of edaphic 

drivers differ between sub regions (figs. 2A and B). Overall, edaphic factors and 

competition were important in determining species biomass at the plot level in both 

regions. For the central redwood forest sub-region, all species out-compete coast 

redwood at relatively higher elevation sites with edaphic factors determining the 

abundance of alternative dominant species. Bay laurel and other species occurred at 

greater basal area in sites with higher soil clay content. At these sites, tanoak was 

more abundant where TMI values were lower suggesting that tanoak may outcompete bay laurel in relatively drier high elevation sites. Low resource sites are 

known to support high species richness in Coastal scrub ecosystems (Moody and 

Meentemeyer 2001). Figure 1 shows strong tanoak association with all other 

overstory species that commonly occur in our plots. This pattern may reflect resource 

poor growing conditions in these plots and suggests that tanoak is adapted to more 

resource poor conditions compared to redwood and bay laurel. Tanoak and other 

species were associated with fine texture soils at middle elevation and moderate 

moisture sites. Within these middle elevation areas, in which solar radiation output 

was high, tanoak and bay dominated over redwood and other species where there was 

relatively more clay in the soil (fig. 2A). In the southern redwood forest sub region 

bay laurel was dominant at low elevation, high solar radiation sites that were 

relatively wet and comprised of fine textured soils. At moderate elevations, redwood 

dominated bay laurel on fine textured soils. Redwood was dominant over 

other species at lower elevation sites that had soils with less clay content. In 

this subregion, bay laurel and tanoak were more dominant over other species 

in sites with relatively lower solar radiation and high soil clay content (fig.

2B). 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 299 

Figure 2—Edaphic dependencies of species dominance in central and 

southern redwood study sites. Edaphic-species relationships are based on 

dominant eigenvalues from a PCA analysis of vegetation abundances and 

edaphic factors including soil texture, elevation, and topographic moisture 

index (TMI). 

Host community structure was an important factor influencing the intensity of disease 

impacts in coastal California redwood forests. Amount of bay laurel infected basal 

area was best described as a simple linear function of bay laurel basal area (table 1). 

Very little residual variation occurred in this model (r2

adj = 0.82) suggesting that 

additional factors such as host and pathogen genetic structure did not dominate bay 

laurel infection dynamics within our study sites. Tanoak infection levels were linear 

functions of tanoak and bay laurel basal area (table 1) but had considerably more 

residual variation compared to bay laurel. This difference in model performance may 

reflect greater sensitivity to historical, host genetic, and pathogen genetic effects but 

the overall slower rate of tanoak infection compared to bay laurel likely accentuates 

these effects. Consequently, our understanding of P. ramorum infection will be 

improved with better understanding of pathogen invasion dynamics including the 

effects of tanoak and pathogen genetics. Tanoak mortality was also a linear function 

of tanoak and bay laurel basal area (table 1). The overall dependency of tanoak 

infection and mortality on tanoak and bay laurel basal area reflects transmission 

dynamics of P. ramorum. Bay laurel produces substantially higher inoculum levels 
GENERAL TECHNICAL REPORT PSW-GTR-221

 300

compared to tanoak; therefore, higher levels of infected bay laurel basal area should 

increase tanoak infection levels at the stand level (Davidson and others 2008, Rizzo 

and others 2005). High inoculum is likely to increase rate of secondary infection and 

increase the frequency of tanoak mortality (Gilligan 2006). Inoculum loads have been 

recovered from rainwater in significantly larger proportions under bay laurel 

canopies compared to mixed canopies at Jack London State Park (Davidson and 

others 2008). Assuming a linear relationship between infected basal area of inoculum 

supporting species and stand level inoculums load, we would expect to find the 

overall linear relationships between amount of infection and mortality present in our 

dataset (Table 1). In our system of study plots, highest inoculum pressures likely 

occur in stands with high bay laurel basal area; however, sudden oak death impacts 

are likely highest in stands at the edaphic and competitive interface between bay 

laurel and tanoak as these stands will have the highest inoculums loads on tanoak and 

the most frequent opportunities for secondary infection. 

Discussion 

Implications for stand level management 

Understanding the biology and ecology of Phytophthora ramorum as well as the 

ecology of the forests it invades are necessary for managing sudden oak death on a 

landscape level (Rizzo and others 2005). This study assesses host distribution and 

community structure of redwood forests invaded by P. ramorum from a 

comprehensive data set comprising of nearly six thousand trees. Edaphic factors in 

conjunction with historical disturbance are likely major drivers of current species 

distribution and abundance in these forests (Foster 1988, Meentemeyer and others 

2008b). Compounded with the fact that host community composition is 

Table 1—Results from multiple regression modeling of 

vegetation structure on infection and mortality caused by 

P. ramorum. 

Standardized regression coefficients are reported when the effects of a 

vegetation factor were significant at the p <0.05 level. Degrees of 

freedom for calculation of statistical effects (DF) and adjusted r square 

values are listed at the bottom of the table for each an alysis.
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 301 

heterogeneous within the central and southern distributions of the redwood forest 

type in coastal California, management actions may be targeted differently depending 

on forest composition. 

The finding that P. ramorum most readily establishes in stands with more tanoak and 

laurel bay basal area follows predictions based on ecological niche modeling and 

community level investigations of pathogen establishment (Davison and others 2003, 

Maloney and others 2005, Rizzo and others 2005, Meentemeyer and others 2008a). 

These data also demonstrate that community level vegetation manipulation may be 

risky at sites where edaphic factors enhance the dominance of sporulation supporting 

species. For example, results from PCA suggest that bay laurel removal efforts would 

be most successful in sites where tanoak has a competitive advantage over bay: drier 

sites at higher elevations in the central sub region and wetter sites at moderate 

elevations within forests of the southern redwood sub region. These efforts should be 

avoided in the south where soil silt content is relatively high at lower elevations and 

in the central region at mid-elevations where soils are fine textured at moderate 

moisture levels because of likely reestablishment of bay laurel and consequently P. 

ramorum over a decadal time scale. Areas in which bay and tanoak coexist and 

dominate over redwood and other species may require further study to identify the 

outcome of stand manipulation. Pruning lower branches of bay laurel and thinning of 

understory tanoak may be effective in reducing infection due to an overall higher 

susceptibility of these individuals. Pruning may be most effective at moderate 

elevation sites where the soil clay content is high for both regions and solar radiation 

is low in the southern region. Because tanoak supports pathogen sporulation, thinning 

of tanoak in the understory would be prudent in any case to decrease P. ramorum 

dispersal. Future research efforts will use pre-disease vegetation structure to make 

predictions of future forest structure and improve disease and forest management 

strategies. 

Literature Cited 

Davidson, J.M.; Patterson, H.; Rizzo, D.M. 2008. Sources of inoculum for Phytophthora 

ramorum in a redwood forest. Phytopathology 98:860-866. 

Davidson, J.M.; Werres, S.; Garbelotto, M.; Hansen, E.M.; Rizzo, D.M. 2003. Sudden 

oak death and associated diseases caused by Phytophthora ramorum. Online. Plant Health 

Prog. (On-line) doi:10.1094/PHP-2003-0707-01-DG. 

 

Ivors, K.; Garbelotto, M.; Vries, I.D.; Ruyter-Spira, C.; Hekkert, B.; T.E.; Rosenzweig, 

N.; Bonants, P. 2006. Microsatellite markers identify three lineages of Phytophthora 

ramorum in US nurseries, yet single lineages in US forest and European nursery populations. 

Molecular Ecol. 15: 1493–1505. 

 

O’Green, A.T.; Dahlgren, R.A.; Sánchez-Mata, D. 2007. California soils and examples of 

ultramafic vegetation. In: Terrestrial vegetation of California. Barbour, M.G.; Keller-Wolf, T.; 

and Shoenherr, A.A. Eds. Third edition. University of California press. 

 

Maloney, P.E.; Lynch, S.C.; Kane, S.F.; Jensen, ; Rizzo, D.M. 2005. Establishment of an 

emerging generalist pathogen in redwood forest communities. J. of Ecol. 93: 899–905. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 302

Moody, A.; Meentemeyer, R.K. 2001. Environmental factors influencing spatial patters 

of shrub diversity in chaparral, Santa Ynez Mountains, California. J. of Vegetation Science. 

12: 41–52. 

 

Meentemeyer, R.K.; Anaker, B.; Mark, W.; Rizzo, D.M. 2008a. Early detection of 

emerging forest disease using disperasal estimation and ecological niche modeling. Ecol. 

App. In press. 

 

Meentemeyer, R.K.; Rank, N.E.; Anacker, B.L.; Cushman, J.H. 2008b. Influence of 

land-cover change on the spread of an invasive forest pathogen. Ecol. Appl. 18: 159–171. 

 

Meentemeyer, R.; Rizzo, D.M., Mark, W.; Lotz, E. 2004. Mapping the risk of 

establishment and spread of sudden oak death in California. For. Ecol. Manage. 

200: 195–214. 

 

Rizzo, D.M.; Garbelotto, M.; Hansen, E.M. 2005. Phytophthora ramorum: integrative 

research and management of an emerging pathogen in California and Oregon forests. Ann. 

Rev. Phytopathol. 43: 309–335. 

 

Sawyer, J.O. 2007. Forests of northwestern California. In: Terrestrial vegetation of 

California. Barbour, M.G., Keller-Wolf, T., and Shoenherr, A.A. Eds. Third edition. 

University of California press. 

 

Sawyer, J.O.; Sillett, S.C.; Popenoe, J.H.; LaBanca, A.; Sholars, T.; Largent, D.L.; Noss, 

R.F.; Van Pelt, R. 2000. Characteristics of Redwood Forests. In: The Redwood Forest: 

History, Ecology, and Conservation of the Coast Redwoods. Noss, R.F. Ed. First edition. 

Island Press. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

303 

Epidemiology of the Alder Phytophthora1

Benoit Marçais,2 Claude Husson,2 Jaime Aguayo,2 and 

Fabrice Elegbede2 

Abstract 

Since the beginning of the nineties, Phytophthora alni, a new hybrid species, has been 

inducing a major decline of alder trees along European watercourses. To evaluate the longterm impact of the disease on the alder population, we set up a permanent survey area in 2002 

along the Sarre River (Moselle). All alder over 1.3m high were inventoried and mapped along 

approximately four kilometers of river, and their health status and growth was annually 

determined. Each year, new trees reaching 1.3m high were recruited in the sample. 

In this study, we used the collected data to document the speed of disease development on the 

trees, based on survival analysis. Additionally, we proposed an estimation of local tree 

infection probability based on kernel estimators. The disease is characterised by presence of 

trunk cankers and decline of the crown. The trunk cankers might better reflect the present 

infection activity while the crown decline might more reflect past infection, being slower to 

appear. We thus based local tree infection probability on the likelihood of new canker cases. 

We then checked whether or not it is important to take into account the lag between infection 

and decline of the crown. For that, we estimated the likelihood that a tree with a healthy 

crown would decline, given trunk dbh and the local likelihood of previous year’s new canker 

cases. The lag depends on the tree size, with seedlings showing very quick crown decline in 

area with high likelihood of new canker cases while large trees decline more slowly. We used 

the fitted function to identify trees that appeared healthy, but nevertheless had a high 

likelihood of being already infected. These trees were compared to healthy trees with little 

risk of being already infected according to our model, slightly declining trees, severely 

declining trees and dead trees. Phytophthora alni presence in the soil was estimated by baiting 

followed by PCR for each of the studied trees in order to determine which are major inoculum 

producers. 

The results show that P. alni inoculum is detected in larger amounts with healthy trees that 

have a high probability of being already infected and moderately declining trees than with 

severely declining trees. By contrast, P. alni is seldom detected in association with healthy 

trees that our model predict to be uninfected or with dead trees. We conclude that it is very 

important for accurately modelling disease evolution to take into account asymptotic 

uninfected trees as they can potentially be major inoculum producers. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 UMR1136 INRA/UHP Interactions arbres/microorganismes, INRA-Nancy, 54280 Champenoux, France. 

Corresponding author: marcais@nancy.inra.fr. 
GENERAL TECHNICAL REPORT PSW-GTR-221

304 

Monitoring Phytophthora ramorum 

Distribution in Streams Within 

Coastal California Watersheds1

S.K. Murphy,2 C. Lee,3 Y. Valachovic,3 W. Mark,4 A. Jirka,4 

D.R. Owen,5 and D.M. Rizzo2

Abstract 

One hundred eighty-seven sites were established in perennial watercourses and sampled for 

one to four years between 2004 and 2007 to monitor for the presence of Phytophthora 

ramorum throughout coastal central and northern California watersheds as well as portions of 

the Sierra Nevada mountain range. In 2007, 132 sites were monitored, including 65 new sites. 

The majority of the monitored watersheds have limited or no Pr at this time, but are near the 

epidemic range of P. ramorum and/or are considered high-risk for invasion by P. ramorum. 

Three currently infested watersheds in Sonoma, Humboldt, and Monterey Counties were 

included as a baseline for successful recovery of Pr. Rhododendron leaves were placed in 

mesh bags and secured in watercourses for one- to three-week intervals to bait for 

Phytophthora species. Recovered symptomatic leaves were plated on Phytophthora-selective 

medium (PARP-H) and monitored microscopically. 

Phytophthora ramorum has been detected at 37 total stream monitoring sites, including all 

sites with a priori knowledge of nearby forest infestation. Phytophthora ramorum was 

detected at 23 streams sites without prior knowledge of adjacent forest infestation in 

Humboldt, Contra Costa, Mendocino, Monterey, and Santa Cruz counties. Forest infestations 

have thus far been confirmed at only nine of these sites; surveys are underway to identify the 

source(s) of inoculum for the other sites. Additionally, P. ramorum was recovered as far as 

25km downstream from known forest infestations. This year (2007) was an unusually dry 

year in California which impacted our recovery of P. ramorum from watercourses. 

Phytophthora ramorum was detected in only 15 streams this year; seven of those were new 

sites for 2007. We recovered no P. ramorum from four streams that were positive for P. 

ramorum in 2006. At the most heavily infested sites, the frequency and quantity of recovery 

of P. ramorum was greatly reduced in 2007. 

Stream monitoring has extended the southern range of Pr in Monterey County and the 

northern range in Humboldt County. All sites in the Sierra Nevada remain negative for Pr. 

With culturing and molecular sequencing we have identified several other Phytophthora 

species within these watersheds; P. gonapodyides is the most commonly detected species and 

was isolated from at least 60 sites. Streams were monitored year-round in 2004 and 2005 and 

revealed a distinct seasonality associated with P. ramorum recovery. Therefore, in 2006 and 

2007 watersheds were monitored monthly, February through June, during the peak seasonal 

period. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Department of Plant Pathology, University of California, Davis, CA 95616, USA. 3 UC Cooperative Extension Humboldt and Del Norte Counties, Eureka, CA 95503, USA. 

4 Cal Poly State University, NRM Department, 1 Grand Ave, San Luis Obispo, CA 93407, USA. 

5

 California Dept. of Forestry and Fire Protection, 6105 Airport Road, Redding, CA 96002, USA. 

Corrresponding author: skmurphy@ucdavis.edu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

305 

Real Time PCR Protocols for Environmental 

Monitoring of Phytophthora alni 

and Its Three Subspecies1

Tomasz Oszako,2 Lassaad Belbahri,3 Gautier Calmin3, and 

François Lefort3

Abstract 

Appearing in the U.K. at the end of the 1980’s, Phytophthora alni has severely reduced alder 

populations in Western Europe and has been emerging as a major threat to alder stands in 

Poland in the past years. For addressing Phytophthora epidemics, which spread in the wild, 

environmental monitoring must be made easier, quicker and cheaper than classical description 

or PCR methods. The objective of the work presented here was to provide with a set of real 

time PCR assays for environmental monitoring of Phytophthora alni, fulfilling the desired 

requirements. We present here the development and evaluation of a set of real-time PCR 

assays for the LightCycler real time hot air thermocycler (Roche Diagnostics), using primers 

that target the Sequence Characterised Amplified Regions (SCARs) previously developed for 

P. alni. The real time PCR format allowed to perform a melting curve analysis of the PCR 

products once amplification was completed and to offer an additional characterization of the 

targeted SCARs. The availability of rapid and specific assays for the detection and 

characterization of P. alni, such as provided in the present study, should facilitate the 

detection, control of outbreaks and the occurrence and distribution of the different subspecies 

of this pathogen in Poland. However, care should be taken when conducting environmental 

studies using these oligos since we show a clear cross contamination with at least P. 

gonapodyides and P. quercina, two species frequently encountered in alder stands. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California.

2 Forest Research Institute, Department of Forest Phytopathology, Sekocin Las, 05-090 Raszyn, Poland. 3 Laboratory of Applied Genetics and Plant Health, Institute Earth Nature and Landscape, Univ. of Applied Sciences 

of Western Switzerland, 150 Route de Presinge 1254 Jussy, Switzerland. 

Corresponding author: T.oszako@ibles.waw.pl.
GENERAL TECHNICAL REPORT PSW-GTR-221

306 

Stand Structure and Local Distribution of 

Phytophthora ramorum in Oregon Forests1

Ebba Peterson,2 Molly Botts,2 Everett Hansen2

Introduction 

The Phytophthora ramorum eradication program in effect in Oregon has allowed for 

the rapid detection of new infection foci, typically before they develop within each 

stand and expand into adjacent sites. Yet despite gallant efforts, new locations that 

previously harbored no apparent infection have been identified each year since the 

original detection in 2001. Multiple factors may influence why and where each new 

infection occurs including, but not limited to, stand characteristics such as dominant 

understory and upper canopy vegetation, age and stem density (which influence 

microclimate variations within the stand), the distribution of host and non-host 

species, and the various possible dispersal mechanisms of the pathogen (Shaw 2004). 

Previous studies in California have found that landscape patterns of disease severity 

are poorly explained by variation at small scales (Condeso and Meentemeyer 2007); 

however detailed work on infestations at their origin, prior to local intensification, 

has not yet been performed. This study examined the stand and canopy characteristics 

and the local distribution of P.ramorum around the first trees detected, if not the first 

trees infected, in a remote location. We further set out to document and characterize a 

site with infestation that has developed to an extent previously unseen in Oregon. 

Methods 

The two sites of this study, site 2748 and North Bean Creek (NBC), were located 

within a mile of each other on land privately owned by South Coast Lumber 

Company near Brookings, OR. Both sites were composed of mixed Douglas fir 

(Pseudotsuga menziesii), alder (Alnus rubra), and tanoak (Lithocarpus densiflorus), 

and bordered fir plantations. The topography of the sites is similar; both are uphill 

from the nearest road and occupying east facing slopes with similar pitches (35-45 

degrees), elevations (approximately 320-365 m above sea level), and locations 

relative to the top of their respective ridges. Both sites were first detected and 

confirmed positive for P.ramorum in summer 2007. 

To assess the distribution of understory vegetation likely to contribute to the lateral 

spread of P.ramorum, notably Rhododendron macrophyllum and Vaccinium ovatum, 

a grid was established either around positive trees (site 2748; 30m by 40m) or 

through the area of worst infestation (NBC; 25m by 60m). For each 5m2

 block within 

the grid presence or absence of foliar hosts was noted (R.macrophyllum, V.ovatum, 

L.densiflorus sprouts, and Umbellularia californica only). Symptomatic tissue 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331. 

Corresponding author: petersoe@science.oregonstate.edu. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

307 

samples were taken from every block and P.ramorum presence was confirmed 

through culture on cornmeal, ampicillin, rifamycin, pimaricin (CARP) medium. 

Stand composition and canopy structure was assessed by running a belt transect (10m 

x 50m for NBC and 10m x 40m for site 2748) nested within the vegetation plot. For 

all tree species, tree height and length of crown were measured, as well as distance 

traveled along the length and width of the transect. Distances were later corrected for 

changes in elevation for depiction. Disease severity on tanoak was rated by 

estimating crown condition (percent browned to the nearest 10 percent), and 

recording presence or absence of symptoms associated with P.ramorum (bleeding, 

frass, symptomatic basal or stem sprouts). The stands were surveyed between July 

25th and August 9th, 2007. 

Results 

Stems were denser at site 2748 though individual trees were much smaller, especially 

amongst tanoak (Table 1). Tanoak comprised a greater proportion of the upper 

canopy and total basal area in NBC (Table 1). In contrast, at site 2748 there was a 

marked stratification between the emergent Douglas fir, which were distributed 

throughout the transect, and the codominant tanoaks. While many of these stems 

were dominant, nearly all tanoak crowns were adjacent to taller Douglas fir crowns; a 

notable exception is the few known positive trees. At site 2748, of the 91 tanoaks 

measured there were only two symptomatic, brown-canopied trees and, directly 

adjacent to these, three bleeding stems. In contrast, within NBC 35 percent of the 78 

tanoaks surveyed were bleeding, though 85 percent had some symptoms associated 

with P.ramorum, predominantly symptomatic, succulent basal sprouts. 

Table 12Stand characteristics for the three tree species measured at North 

Bean Creek (NBC) and site 2748. 

 

No. live 

trees 

counted a

Tree density 

(stems/ha)

Total basal 

area 

(m2

/ha) 

Mean tree 

height (m) 

 Species NBC 

site 

2748 NBC 

site 

2748 NBC

site 

2748 NBC

site 

2748

L.densiflorus 78 91 1520 2275 35.6 16.7 13.8 7.6 

A.rubra 8 0 160 0 7.1 0 20.5 0 

P.menziesii 3 23 60 575 5.7 19.1 24.0 14.8 

Total for all 

species 89 114 1740 2850 48.4 35.8 

a NBC: 500m2 plot, all trees greater than 7cm DBH; site 2748: 400m2 

plot, all trees greater than 5cm DBH 

Both sites had a continuous understory of host vegetation (Table 2). Umbellularia 

californica was present only at NBC, though only as small understory saplings in 

four blocks. In previous studies in California recovery of the pathogen from attached 

leaves declined dramatically over the summer months (Fitchner and others 2007), 

however P.ramorum was readily recovered from the highly infested site in Oregon. 

P.ramorum was isolated from leaf tissue in all but two 5m2 blocks within NBC (96 
GENERAL TECHNICAL REPORT PSW-GTR-221

308 

percent of 69 samples taken, Table 2). In contrast, at site 2748 despite extensive 

sampling within the grid P.ramorum was only recovered within 5m of bleeding trees 

(4 percent of 80 samples taken, Table 2). 

Table 22Prevalence of host vegetation within the vegetation grid, number of 

samples taken, and proportion of samples positive for P.ramorum at site 2748 

(2a; low incidence) and North Bean Creek (2b; high incidence). 

 

Proportion of blocks 

with species present 

a 

# vegetation 

samples tested 

Proportion 

positive 

samples 

 Species NBC site 2748 NBC 

site 

2748 NBC 

site 

2748 

L.densiflorus 1 1 55 26 0.98 0.08 

R.macrophyllum 0.93 0.96 11 45 0.91 0.02 

V.ovatum 0.9 1 1 9 0 0 

U.californica 0.07 0 2 - 1 - 

 Total for all species: 69 80 0.96 0.04 

a 500m2 (NBC) or 400m2 (site 2748) plot divided into 5m2

 blocks 

Discussion 

Our results are consistent with previous observations that the development of disease 

in Oregon is dependant upon the infection of mature tanoak. Site 2748 is a typical 

location: highly restricted, local infection centered around isolated trees whose 

disease developed from inoculum produced at an unknown source. While other 

studies have focused upon movement of infested soil to explain the original 

introduction of inoculum into remote areas (Davidson and others 2005), the location 

of this site is not consistent with movement by either people or vehicles. Other 

possibilities include movement by animals, either from below or above, or the aerial 

dispersal of inoculum. The crown exposure of the initially infected trees, as well as 

the lack of any observable infection on surrounding vegetation is suggestive of aerial 

dispersal. Further studies are needed to determine whether the observed pattern 

reflects the dispersal method of the pathogen or microclimate variation that favors 

initial establishment (or both). 

It is likely not for the lack of host continuity or topographical differences that the 

severity of disease in either site differs so drastically, and which other factors may 

best explain this remain unclear. Nevertheless, this study highlights the potential for 

the diseases caused by P.ramorum to reach epidemic proportions in Oregon. 

Literature Cited 

Condeso, T.E., Meentemeyer, R.K. 2007. Effects of landscape heterogeneity on the 

emerging forest disease sudden oak death. Journal of Ecology 95:364-375 

Davidson J.M., Wickland A.C., Patterson, H., Falk K., Rizzo D.M. 2005. Transmission of 

Phytophthora ramorum in mixed-evergreen forests of California. Phytopathology 95:587–96 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

309 

Fichtner, E., Rizzo, D., Lynch, S., Davidson, J., Buckles, G., Parke, J. 2007. Summer 

Survival of Phytophthora ramorum in California bay laurel leaves [Poster]. In: sudden oak 

death science symposium III; 2007 March 5-7; Santa Rosa, CA. 

Shaw, D.C. 2004. Vertical organization of canopy biota. In: Lowman, M.D., Rinker, H.B., 

ed.s, Forest Canopies 2nd ed. San Diego: Academic Press: 73-101. 
GENERAL TECHNICAL REPORT PSW-GTR-221

310 

Physiological Changes and Gene 

Expression on European Beech 

(Fagus sylvatica) Infected With 

Phytophthora citricola1

Roberto L. Portz,2 Julia Koehl,2 Frank Fleischmann,2 

and Wolfgang Oßwald2 

Abstract 

In the last decade Phytophthora citricola Saw. was described as an important and aggressive 

pathogen on European beech (Fagus sylvatica L.). Inoculated beech seedlings, grown under 

controlled conditions, showed a significant decrease of photosynthesis, transpiration and 

water potential after three days. Typical wilt symptoms on leaves were observed only after a 

consistent reduction of these parameters. Sugar contents of roots of infected plants were 

significantly lower in infected plants as compared to control seedlings. A negative correlation 

between photosynthesis, transpiration and water potential and the increase of P. citricola in 

roots was demonstrated. In addition synthesis of ethylene of infected plants was increased two 

days after inoculation compared to controls. Expression of genes of the primary and 

secondary metabolism, as well as of defense-related genes, is ongoing in order to get more 

detailed information of the studied host-parasite interaction. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Section Phytopathology of Woody Plants, Technische Universität München, Am Hochanger 13, 85354 Freising, 

Germany 

Corresponding author: portz@bot.forst.tu-muenchen.de 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

311 

Characterizing the Community of 

Phytophthora Species in an Oregon Forest 

Stream1

Philippe Remigi,2 Wendy Sutton,3 Paul Reeser,3 and Everett 

Hansen3

Introduction 

Phytophthora species are best known as pathogens of agricultural crops, or invasive 

pathogens destroying forests. Little is known about indigenous species, especially in 

wild ecosystems. Previous work showed that Phytophthora species are relatively 

abundant in natural streams in forests, but the species present are poorly 

characterized, and their ecology is essentially unknown. The aim of this work was to 

compare methods for the collection, identification, and enumeration of Phytophthora 

spp. in streams. Three methods of isolate collection were compared (leaf baits, pear 

baits, and filtration), and species identification was carried out using morphological 

and growth characters, and by DNA sequencing two regions of the genome (ITS 

rDNA, mitochondrial COX spacer region). Stream sampling was conducted at five 

locations in the Oak Creek drainage near Corvallis Oregon. 

Oak Creek 

Oak Creek starts in forest, then runs through an agricultural valley and an urban area, 

where it joins Marys River and then the Willamette River. Sampling Site 1 was at the 

beginning of the stream, in forest. Site 2 was downstream at the edge of the forest, 

before the first habitations. Site 3 was located in the plain with scattered housing, a 

few orchards, meadows and crop fields. Site 4 was in Marys River, just after Oak 

Creek flows into it. Site 5 was in the Willamette River, which flows through a fertile 

valley with many orchards, fields and forests. Tree species present at all sites 

included Douglas-fir, grand fir, big leaf maple, and red alder. 

Methods 

 Isolations were made on CARP + (CMA with 25ppm hymexazol (99 

percent), 20ppm delvocid (50 percent natamycin salt), 200ppm ampicillin 

sodium salt, 10ppm rifampicin SV sodium salt and 30ppm benlate (benomyl 

50WP)) then transferred to carrot agar. 

 Isolates were collected every other week for 12 weeks, by filtration, leaf 

baiting, and pear baiting. 700 ml of stream water, in 50, 100, and 200ml 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 INRA, Toulouse, France. 

3 Department of Botany and Plant Pathology, Oregon State University, Corvallis OR 97331, USA. 

Corresponding author:remigi@toulouse.inra.fr 
GENERAL TECHNICAL REPORT PSW-GTR-221

312 

portions, was passed through 5 μm nitrocellulose filters using a portable 

vacuum filtration unit. Filters were plated on CARP +. 

 Two Rhododendron leaves, one tanoak leaf and three small pieces of PortOrford-cedar foliage were placed in a mesh bag and immersed in the stream. 

After two weeks, leaves were removed, washed, and plated on CARP +. One 

d'Anjou Pear was also immersed in the stream. After three days, pears were 

removed, washed and tissues with lesions were plated on CARP +. All 

isolates were grown up on CMA and DNA was extracted from colonized 

agar plugs using a CTAB buffer protocol. The COX gene spacer region was 

PCR amplified using primers FMPH8 and 10, and sequenced in one 

direction. These single strands were aligned with other known (reference) 

sequences for initial species identification. Isolates not matching a reference 

isolate were then amplified and sequenced using ITS4 and 5, and blasted 

against the GenBank database. 

Results 

A total of 514 isolates were recovered from baits or filters. About 57 of these proved 

to be Pythium or zygomycetes. Another 20 isolates were not characterized because of 

sequencing problems or other difficulties. 437 Phytophthora isolates were analyzed. 

An average of 13 Phytophthora colonies were grown from each liter of stream water 

filtered. 

Phytophthora Diversity 

ITS and COX spacer DNA analysis distinguished at least 18 groups of isolates. These 

18 groups formed nine clusters. 

 Four of the clusters matched known species (P. citricola, P. 

gonapodyides, P. pseudosyringae and P. syringae). 

 Three clusters contain groups of isolates that match and/or whose 

sequence is very similar to a known species (P. cambivora, P. 

megasperma and P. sp. “Pg chlamydo”). 

 Two clusters contain groups of isolates that are similar but not identical 

to a known or unnamed species (P. europaea and Clade 6 “Kalamazoo”). 

 More than 90 percent of all isolates were in ITS clade 6: P. 

gonapodyides, P. megasperma, P.sp. “Pg chlamydo”, and unnamed 

related groups (“Kalamazoo” types). 

Capture Method 

Water filtration yielded more different species groups than either leaf baits or pears 

baits. P. megasperma and related isolates were only recovered from filters. On the 

other hand, P. cambivora and variant group and P. syringae were only recovered 

from baits (Table 1). 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

313 

Table 12Recovery of Phytophthora species groups/clusters on filters, leaf 

baits, and pears. 

 Filter Leaf Pear Total 

P. cambivora cluster 0 3 6 9 

P. citricola 7 11 5 23 

Clade 6 “Kalamazoo” cluster 41 15 26 82 

P. europaea cluster 2 0 1 3 

P. gonapodyides 92 74 20 186 

P. megasperma cluster 7 0 0 7 

P. sp. “Pg chlamydo” cluster 48 66 8 122 

P. pseudosyringae 0 0 1 1 

P. syringae 0 1 3 4 

All Phytophthoras 197 170 70 437 

Sample Time and Location 

Phytophthora species varied by sample time and location within the watershed 

(Tables 2 and 3). Phytophthora syringae was recovered from 3 different locations 

but only at one June sample time. P. cambivora and cluster isolates and P. europaea 

cluster isolates came only from sample sites 1 and 2. In contrast, P. citricola was 

recovered only from downstream sites, late in the season, while P. megasperma and 

cluster isolates came from downstream sites early in the sampling period. The clade 6 

groups (P. gonapodyides, P.sp. “Pg chlamydo”, and the cluster of variants similar to 

P. “Kalamazoo”) tended to dominate at all locations and sample times. 

Table 22Recovery of Phytophthora species group/clusters by sampling time, 

June±August 2006. 

Site1 Site2 Site3 Site4 Site5 Total 

P. cambivora cluster 7 2 0 0 0 9 

P. citricola 0 0 4 17 2 23 

Clade 6 “Kalamazoo” cluster 1 9 30 26 16 82 

P. europaea cluster 3 0 0 0 0 3 

P. gonapodyides 37 55 52 32 10 186 

P. megasperma cluster 0 2 2 2 1 7 

P. sp. “Pg chlamydo” cluster 4 58 25 30 5 12 

P. pseudosyringae 0 1 0 0 0 1 

P. syringae 0 1 1 2 0 4 

All Phytophthoras 52 128 114 109 34 437 
GENERAL TECHNICAL REPORT PSW-GTR-221

314 

Table 32Recovery of Phytophthora species group/clusters by sampling 

location in Oak Creek and downstream sites. 

Time1 Time2 Time3 Time4 Time5 Time6 Total 

P. cambivora cluster 0 0 0 1 6 2 9 

P. citricola 0 0 1 1 7 14 23 

Clade 6 “Kalamazoo” cluster 6 2 7 22 14 31 82 

P. europaea cluster 0 0 0 2 0 1 3 

P. gonapodyides 49 33 29 21 16 38 186 

P. megasperma cluster 4 2 1 0 0 0 7 

P. sp. “Pg chlamydo” cluster 10 16 22 21 23 30 122 

P. pseudosyringae 0 0 1 0 0 0 1 

P. syringae 0 4 0 0 0 0 4 

All Phytophthoras 69 57 61 68 66 116 437 

Conclusions: 

 Water filtration provided more isolates and more species than baiting and 

also enabled quantification of propagules. 

 12 to 20 Phytophthora propagules/liter of water were usually detected. 

 At least 18 different groups of isolates were distinguished. 

 12 groups did not match named species. 

 P. gonapodyides was most numerous, followed by P.sp. “Pg chlamydo” and 

its variants (cluster), and another unidentified group of taxa from ITS clade 

6. 

 The Phytophthora community changed through the season, and differed at 

different locations in the watershed. 

Acknowledgements 

We would like to thank Frank Martin for suggesting the use of COX spacer single 

strand alignments as a species screening tool. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

315 

Phytophthora ramorum Tissue Colonization 

Studied With Fluorescence Microscopy1

Marko Riedel,2 Stefan Wagner,2 Monika Götz,2 Lassaad Belbahri,3

François Lefort,3

 and Sabine Werres2

Abstract 

The proceeding worldwide spread and the expanding host spectrum of P. ramorum has 

become a serious threat to natural plant communities. To encounter this threat detailed 

knowledge about infection pathways and tissue colonization is essential. To analyze these 

issues, histological studies of infected tissue with epifluorescence microscopy have been 

started. For first infection tests Rhododendron has been taken as a model host. Root infection 

of potted Rhododendron cuttings with the P. ramorum isolate BBA 9/95 (ex-type strain) were 

started. Inoculation of the non injured plants was done by application of a zoospore 

suspension onto the surface of the pot soil. The plants were then incubated with 16 hour light 

at 20°C in a quarantine chamber. Samples of healthy looking plants and plants with typical 

symptoms were taken and fixed. Unstained hand-cuttings were analyzed with fluorescence 

microcopy. The development of zoosporangia on the leafsurface was analyzed using the vital 

stain FUN®1 (Molecular Probes). Natural autofluorescence of P. ramorum and plant tissue is 

enhanced using the fixing fluid FAA (formaline-aceto-alcohol). Epifluorescence images 

showing P. ramorum structures in different tissues and in different stages of disease 

development are presented. An overview of the development of P.ramorum in Rhododendron 

root, twig and leaf tissue is offered for discussion. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Institute for Plant Protection in Horticulture, Federal Biological Research Centre for Agriculture and Forestry 

(BBA), Messeweg 11/12, D-38104 Braunschweig, Germany. 

3

 Laboratory of Applied Genetics and Plant Health, Institute Earth Nature and Landscape, Univ. of Applied Sciences 

of Western Switzerland, 150 Route de Presinge 1254 Jussy, Switzerland. 

Corresponding author: m.riedel@bba.de. 
GENERAL TECHNICAL REPORT PSW-GTR-221

316 

Phytophthora Species in Finland1

Anna Rytkönen,2 Arja Lilja2, Päivi Parikka3, Asko Hannukkala3, 

Mirkka Kokkola4

 and Jarkko Hantula2 

Abstract 

Potato late-blight epidemics caused by Phytophthora infestans have been reported in Finnish 

newspapers since 1847. The pathogen has caused considerable losses in potato fields in 

southern Finland. Before the late 1970's only the A1 mating type of the heterothallic pathogen

was present outside Mexico. At the early 1980's a new potato late blight population migrated 

from Mexico to Europe and replaced the old asexual population (Fry eand others 1993). The 

first indication of the change was the appearance of A2 mating type of P. infestans in Europe 

(Hohl and Iselin 1984). The monitoring of the occurrence of A2 mating type in Finland was 

started in 1992 when mating type A2 proved to be present in a very low proportion in the 

potato late blight population. Unfortunately there were no older isolates of the pathogen 

available and the exact time of migration of A2 remains unsolved (Hermansen and others 

2000). In the Northern climate the cold winters limit the survival of the asexual stage of 

potato late blight fungus in infected tubers in frozen soil. Therefore the build up of blight 

epidemics derived from asexual sources of primary inoculum usually was delayed until the 

end of August in Finland (Mäkelä, 1966). The oospores formed in a sexually reproducing 

population provide a new serious overwintering source of primary inoculum independent of 

the survival of the tubers during cold winters in Finland. 

Phytopthora cactorum was first time isolated in Finland in the early 1990's from strawberries 

suffering from leather rot (Parikka 1991). Since then the pathogen has caused losses in 

strawberry production as an agent of crown rot as well as causing stem lesions on silver birch 

in forest nurseries. Fosetyl-Al used to control the pathogen on both host plants. The genetic 

variation on P. cactorum in Finland has been studied by Random Amplified Microsatellite 

(RAMS) and Random Amplified Polymophic DNA (RAPD) analyses. The Finnish isolates 

from birch have been shown to contain considerable genetic variation, in contrast to the 

strawberry crown rot isolates, which are found by fingerprinting analyses to be largely clonal 

in Europe (Lilja and others 1998, Hantula and others 1997, 2000, Eikemo 2004). As well as 

separating genetically in fingerprinting analyses, the strains causing crown rot on strawberry 

and stem lesions on birch in Finland have also shown to be pathogenically separate. P. 

cactorum isolates from strawberry have been able to infect birch, but strawberry seedlings 

have not been affected by isolates from birch in pathogenity tests (Hantula and others 2000). 

We have also baited P. cactorum from a natural pond from which a forest nursery is taking its 

water for irrigation. The pathogen was present in the pond every year during the 3-year study. 

The P. cactorum strains in the pond were tested by RAMS analysis to be genetically similar to 

those causing symptoms on birch in the nursery. 

Phytopthora ramorum was described in 2001 in Germany (Werres and others) causing twig 

blight on Rhododendron and Viburnum, and it has later been found to be the cause of sudden 

oak death (SOD) disease in Quercus and Lithocarpus spp. in California (Rizzo and others 

2002). The pathogen was recognized worldwide as a threat both to the nurseries and wild 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Finnish Forest Research Institute, Vantaa Research Centre, P.O. Box 18, FI-01301 Vantaa, Finland. 3 MTT Agrifood Research Finland, FI-31600 Jokioinen, Finland. 4

 Finnish Food Safety Authority EVIRA, Plant Protection Department, P.O. Box 42, FI-00501 Helsinki, Finland. 

Corresponding author: anna.rytkonen@metla.fi. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

317 

plant populations, and many countries created quarantine programs in order to limit its spread. 

Phytopthora ramorum is a regulated pathogen also in Finland. In the spring 2004 P. ramorum

was isolated from marketed Rhododendron plants imported from other EU countries (Lilja 

and others 2007). In August 2004 the pathogen was also found in one Finnish nursery on 

German Rhododendron catawbiense plants and on several Finnish Rhododendron spp.

cultivars. P. ramorum was detected by species specific PCR and isolated. Phytopthora 

ramorum has been found every year since 2004 on plant material transported to Finland from 

other EU countries. 

In surveys for P. ramorum on Finnish Rhododendron cultivars, another Phytophthora sp. was 

isolated in 2004 and 2005 from Rhododendron spp. It was confirmed as Phytopthora inflata

by morphological characteristics and beta tubulin gene sequencing, where one identical 

sequence was found in the GenBank. It is likely that our P. inflata strain has been imported 

with the ornamental plant trade, since the reference strain has been isolated from Syringa sp. 

in the U.K. in 1990 (Hall and others 1992). 

The pathogenicity of P. inflata and P. ramorum isolates to the most common Finnish tree 

species and ornamentals was tested by stem-wound inoculation tests. Both P. inflata and P. 

ramorum caused necrotic lesions on Alnus glutinosa, A. incana and Betula pendula, but P. 

ramorum was less pathogenic than P. inflata. P. inflata was also able to infect Picea abies, 

Vaccinium myrtillus, V. uliginosum, V. vitis-idaea, V. angustifolium and Fragaria x ananassa. 

Viburnum lentago was the only plant tested showing susceptibility to P. ramorum while being 

resistant to P. inflata. Pinus sylvestris was resistant to both Phytophthora species. Survival at 

low temperatures was tested by inoculating apples with Phytophthora isolates and incubating 

them at -5 oC for two weeks. The growth was evaluated after plating on MA. P. ramorum 

isolates remained viable after two weeks at -5 oC in laboratory conditions, but isolates of P. 

inflata did not. However, we have been able to isolate viable P. inflata from host plants 

(Rhododendron 'Cunningham's White' and Vaccinium angustifolium) in a private garden one 

year after first detection, which indicates that P. inflata is able to survive in Finnish winter 

conditions.

Literature Cited 

Fry, W.E.; Goodwin, S.B.; Dyer, A.T.; Matustsak; J.M., Drenth, A.; Tooley, P.W.; 

Sujkovski L.S.; Koh, Y.J.; Cohen, B.A.; Spielman, L.J.; Deahl, K.L.; Inglis, D.A.; 

Sandlan, K.P. 1993. Historical and recent migrations of Phytophthora infestans - chronology, 

pathways and implications. Plant Disease 77: 653-661. 

Hall, G.; Dobson, S.; Nicholls, C. 1992. First record of Phytophthora inflata in the United 

Kingdom. Plant Pathology 41: 95-97. 

Hantula, J.; Lilja, A.; Parikka, P. 1997. Genetic variation and host specificity of 

Phytophthora cactorum isolated in Europe. Mycological Research 101 (5): 565-572. 

Hantula, J.; Lilja, A.; Nuorteva, H.; Parikka, P.; Werres, S. 2000. Pathogenicity, 

morphology and genetic variation of Phytophthora cactorum from strawbeyy, apple, 

rhododendron and silver birch. Mycological Research 104 (9): 1062-1068. 

Hermansen, A.; Hannukkala, A.; Naerstad, R.H.; Brurberg; M.B. 2000. Variation in 

populations of Phytophthora infestans in Finland and Norway: mating type, metalaxyl 

resistance and virulence fenotype. Plant Pathology 49:11-22 

Hohl, H.R.; Iselin, K. 1984. Strains of Phytophthora infestans from Switzerland with A2 

mating type behaviour. Transactions of the British Mycological Society 83: 529. 
GENERAL TECHNICAL REPORT PSW-GTR-221

318 

Eikemo, H.; Klemsdal, S.S.; Riisberg, I.; Bonants, P.; Stensvand, A.; Tronsmo, A.M. 

2004. Genetic variation between Phytophthora cactorum isolates differing in their ability to 

cause crown rot in strawberry. Mycological Research 108(3): 317-324. 

Lilja, A.;, Karjalainen, R.; Parikka, P.; Kammiovirta, K.; Nuorteva, H. 1998. 

Pathogenity and genetic variation of Phytophthora cactorum from silver birch and strawberry. 

European Journal of Plant Pathology 104: 529-535. 

Lilja, A.; Rytkönen, A.; Kokkola, M.; Parikka, P.; Hantula, J. 2007. Report on the first 

finding of Phytophthora ramorum and P. inflata in ornamental rhodorendrons in Finland. 

Plant Disease 91(8): 1055. 

Mäkelä, K. 1966. Factors influencing the epidemics of Phytophthora infestans (Mont.) de 

Bary in Finland. Suomen Maataloustieteellisen Seuran Julkaisuja, Acta Agralia Fennica. No 

104. 

Parikka, P. 1991. Phytophthora cactorum on strawberry in Finland. Nordisk 

Jordbruksforskning 73, 121. 

Rizzo D.M.; Garbelotto, M.; Hansen, E.A.; 2005. Phytophthora ramorum as the cause of 

extensive mortality of Quercus spp. and Lithocarpus densiflorus in California. Plant Disease 

86: 205- 

Seppänen, E. 1971. Influence of weather conditions and late blight on the yields of potatoes 

in Finland, 1931-62. Annales Agriculturae Fenniae 10: 69-109. 

Werres, S.; Marwitz, R.; Man In't Veld, W.A.; De Cock, W.A.M.; Bonants, P.J.M.; De 

WeertThemann, K.; Ilieva, E.; Baayen, R.P. 2001. Phytophthora ramorum sp. nov. a new 

pathogen on Rhododendron andViburnum. Mycological Research 109: 115-1165. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

319 

Screening Wild Cherry (Prunus avium ) 

Micropropagated Clones for Resistance to 

Four Phytophthora Species1

Alberto Santini,2 Luisa Ghelardini,2 Anna De Rogatis,3 Serena 

Guerri,3 and Stefano Vedele3

Abstract 

Prunus avium L., wild cherry, is a valuable component of diversity in mixed coniferous 

forests and temperate broadleaf forest ecosystems in Europe. Wild cherry is also one of the 

most prized species for wood production, and as a result it has been increasingly planted both 

in artificial stands and in natural forests. A number of Phytophthora species have been 

associated with root rot of wild and cultivated cultivars of cherry trees under different 

environmental conditions. In vitro Phytophthora pathogenicity assays are commonly used in 

order to avoid soil contamination and to speed up the selection procedure to obtain resistant 

plants. For these assays the cited Authors used callus tissue cultures, however, the symptom 

assessment could lead to questionable results far from the natural host-pathogen interactions. 

Thus, the purpose of this paper was to determine in vitro the pathogenicity of four 

Phytophthora isolates of several species (P. alni ssp. uniformis, P. megasperma var. 

megasperma, P. citrophthora, and P. cinnamomi) on micropropagated wild cherry genotypes, 

previously selected for having superior phenotypic characters and adapted to different Italian 

environments, by using plantlets 10 cm high cultivated in sterile environment. Host 

susceptibility was evaluated in accordance with a disease score scale, taking into account the 

percent of yellowing/wilting. All the wild cherry clones resulted highly susceptible to P. 

citrophthora. The P. cinnamomi virulence varied according to the challenged clone, while P. 

alni ssp. uniformis and P. megasperma var. megasperma were able to cause only modest 

symptoms. Three of the clones showed resistance to the last three pathogens. The results are 

consistent with what reported in literature. The method is functional and quick, moreover any 

contamination risk of the environment by the pathogens spores is avoided. For these reasons it 

could be considered for early resistance screening tests. On the other hand, the need of a 

specific protocol for regeneration and multiplication of each clone acted as a limiting factor in 

terms of number of challenged clones and replicates of each clone. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Istituto per la Protezione delle Piante C.N.R. - Via Madonna del Piano 50019, Sesto fiorentino, Italy. 3

 Istituto Sperimentale per la Selvicoltura C.R.A. - Viale Santa Margherita 80, 52100 Arezzo, Italy. 

Corresponding author: a.santini@ipp.cnr.it. 
GENERAL TECHNICAL REPORT PSW-GTR-221

320 

Involvement of Phytophthora Species in the 

Decline of Beech (Fagus sylvatica) in the 

Southern Part of Belgium1

S. Schmitz,2 J. Zini,2 and A. Chandelier2 

Abstract 

During the last decade, typical symptoms of Phytophthora diseases were observed in beech 

stands of several European countries. The main symptoms were the presence of bleeding 

cankers on the stem, a low crown density as well as the yellowing of foliage and the small 

size of leaves. Several species of Phytophthora, such as Phytophthora citricola, P. cambivora

and P. cactorum, were reported as the causal agents. 

In order to evaluate the implication of the different Phytophthora species in beech decline in 

the southern part of Belgium, a monitoring was undertaken with the help of managers of 

public and private forests. Phytophthora strains isolated from beech of different stands as well 

as from soil were characterized through morphological and molecular analyses (PCR-RFLP of 

ITS). All the isolated strains were identified as P. cambivora which is considered as one of 

the most aggressive Phytophthora species involved in beech decline in Europe. 

Molecular analysis was also directly applied to necrosed tissues of bleeding beeches and 

enabled the detection of additional cases. All positive cases exhibited a profile characteristic 

of the P. cambivora species, except for one of the sampled trees showing a different RFLP

profile. Identification of the involved species is ongoing. 

Regarding P. cambivora, both mating types (A1 and A2) were identified, sometimes in the 

same sampling site. Ornamented oogonia characteristic of the P. cambivora species were 

produced by pairing A1 and A2 strains isolated from the same site. Oospores from 

intraspecific pairing were characterized in terms of viability and germinability. 

Introduction 

During the last decade, typical symptoms of Phytophthora diseases were observed in 

beech stands of several European countries (Jung and others 2005). The main 

symptoms are the presence of bleeding cankers on the stem and typical necroses of 

inner bark tissues. A low crown density, as well as yellowing of foliage dectreased 

leaf size, are also observed. Several species of Phytophthora, such as Phytophthora 

citricola, P. cambivora and P. cactorum, were reported as the causal agents. In order 

to evaluate the involvement of the different Phytophthora species in beech decline in 

the southern part of Belgium (Wallonia), trees were sampled on the basis of 

notification from the managers of public and private forests. Inner bark tissues 

displaying necroses were collected at the margin between infected and healthy areas. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2 Walloon Agricultural Research Centre, Department Biological Control and Plant Genetic Resources, Rue de Liroux 

4, 5030 Gembloux, Belgium. 

Corresponding author: schmitz@cra.wallonie.be. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

321 

Soil samples were also collected at the foot of some of the bleeding beeches using an 

auger. 

Methods 

Identification

Microbiological and molecular methods were used to determine the occurrence of a 

Phytophthora disease. 

Nineteen Phytophthora strains were isolated from necroses of the inner bark of 

bleeding beeches. Isolations from rhododendron leaves used as baits enabled the 

recovery of one additional strain from a soil sample. Among these twenty strains, 

nineteen showed identical morphological characteristics: uniform colonies on V8 

juice agar; no chlamydospore; nonpapillate, nondeciduous, ovoid sporangia; no 

oogonium in pure culture. They were identified as P. cambivora. Only one strain 

presented different morphological characteristics, notably corresponding to P. 

gonapodyides: petaloid colony on V8 juice agar; no chlamydospore; nonpapillate, 

nondeciduous, ovoid sporangia; nesting of sporangia; no oogonium in pure culture or 

after pairing with tester strains. 

Molecular analysis was used to confirm the specific identification of the isolated 

strains but also to detect additional positive cases from beech necrotic tissues. Total 

DNA was extracted from the mycelium of the isolated strains or from necrotic tissues 

using the HighPure PCR Template Preparation kit (Roche Diagnostic). PCR-RFLP 

was carried out on total DNA using the test developed by Cooke and others (2000) 

targeting the ITS rDNA and the test of Martin and Tooley (2004) on the mtDNA. 

With both methods, the identity of P. cambivora was confirmed for the nineteen 

collected isolates. Six additional cases of infection by this Phytophthora species were 

detected from necrotic tissues. The identity of the P. gonapodyides strain was 

established by sequencing the ITS rDNA. Sequence similarity searching was 

achieved using the FASTA program. One additional case of infection caused by P. 

gonapodyides was revealed by PCR-RFLP of the ITS rDNA from necrotic tissues. 

Geographic Distribution 

A total of 83 beeches presenting bleeding cankers on the trunk were sampled in 49 

different sites in the southern part of Belgium. Isolation proved the infection of 

nineteen beeches by a Phytophthora: eighteen by P. cambivora and one by P. 

gonapodyides. PCR-RFLP from necrotic tissues enabled the detection of a 

Phytophthora on seven additional beeches: six infected by P. cambivora and one by 

P. gonapodyides. These 26 positive cases were identified in nineteen different sites 

mainly distributed around the French border (fig 1). However, the non-uniform 

character of sampling makes interpretation difficult. 

No preferential orientation of necroses was observed among affected trees. 

Dimensions of trees infected by a Phytophthora were highly variable but the smallest 

tree had a circumference of 80 cm. For half of the affected trees, bleeding cankers 

appeared from collar and reached 6 to 10m high in 27 percent of the cases. 
GENERAL TECHNICAL REPORT PSW-GTR-221

322 

Figure 12Geographic distribution of the sampling sites in Wallonia: ○

Phytophthora-free beeches; ● beeches from which a Phytophthora was 

isolated; ▲ beeches from which a Phytophthora was detected by PCR-RFLP 

Characteristics of the Isolated Strains 

Mating Type and Viability of Oospores 

Proportions of each mating type (A1 and A2) were evaluated among P. cambivora

isolates by pairing with reference strains of known mating type (Dr Werres – BBA, 

Germany). It was established that, among the nineteen P. cambivora strains, 

seventeen were from A2 type and two were from A1 type. Both mating types were 

identified in the site of Aublain. Mating between A1 and A2 strains of this site 

produced ornamented oogonia. So far, attempts to obtain germinated oospores from 

mating between Belgian isolates of P. cambivora were unsuccessful but their 

viability was evaluated using the staining method of Sutherland and Cohen (1983). 

Most of the oospores (76 percent) presented a purple coloration corresponding to 

dormant structures. Unstained oospores (non viable) were also frequently 

encountered (22 percent). Blue staining associated with active structures was 

observed for 1 percent of the oospores. 

Pathogenicity Tests 

One isolate of each mating type of P. cambivora was tested for pathogenicity on 

woody twigs of three forest species (Fagus sylvatica, Castanea sativa and Quercus 

petraea). Inoculations (six replicates) were performed by inserting agar plugs into 

bark flaps at the middle of the twig. Necroses were observed on the three forest 

species regardless of the strain inoculated. No symptom developed on negative 

controls. Moreover, it was noticed that necroses on Fagus sylvatica were particularly 

scattered while a continuous necrosis of the whole twig was observed for Castanea 

sativa and Quercus petraea. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

323 

Conclusions 

The study demonstrates the involvement of the genus Phytophthora in the decline of 

beech stands in Wallonia. A sampling was conducted on 83 bleeding beeches and P. 

cambivora – known to be the most aggressive species among the different 

Phytophthora involved in the decline of beech in Europe (Jung and others 2005), was 

detected 24 times in 18 different sites by combined use of morphological and 

molecular analyses. Moreover two affected trees proved to be infected by P. 

gonapodyides. Only five sites show multiple infections of beeches by a Phytophthora

(from two to five affected trees). In the other sites, single infections were observed 

and affected trees were generally surrounded by many beeches without symptom. 

Both mating types of P. cambivora are present in Wallonia, sometimes in the same 

site, and pairing between A1 and A2 isolates produced oospores in vitro. 

Literature Cited 

Cooke, D.E.L.; Duncan, J.M.; Williams, N.A.; Hagenaa-de Weerdt, M.; Bonants, P. 

2000. Identification of Phytophthora species on the basis of restriction enzyme fragment 

analysis of the internal transcribed spacer regions of ribosomal RNA. EPPO Bulletin. 30: 519-

523. 

Jung, T.; Hudler, G.W.; Jensen-Tracy, S.L.; Griffiths, H.M.; Fleischman, F.; Osswald, 

W. 2005. Involvement of Phytophthora species in the decline of European beech in Europe 

and the USA. Mycologist. 19: 159-166. 

Martin, F.N.; Tooley, P.W. 2004. Identification of Phytophthora isolates to species level 

using restriction fragment length polymorphism analysis of a polymerase chain reactionamplified region of mitochondrial DNA. Phytopathology. 94 (9): 983-991. 

Sutherland, E.D.; Cohen, S.D. 1983. Evaluation of tetrazolium as a vital stain for fungal 

oospores. Phytopathology. 73 (11): 1532-1535. 
GENERAL TECHNICAL REPORT PSW-GTR-221

324 

Phosphite Application as an Explorative 

Tool for Understanding and Controlling 

Eucalyptus gomphocephala (Tuart) Decline 

in Southwest Western Australia1

Peter M. Scott,2,3 Harry T. Eslick,3 Paul A. Barber,2,3 Michael C. 

Calver,3 Giles E.St.J. Hardy,2,3 and Bryan L. Shearer4

Abstract 

Eucalyptus gomphocephala is a Mediterranean forest canopy species endemic to a narrow (5-

10 km wide) coastal strip approximately 300 km in length in south-west Western Australia. 

Eucalyptus gomphocephala is undergoing a significant decline that was first identified as a 

spot decline in 1994 and now occurs throughout large sections of its remnant distribution 

within Yalgorup National Park, in some areas resulting in 100 percent mortality. The 

reduction of this keystone species represents a significant modification to the associated 

ecosystem. Modifications to hydrology, fire regimes, entomological pressures, and fungal and 

Pythiaceous soil pathogens have been identified as possibly contributing to the decline 

syndrome. The potential of phosphite (phosphonate), nutrient and insecticide treatments to 

reverse the decline in tree health was assessed as (a) a method for controlling the decline and 

(b) a method for diagnosing possible causal agents. Phosphite has been successfully used to 

control Phytophthora and Pythiaceous soil pathogens by inducing a host defense response 

within the plant. Stem injection of declining Eucalyptus gomphocephala in the present study 

has resulted in improved canopy health and vigor, indicating that Phytophthora and/or other 

Pythiaceous microorganisms may be playing a role in the decline. The impact of phosphite 

application on nutrient uptake and fine feeder root concentration was also assessed. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Centre of Phytophthora Science and Management, School of Biological Science and Biotechnology, Murdoch 

University, Murdoch, Western Australia 6150, Australia. 

3

 Tuart Health Research Group, School of Biological Science and Biotechnology, Murdoch University, Murdoch, 

Western Australia 6150, Australia. 

4

 Science Division, Department of Environment and Conservation, Locked Bag 104, Bentley Delivery Centre, WA 

6983, Australia. 

Corresponding author:P.Scott@murdoch.edu.au. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

325 

An Evaluation of Stream Monitoring 

Techniques for Surveys for Phytophthora 

Species in Victoria, Australia1

Benjamin Smith,2 Ian W. Smith,2 Rodney H. Jones,3 

and James Cunnington3

Abstract 

In Australia, surveys for Phytophthora species generally rely on either soil or plant testing to 

confirm their presence in an area. Stream monitoring has become an important part of early 

detection surveillance systems for Phytophthora ramorum in the USA. In the present study, 

water monitoring methods used for the detection of Phytophthora species in streams in the 

USA were evaluated in surveys in June 2006 and March 2007 at 8 sites and 4 locations to the 

east of Melbourne, Australia. 

At each site, five leaves from four different bait types were floated in mesh bags in the 

streams for periods varying from one to three weeks depending on the bait type (total 160 

baits). In the first survey in June, bait types included Rhododendron leaves (undamaged and 

cut in a herringbone pattern), mature Eucalyptus regnans leaves and E. sieberi cotyledons, a 

bait used often for isolation of Phytophthora cinnamomi from soil in Victoria. In the March 

survey, Pittosporum undulatum leaves were added as baits and only undamaged 

Rhododendron leaves used, along with the other eucalypt baits. One litre of water was 

collected from each site at the end of each baiting period and filtered in the laboratory using 

0.2μm Metricel membrane filters. Lesions that developed on the bait samples, and the 

complete filters, were plated directly onto P5ARP(H) agar. DNA sequencing of the rDNA ITS 

region was carried out on purified cultures of Phytophthora species isolated from the plates. 

Phytophthora species were isolated from all sites, bait types and filters used in the study. 

Isolated species included Phytophthora cinnamomi, P. citricola, P. gonapodyides, 

undescribed Phytophthora species from clades 6 and 7, and an unknown, potentially new 

clade. Species isolated varied with season, bait and filter used. This highlights the need to use 

a variety of parameters for stream monitoring when targeting a wide range of Phytophthora 

species. 

Stream baiting has potential as a valuable method to determine the presence of a range of 

Phytophthora species in catchments across Australia. It also provides a valuable tool for early 

detection of Phytophthora species, and needs to be included in Australia’s network of 

quarantine surveillance activities. The method may also be potentially applicable to the 

monitoring of stream and dam water for use in irrigation and fire fighting activities, and of 

water runoff from gravel pits and nurseries so as to identify the presence of the pathogens, and 

reduce their spread across Australia. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 School of Forest and Ecosystem Science, University of Melbourne, 123 Brown St (PO Box 137), Heidelberg 

Victoria, 3084, Australia. 

3

 Primary Industries Research, Victoria, Department of Primary Industries, Private Mail Bag 15, Ferntree Gully 

Delivery Centre, Victoria 3156, Australia. 

Corresponding author: ismith@unimelb.edu.au. 
GENERAL TECHNICAL REPORT PSW-GTR-221

326 

Molecular Testing Uncovers New 

Phytophthora Taxa From Natural 

Ecosystems in Western Australia1

M.J.C. Stukely,2 J.L. Webster,2 J.A. Ciampini,2 T.I. Burgess,3 D. 

White,3 W.A. Dunstan,3 and G.E.St.J. Hardy3

Abstract 

Verification of mapping of the extent of Phytophthora dieback disease, based on shadowless 

colour aerial photography, involves the routine testing of soil and root samples collected from 

beneath dying, Phytophthora-sensitive native plant “indicator species” for the presence of the 

pathogen. In addition to P. cinnamomi, other isolates have been recovered on selective agar 

following the baiting of soil, or the direct plating of plant tissue, during these operations. 

These have been identified, using morphological characters, as P. citricola, P. megasperma, 

P. cryptogea, P. drechsleri, P. nicotianae, and P. boehmeriae. 

The recent advent and availability of DNA sequencing techniques for the identification of 

Phytophthora species has enabled the testing of new isolates that were difficult to identify 

from their morphology, as well as a range of historical isolates dating back to the 1980s from 

the Department of Environment and Conservation culture collection. 

DNA was extracted from pure cultures grown on cornmeal agar and the Internal Transcribed 

Spacer (ITS) regions of the rRNA were amplified using primers ITS6 and ITS4. BLASTn 

searches of sequence data were conducted in GenBank to determine the most closely related 

Phytophthora spp. Sequences were then aligned and parsimony and distance analyses 

conducted in PAUP. Based on phylogenetic analysis, seven potentially new and undescribed 

taxa of Phytophthora can be distinguished. Several of these are morphologically 

indistinguishable from known species (eg P. citricola, P. megasperma, and P. cryptogea). In 

some cases the new taxa are indeed most closely related to the known species (eg P. 

citricola), but in others their DNA sequences show that they are not closely related to the 

morphologically similar species (eg P. megasperma). 

Phytophthora inundata, described in Europe in 2003, has been identified based on 

phylogenetic analysis from several locations in the south-west where it has been associated 

with dying native plants. Some of these isolates have been stored since the 1980s. One of the 

new species, with morphology similar to P. citricola, but most closely related 

phylogenetically to P. bisheria and P. multivesiculata, has been isolated from dying 1- to 2-

year-old jarrah (Eucalyptus marginata) seedlings in rehabilitated open-cut bauxite mine pits. 

Further work is planned to describe the new taxa and their relationships, and to test their 

pathogenicity, so that an estimate of the level of threat they pose to native vegetation can be 

made. 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Department of Environment and Conservation, Science Division, Locked Bag 104, Bentley Delivery Centre, WA 

6983, Australia. 

3

 Centre for Phytophthora Science and Management, School of Biological Sciences and Biotechnology, Murdoch 

University, Murdoch, WA 6150, Australia. 

Corresponding author: mike.stukely@dec.wa.gov.au. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

327 

Long-Term Storage of Phytophthora

Cultures in Water1

Wendy Sutton,2 Paul Reeser,2 and Everett Hansen2

Abstract 

Long-term storage of cultures of Phytophthora species is a challenge for any lab managing a 

working collection of isolates. Storage in liquid nitrogen is generally considered to be optimal 

for archival storage, and successful recovery of most species is regularly achieved after many 

years. Nitrogen storage has its drawbacks, however, especially for a working collection. It 

requires species specific freezing conditions, and thawing must be done carefully. Equipment 

is bulky, and regular addition of liquid nitrogen is necessary. Storage in vials in water at room 

temperature is an efficient, effective alternative for many collections. Isolates are grown on 

agar, and plugs are removed from the colony margin and placed in sterile water in 1.5 ml 

plastic tubes, with or without pieces of sterilized hemp seed. Tubes are kept in coded racks at 

room temperature, in the dark. Agar plugs can be removed one at a time as needed and plated 

on selective agar to resume growth. In a recent test, we had nearly 100 percent recovery of 

several Phytophthora species, including P. ramorum, after 7 years in water storage. 

Introduction 

Long-term storage of cultures of Phytophthora species is a challenge for any lab 

managing a working collection of isolates. Storage in liquid nitrogen is generally 

considered to be optimal for archival storage, and successful recovery of most species 

is regularly achieved after many years. Nitrogen storage has its drawbacks, however, 

especially for a working collection. It requires species-specific freezing conditions, 

and thawing must be done carefully. Equipment is bulky, and regular addition of 

liquid nitrogen is necessary. 

In 1976 Boesewinkel described a new storage method for storing Phytophthora using 

colonized agar blocks in bottles (90 x 26 mm) of sterilized distilled water. This 

method eliminated the space and cost issues associated with nitrogen storage. We 

have adapted the method, using smaller vessels and adding hemp seeds. 

Methods 

Culture Storage 

Water vials are 1.5 ml of de-ionized water placed in 1.5 ml cryogenic vials. Hemp 

seed/water vials are one and a half ml of de-ionized water and one hemp seed placed 

in 1.5 ml cryogenic vials. The vials are sealed, sterilized by autoclaving and cooled to 

room temperature. 

 

1 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Department of Botany and Plant Pathology, Oregon State University, Corvallis OR 97331, USA. 

Corresponding author: suttonw@science.oregonstste.edu. 
GENERAL TECHNICAL REPORT PSW-GTR-221

328 

Isolates are grown on agar (usually corn meal agar amended with 20 ppm β

sitosterol) for five to ten days. Each isolate is stored in a pair of vials, one 5mm plug 

placed in a hemp seed/water vial and three to five plugs in a water vial. The vials are 

placed in CryoBoxes and stored in the dark at room temperature. 

Culture selection 

Species selection for this test was based on availability in long term storage 

and interest to the larger community. Cultures that have not been identified to 

species were not included in this test. When possible, five isolates per species 

were included. 

Water Tube Culture Retrieval 

A single plug was retrieved from a water vial and plated on the selective 

medium CARP (17 g BBL Corn Meal Agar, 1 L de-ionized water, 20ppm 

Delvocid (50 percent natamycin salt), 200ppm Ampicillin, sodium salt and 

10ppm Rifamycin SV sodium salt). At 14 days plates were examined for 

growth. If new mycelium had grown into the agar it was scored a success. If 

no new mycelium was present the water storage vial was re-sampled, this time 

plating all remaining plugs. Again if there was new growth in 14 days it was 

scored as a success and if there was no growth it was considered a failure. 

Hemp/water tube culture retrieval 

Both the hemp seed and plug were plated on CARP. Frequently in the hemp tubes a 

mass of mycelium forms and this was plated as well. At 14 days the plates were 

examined for new growth. New mycelium emerging from the plug, hemp seed or 

mycelial mass was scored a success. 

Results 

Cultures were successfully retrieved from 72 of the 78 water vials after three to eight 

years in storage (Table 1). Of the six failures, the four 7-year-old P. lateralis and one 

5-year-old P. ilicis vials had only one remaining plug and so couldn’t be re-sampled. 

The 8-year-old P. megasperma had five non-viable plugs. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

329 

Table 12Water vial culture success ratios (number of successful 

retrievals/number of vials sampled) by years stored. 

YEARS IN STORAGE 

SPECIES 8 7 6 5 4 3 Total 

P. cactorum 1/1 5/5 6/6 

P. cambivora 2/2 2/2 4/4 

P. cinnamomi 3/3 3/3 

P. citricola 2/2 1/1 1/1 4/4 

P. cryptogea 2/2 2/2 4/4 

P. sp. “DF1” 5/5 5/5 

P. europaea 1/1 1/1 

P. gonapodyides 2/2 5/5 7/7 

P. ilicis 0/1 1/1 1/2 

P. lateralis 0/4 3/3 3/7 

P. megasperma BHR 7/8 7/8 

P. nemorosa 5/5 5/5 

P. sp. “Pg chlamydo” 5/5 5/5 

P. pseudosyringae 4/4 4/4 

P. pseudotsugae 1/1 1/1 

P. ramorum 7/7 7/7 

P. siskiyouensis 5/5 5/5 

Totals 21/22 5/9 19/19 14/15 11/11 2/2 72/78 

Cultures were successfully retrieved from 34 of the 38 hemp/water vials after three to 

six years in storage (Table 2). Of the four failures, two had bacterial contamination. 

Of the successes, in all but one case, both the hemp/water plug and the hemp 

seed/mycelium mass were viable. 

Table 22Hemp seed/water vial culture success ratios (number of successful 

retrievals/number of vials sampled) by years stored. 

YEARS IN STORAGE 

SPECIES 6 5 4 3 Total 

P. cactorum 

P. cambivora 5/5 2/2 7/7 

P. cinnamomi 3/3 3/3 

P. citricola 1/1 1/1 2/2 

P. cryptogea 2/2 2/2 4/4 

P. sp. “DF1” 

P. europaea 

P. gonapodyides 2/2 4/5 6/7 

P. ilicis 0/1 0/1 

P. lateralis 

P. megasperma BHR 

P. nemorosa 

P. sp. “Pg chlamydo” 

P. pseudosyringae 3/4 3/4 

P. pseudotsugae 

P. ramorum 2/2 3/3 5/5 

P. siskiyouensis 4/5 4/5 

Totals 8/8 16/18 8/10 2/2 34/38 
GENERAL TECHNICAL REPORT PSW-GTR-221

330 

Comparing the success of the two methods was possible in 31 cases, where isolates 

had been stored in both water and hemp/water vials at the same time (Table 3). 

Table 3—Culture success comparing water vial to hemp/water vial. 

 Water Vial 

Hemp/Water Vial Success Failure 

Success 27 0 

Failure 3 1 

Discussion 

There are many advantages to this type of culture storage, cost, ease and high 

success rate to name a few. The ability to remove one plug at a time is more 

efficient than thawing and plating an entire vial. However, there are a few 

cautions. Repeated visits to the same tube increase the odds of contamination. 

Our “room temperature” ranges from about 18-24oC, but some species may 

need special conditions, especially cooler or more controlled temperatures. 

After retrieval of a culture it’s a good idea to restore anything that’s been in 

storage more than two years. 

We have been using water storage exclusively for at least 12 years. Recently, 

P. lateralis was successfully cultured from 4 hemp vials stored in 1995. While 

there is no way of knowing which isolates are still viable until they are 

retrieved, over the course of the 12 years our success rate has been high. 

Literature Cited 

Boesewinkel, H.J. 1976. Storage of Fungal Cultures in Water. Trans. Br. Mycol. Soc. 

66:183-185. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 331 

Occurrence and Impact of Phytophthora 

Species in Forest Trees in Hungary1

Ilona Szabó2 and Ferenc Lakatos2

Abstract 

Occurrence of Phytophthora species and their phytopathological role have been investigated 

in forest stands in Hungary since 1999. Decline symptoms, specific stem lesions and 

unspecific top drying signs were surveyed in forest stands of different tree species. 

The isolation of Phytophthora was carried out from soil samples taken from around the 

diseased trees by baiting with Prunus laurocerasus leaves on selective medium PARPNH. 

The isolates were identified by morphological and molecular way. The morphological 

characters were observed in cultures growing on carrot agar medium. The formation of 

sporangia was induced by flooding the cultures with soil extract. The molecular identification 

was performed by sequencing the ITS regions of rDNA and comparing the sequences with the 

known Phytophthora sequences accessible in GenBank database. The pathogenicity of the 

isolates was tested by wound inoculation in the stem of seedlings and by root infections. 

Phytophthora species were found in Alnus glutinosa with bleeding stem lesions and crown 

drying symptoms, in Juglans nigra, Quercus petraea and Q. cerris with crown drying 

symptoms. The morphological and molecular identification resulted in 8 Phytophthora

species in Alnus (P. alni, P. citricola, P. gonapodyides, P. inundata, P. megasperma, P. sp.1, 

P. sp. 2., P. sp. 3.), 4 in Juglans (P. cactorum, P. cirticola, P. hedraiandra, P. sp.1) and 2 in 

Quercus (P. citricola, P. gonapodyides). The artificial inoculations caused well-delimited 

bark necrosis in the stem of the seedlings, the largest by P. alni in alder and P. citricola in 

black walnut, but generally not exceeding 3-4 cm length. Root infections resulted in lesion 

and reduction of the fine roots, most pronounced by P. citricola in black walnut. The impact 

of Phytophthora species on the healthy condition of the forest trees proved to be most 

important in planted Alnus glutinosa and Juglans nigra stands situated in wet sites and flood 

areas respectively. A community of Phytophthora species occurs in the rhizosphaera of these 

trees causing root and collar rot and bleeding stem lesions in alder and fine root reduction 

manifesting by crown drying in black walnut. The appreciation of the role of Phytophthoras 

in oak decline needs further research because of their fewer occurrences in the oak drying 

cases. The identified Phytophthora species, except of P. cactorum, were recorded first time 

in Hungary during this research work. 

Introduction 

Phytophthora root and collar rot of alder was found first time in Hungary in 1999 in 

some planted alder forests of medium age situated in Northwestern area of the 

country (Szabó et al. 2000). Typical bleeding stem lesions and crown drying 

symptoms also were common. The rate of symptomatic trees varied up to 20.6 

 

1

 A version of this paper was presented at the Fourth Meeting of IUFRO Working Party S07.02.09, Phytophthoras in 

Forests and Natural Ecosystems, August 26-31, 2007, Monterey, California. 

2

 Institute of Silviculture and Forest Protection, University of West Hungary, 5 Ady Endre Street, H-9400 Sopron, 

Hungary. 

Corresponding author: szaboi@emk.nyme.hu. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 332

percent and the mortality reached 10.7 percent in five forest subcompartments 

evaluated in 2000. The annual ring analysis showed that the disease has begun a few 

years earlier, and up to 20.7 percent dead trees were already removed during the 

precedent years. This first record of a destructive Phytophthora disease promoted the 

research of this group of pathogens in Hungary. Since 1999 long time investigations 

have been started in order to detect the occurrence of Phytophthora species and to 

clarify their phytopathological role in forest decline processes. 

Materials and Methods 

Decline symptoms, specific stem lesions and unspecific crown drying signs were 

surveyed in forest stands of different tree species all over the country. The 

Phytophthoras were isolated from soil samples taken from around the diseased trees, 

by baiting method using Prunus laurocerasus leaves as baits. The isolations were 

performed on selective medium, than the colonies were passed on PDA in tubes for 

long time storage. The isolates were identified by morphological and molecular way. 

The morphological characters were observed in cultures growing on carrot agar 

medium (Kröber 1985). The colony patterns and growth rate were recorded (Erwin 

and Ribeiro 1996). Formation of sporangia was induced by flooding the cultures with 

soil extract (Nagy and others 2000) after that the microscopical structures were 

observed and measured. The molecular identification was performed by sequencing 

the ITS1 and ITS2 regions of rDNA and comparing with the known Phytophthora

sequences accessible in GenBank. The DNA was extracted from pure culture using 

REDExtract-N-Amp kit from Sigma, PCR was performed with specific primers ITS6 

and ITS4 (Cooke and Duncan 1997). The pathogenicity of the isolates was tested by 

wound inoculation with mycelium in the stem of seedlings and by root infections.

Results 

Decline symptoms occurred in alder, oaks, beech, poplars and black walnut stands, as 

well as in silver birch, chestnut and horse chestnut individual trees. Phytophthora

species were found in Alnus glutinosa showing bleeding stem lesions and crown 

drying symptoms, in Juglans nigra, Quercus petraea and Q. cerris with crown drying 

symptoms. Between 1999 and 2005 a number of 376 isolates were collected and 

investigated, 98 of them were identified as Pythium by their morphology. The 

morphological and molecular identification resulted in eight Phytophthora species in 

Alnus (P. alni, P. citricola, P. gonapodyides, P. inundata, P. megasperma, P. sp.1, P. 

sp. 2, P. sp. 3), four in Juglans (P. cactorum, P. cirticola, P. hedraiandra, P. sp.1) 

and two in Quercus (P. citricola, P. gonapodyides). 

Alder stands were investigated in five different areas of Hungary. The host specific 

Phytophthora alni was isolated in stands of two areas. This species was found less 

frequently and only in the active disease processes of typical symptoms of fresh 

exudates at the stem basis of the trees. Phytophthora inundata, P. gonapodyides, and 

P. sp.1 were found the most common wide-spread species in alder stands. 

Phytophthora citricola, P. megasperma, P. sp.2 and P. sp. 3 occurred more rarely in 

alder stands, they are represented by only a few isolates. 
Proceedings of the Fourth Meeting of IUFRO Working Party S07.02.09 

 333 

Black walnut is cultivated in Hungary mostly in flood areas of Danube and some 

other wet lowland sites. Decline symptoms as yellowing the leaves, crown drying 

then death of the trees have been observed by the foresters some years ago. The 

investigation of the role of Phytophthora species started in 2004 in a heavily affected 

stand where the rate of the symptomatic and dead trees reached 42 percent. Bleeding 

lesions were not observed, unspecific crown drying occurred only. P. citricola was 

common in the soil around these trees, and P. cactorum also was less frequently 

detected. P. hedraiandra and P. sp. 1 are represented by only one isolate each from 

walnut. 

Phytophthora species were not found in the most of studied oak decline processes in 

Hungary. P. citricola and rarely P. gonapodyides were isolated in one site only, in a 

mixed forest near to Sopron, in soil samples taken from declining Q. petraea and Q. 

cerris trees. 

Pathogenicity tests were conducted in alder and black walnut seedlings using isolates 

of different Phytophthora species. The artificial inoculations caused well-delimited 

bark necrosis in the stem of seedlings, the largest by P. alni in alder and P. citricola

in black walnut. The lesions generally not exceeded 3-4 cm length, but some 

inoculations with P. citricola in black walnut resulted in large diffuse cankers 

growing along the stem and destroying the seedlings. Root infections resulted in 

lesion and reduction of fine roots, most pronounced by P. citricola in black walnut. 

Conclusions 

The impact of Phytophthora species on the healthy condition of forest trees proved to 

be most important in planted Alnus glutinosa and Juglans nigra stands situated in 

lowland sites and flood areas. A community of Phytophthora species occurs in the 

rhizosphaera of these trees causing root and collar rot and bleeding stem lesions in 

alder and fine root reduction manifesting by crown drying in black walnut. The 

appreciation of the role of Phytophthoras in oak decline needs further research 

because of their fewer occurrences in the oak drying cases. The identified 

Phytophthora species, except of P. cactorum, were recorded first time in Hungary 

during this research work (Szabó et al. 2000, Szabó 2003, 2005, Szabó and Lakatos 

2007). 

Acknowledgement

The research was supported by the Hungarian Research Found, grants OTKA T 

037352 and OTKA T 049077. 
GENERAL TECHNICAL REPORT PSW-GTR-221

 334

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