The 12th NIAS International

Workshop on Genetic Resources

Genetic and Functional
Agricultural
In conjunction

Diversity

Microorganisms

with the 10th International

Congress for Culture

ll-16 OCTOBER 2004
TSUKUBA, JAPAN

NationalofInstituteof Agrobiological

Sciences

Collections

Genetic and Functional
Proceedings

EditedDiversityof the 12th NIAS Internationalof Agricultural

Microorganisms

Workshop on Genetic Resources

by

KURISAKI, Jun-ichi
KAKU, Hisatoshi
SATO, Toyozo
AOKI, Takayuki
and

NAGAI, Toshirou

Publishedby
National

Instituteof Agrobiological

3 1 January 2005
Printedby
Asahi Printing

ISBN

4-931511-11-2

Co., Ltd, Tsukuba, Japan

Sciences,

Tsukuba,Japan

CONTENTS
Introduction

to the workshop

Jun-ichi Kurisaki and Taro Obata
1. Genetic

and functional

diversityof agricultural

microorganisms

Preface
Hisatoshi

Kaku and Kerry O 'Donnell

Mobile genetic elements contribution

to the differentiationof closely

relatedXanthomonas genomes
Marie-Anne VanSluys and Claudia B. Monteiro-Vitorello
Species recognition

and identificationof agriculturally

importantfiisaria:

current status

and future prospects
Kerry O 'Donnelly DavidM Geiser and Takayuki Aoki
Functional

analysisfor pathogenicity-related

genes of xanthomonads using

mutagenesis approach
Chaozu He and Wei Qian
Genetic diversityof Ralstonia

solanacearum strains in Japan and the Southeast

Asian countries
Kenichi Tsuchiya, Mitsuo Honta, WongBoonsuebsakul, Josehto, E. Villa
and Karden Mulya
Taxonomic analyses of soybean SDS and dry bean root-rot pathogens based on
a MAFF - NRRL collaboration
Takayuki Aoki and Kerry O 'Donnell
Systems biology
Yong-Hwan
Lee

initiatives

in the rice blast fungus, Magnaporthe grisea

2. New paradigms(Reproducedof biologicalresource centersby permission of ICCC- 1 0 Secretariat)

Preface
Hideaki Sugawara, Makoto M WatanabeandJean Swings
The new paradigm of the Biologicalof culture collections

Resource Centres (BRCs)

39
challenges

and opportunities

or BRCs

J. Swings and V.Storms
The criticalrole of Biological

40
Resource Centers in publichealthRaymondH. Cypess and Shung-Chang Jong
National

Biologicalfor life sciences

Resource Center to establish

43
the intellectual

infrastructure

and biotechnology

Ken-ichiro Suzuki
Microbial

diversity49
and pharmaceuticalindustry

culture collections

Dwight Baker

56

Biosafety

demands and the self-image

Christine

Rohde

3. Progressin the Researchof modern BRCs - Global challenges
62

on Agricultural

Microorganisms

Overview of the MAFF Genebank project
Toyozo Sato, Toshirou Nagai, Keisuke Tomioka, Kasumi Takeuchi, Motoko Iida
and MasaeKawada

69

Current status and future prospectsof collection

and use of microbial

resources in ACCC

Ruibo Jiang, Jingang Gu, Bingquan Fan, Xiaoxia Zhang, Xiaotong Ma, Shigui Li
and Zhiyong Ruan

71

Collection

and identificationof plant pathogenic

Apirusht

Somnth, Pattana Sontirat,

Fusarium in Thailand

Niyom Khaimook and Tharntip Bhasabutra.

74

Maintenance of microbial

genetic

resources by cryopreservationand freeze-drying

Toshirou Nagai, Toyozo Sato, Keisuke Tomioka, KasurniTakeuchi, Motoko Iida
and MasaeKawada

76

Long term preservationfor 20 years of entomogenous fungi

Sanae Wada, Ritsuko Murakami, Wataru Mitsuhashi,

Kazuhisa Miyamoto

and Kiyoshi Kawakami

78Grouping of bacteria

isolated

Hirosuke Shinohara,

Jun-ichirofrom leaf sheaths

and panicles

Enya, Shigenobu

of intact rice plants

Yoshida, Takao Tsukiboshi

and Seiya Tsushima
Pathogenic

diversity

80
ofXanthomonas oryzae pv. oryzae in Myanmar

Thein Lwin and Seint San Aye
Phylogenetic
of23S

analysis82

ofXanthomonasrRNA gene and 16S-23S

species based on the nucleotide

sequences

rDNA spacer region

Hirokazu Ochiai, Jean Swings and Hisatoshi

Kaku

86

Development of genome database system for Xanthomonas oryzae pv. oryzae
Masaru Takeya, Hirokazu Ochiai, Masahiro Satoh and Hisatoshi Kaku
PCR-based genomovar identification
complex strains isolated

and characterization

88

of Burkholderia

cepaciafrom diverse origins

Kenichi Tsuchiya, Sang-Tae Seo, Chika Fujitam, Hiroyuki Sawada, Takanobu Yoshida
and MamiTakahashi
Flagellin

glycosylation90
islandin Pseudomonas syringae

Kasumi Takeuchi, Fumiko Taguchi, Chihiro
Etsuko Katoh, Yoshishige

Yasuda, Hanae Kaku, Katsuyosh Murata,

Inagaki, Kazuhiro Toyoda, TomononShiraishi

and Yuki Ichinose

92

Huanglongbing

diseasemanagement in citrus in Indonesia-Current

Siti Subandiyah,

Andi Trisyono, Susamto Somowiyarjo, Andrew Beattie,

Zamir Hossain and Paul De Barro

research
Paul Holford,94

A preliminary

study of a toxin associatedSiti Subandiyah,with Huanglongbing

disease on citrus

Rina Ediati, Achmad Himawan,Andi Trisyono, Arman Wijonarko,

Zamir Hossain, Paul Holford and Toru Iwanami
Phylogeny

and taxonomy of bacterial

97

nitrogen-fixing

legume symbionts

Hiroyuki Sawada, Toshiki Uchiumi, Mikiko Abe, Masakito Hayatsu, Yukan Ichiman,
Katsumi Akutsu, Takanobu Yoshida and Kenichi Tsuchiya
Gene expression

controlled

by a conidiophore

patterning

100
regulator

Acr lin Magnaporthe grisea
Marie Nishimura

102

Study on durabilityof resistancegenes to blast disease(Pyricularia

grisea)in the Mekong delta
Pham VanDu andLe CamLoan
Current status of asparagus

104

diseases

Baharuddin Salleh, Siti Nurdijati,in Southeast

Asia (SEA)

Fachri Djas, Pangeran Insanul, Kasmaland Lahmuddin

1 08

Taxonomic and phylogenic

study on Bipolarisfiingi from Japan and their productivityof ophiobolins
Takao Tsukiboshi,
Studies

WenHsin Chung and Shigenobu

on phytopathological,of Plectosporium

morphological

Yoshida11 1

and molecular variations

tabacinum in Japan

Toyozo Sato, Jun Takeuchi, Hideyuki Nagao and Keisuke Tomioka
Morphology

and molecular taxonomy of Colletotrichum

Glomerella

glydnes)and related(Teleomorph:

species

Jouft Moriwaki, Toyozo Sato, Takao Tsukiboshi,
Pathogenicity

destructivum

11 3

of some Colletotrichum

species

Masako Noguchi and Kazuyuki Hirayae
to petals

Keisuke Tomioka, Jouji Moriwaki and Toyozo Satoof Antirrhinum

1 15

majus
117

Virulenceof Fusicoccum aesculi, Phomopsis phomoides, Fusarium lateritium

Stemphylium

lycopersici

andto sweet pepper fruits

Keisuke Tomioka and Toyozo Sato
Saccharomyces cerevisiae
of yeasts,

Williopsis

1 19

genome-wide mutant screen for antifungal

activities

mrafcii, Kluyveromyces lactis and S. cerevisiae

Hiroko Kuze Kitamoto
Isolation

12 1

and characterizationof bacteria

in fermented vegetables

sold in northernpart of Vietnam
Yasuhiro Inatsu and Shinichi

Kawamoto

A newmethod to obtain plasmid

123

variants from Lactococcus lactis

Miho Kobayashi, Masaru Nomuraand Hiromi Kimoto-NiraEffect of plasmids

ofLactococcus

strains on cytokine

production125
from murine

macrophage cells
Hiromi Kimoto-Nira, Miho Kobayashi, Koko Mizumachi, Jun-icki Kurisaki
and Takashi Okamoto
Effects of a probiotic

strain on the cellular127
immuneresponse to food antigens

Koko Mizumachit Hiromi Kimoto and Jun-ichi Kurisaki
Training

courseSchedule

and participantsin mice1 29

13 1

Introduction

to the workshop

Jun-ichi

Kurisaki and Taro Obata

National

Instituteof Agrobiological

The National

Sciences

Instituteof AgrobiologicalWorkshop on Genetic Resources annually.
research

information

with experts

Sciences

The objectives(NIAS)

holds

an Internationalof these workshops are to exchange

from around the world and to promotecollaboration
for the development of technologyof plant, animal and microorganism geneticof our genebank

international

related to the evaluation, conservation and use
resources These workshops have helped tostimulateactivities

system in Japan and help

scientists

to forge collaborative
linkages with scientists in other countries
This year we are holding the 12th NIAS International Workshop on Genetic Resources

This workshop consisted of two themes
1 Current research on the genetic and functional

genetic

toin the light of developments

inmicrobial genomics and trends related to the Convention on Biological Diversity.
This workshop was held in conjunction with the 10th International
CongressforCollections

wasCulture(ICCC-10)"Innovative roles of biologicalThis book containsof microorganisms

resources

relatedagriculture ;2. Ways to improve the quality

diversity

Japaneseand use of culture collectionsin Tsukuba during October 2004. The theme of ICCC-10
resources centers"
the proceedingsof the 12th NIAS

International

Workshop on

Genetic Resources and consists of four parts The first two parts are the papers presented
two workshop

symposiaorganized

under the titles

agricultural

microorganisms"

the papers

based on the poster presentations

training

techniques

Wegreatly
helpedand "Newparadigms of biologicalcourse was held for Southeast

culture collection

appreciate

agricultural

furtherinternational

microorganisms

are included.

Asian microbiologists

A report on this training
the inputs

to make this multi-objective

stimulate"Geneticof scientists

resources centers"

diversityof

In addition,

At the time of the workshop,
in order to help improving

course is provided

a

their

in this book.

from around the world and Japan thatworkshop a successdevelopments

and functionalat theWe hope that this workshop

and research cooperation

on culture collections

will
for

The 12th NIAS International

Workshop

Organizing
Committee
OBATA, Taro (Chairperson)
KURISAKI, Jun-ichi

on Genetic Resources

Genome and Biodiversity

Genetic Diversity

Division

Department

OKUNO, Kazutoshi Genebank
NAKAMOTO,Yasutoshi : Genebank
Executive Committee
KAKU, Hisatoshi(Chairperson)

Genetic Diversity

SATO, Toyozo: Genebank
HAYASHI, Nagao: Genetic Diversity

Department

AOKI, Takayuki : Genetic

Department

Diversity

TANAKA, Keisuke : Genebank
Secretariat

SATO, Toyozo (Secretary

general)Genebank

TANAKA, Keisuke : Genebank
OCHIAI, Hirokazu : Genetic

Diversity

NAGAI, Toshirou : Genebank
TOMIOKA, Keisuke : Genebank
TAKEUCHI, Kasumi Genebank
IIDA, Motoko: Genebank

Department

Department

1. GENETIC AND FUNCTIONAL
DIVERSITY OF AGRICULTURALMICROORGANISMS

Preface
Molecular

genetic

aspectsof microbiologicalagriculturally

importantPublicaccessibility

advances in virtually

research.

genomes of over thirty

Remarkably,

microorganisms

the completeallhave been completed just within the past half-decade.

to this wealth of genetic data via gene banks such as the DNA Data Bank ofJapan and GenBank has
descriptive

tools have paved the way to significant

phylogenetic

spurredgroundbreaking

and systematicstudiesthese are four phytopathogenic
and evolution

in molecularfunctional

plant pathology.

analyses of these data shouldof plant-microbe

analyses
Included

Xanthomonas species whose genomes either

been sequenced. Comparative phylogenomic
into the ecology

experimental

among

are being or haveprovideand animal-microbe

andnew insights

interactions,

just

to

mention a few.
These advances are also reflected
analysisof fungi,

in phylogenetic-basedeven though the genomes of relatively

have been sequenced

to date. Phylogenetic

analysis

essential

for elucidating

their genetic

systematics

for theirrapid

Phylogenetic

detection
studiesvirulence-associateddiversity,and surveillance

are also providing

associatedsystematic

few agronomicallyof plant pathogenic

has become

molecular markers

with the globalizationof world trade.

a framework for understanding

recent research advances and future prospects

taxonomy and functional
focus is directed

phytopathogenic

fungi

fungi

the evolutionoffactors such as mycotoxins

Resources, highlights
Specialimportantand for providing

This symposium, which is held as a part of NIAS International
diversity,and functional

analysisat phytopathogenicof agronomicallybacteria(i.e.,

Workshop on Genetic
in the genomics,

importantgenetic

microorganisms

xanthomonads and Ralstonia)

andfungi (/ e. , fusaria and Pyricularia)
Hisatoshi
Kerry O'Donnell,Kaku, National
National

Instituteof AgrobiologicalCenter for Agricultural

Utilization

Sciences,Japan

Research,USAMobile genetic elements contribution
related Xanthomonas genomes

to the differentiation

Marie-Anne Van Sluys1 and Claudia

B. Monteiro-Vitorello2

1 Departamento

de Biociencias,

277,

05508-900,

Universidade
Emailde Botanica,

Institute)

Sao Paulo,

SP, Brazil,

2Escola

Universidade
Superiorde Sao Paulo, R. do Matao,de Agriculturade Sao Paulo, Av. Padua Dias, 1 1, 13418-900,

of closely

Piracicaba,

Luiz de Queiroz,

SP, Brazil

mavsluys@ib.usp.br

Whole-genome sequencing and comparison ofX. axonopodis pv. citri (Xac), strain 306, and X
campestris pv. campestris QCcc\ ATCC33913, disclose a high degree of identity and colinearity of thechromosome backbone. Analyses of strain-specificgenes preferentiallylocated at discrete genomic
islands suggest different strategies for adaptation to host and disease development Xcc chromosome
contains a nitrate assimilation operon, higher number of avirulence genes and plant cell wall degrading
enzymes probably facilitating
its systemic dissemination. Xac has two plasmids that contain four copies
of avrBs3lpthA gene, which has been implicated in the induction of plant cell proliferation
associatedwith canker disease. Differences in their type III secretion systems, rpf, type IV fimbriae and LPS Oantigen operons were detected and may be associated with host-specific adaptation. No antibiotic
synthesis was detected as part of the TE units, but some of the insertions are in close association withgenes coding for toxins, drug resistance. And other genes knownto be associated with pathogenicity
arein close association with genetic mobile elements, mainly IS elements Both genomes were invaded bydifferent families of IS elements and in each genome one particular IS proliferated.
In addition, it isnotable that these genetic mobile elements are generally associated the strain specific genes suggestingthat they could be drivers of the genome evolution of this important group of plant pathogens
Introduction
Xanthomonas axonopodis pv. citri and Xanthomonas campestris pv. campestris areimportant necrogenic plant pathogens responsible
for citrus canker and black rot of crucifers,
respectively.
unveilthe strategies

consortium
suggestIn an attempt to identify
undertookthat disease

the genes responsibleof the two pathogens

to colonize

for diseaseplanttissues,genome sequencing

and comparative analyses

phenotypeby the presence

Xanthomonas, most of themis brought
in close

association

chromosomes Two types of genomic rearrangements
are either changes in gene order (colinearity)
these INDEL, we defined twelve regions

withswitched

and major insertion/deletions(INDEL)
Withinthat are larger than ten kbp in Xanthomonas

systems which are similarplaces around the origin of replication,

on both

in these genomes thataxonopodis pv. citri and seven regions in Xanthomonas campestris pv. campestris.
both type II and type III secretion

Results

gene sets in eachrearrangements

were characterized

and tothe ONSA/FAPESP

of both bacteria.

of particularspecific

development

at -90 % nucleotide

all other genomic islands

Except forlevel but havewere different

in genecontent between the two genomes All these 19 INDEL regions, referred to as genomic islands,
carry proteins
genetic

related to genetic mobile elements such as transposases

Phages,plasmids

variability

for bacteria

Mira et al 2002)

and transposable

elements

genome evolution

Most of the prokaryotic

and mtegrases

are considered(Boucherimportant

et al 2003,

organisms experiencesources ofCanchaya et al 2004;

clonalcellulardivision

which genetic exchange can only occur by invasive DNA. Recent reports disclose
these mobile elements in the differentiation
discussion

the conceptof bacterial

chromosome be in order to define
minimal percentage
be raised

species

relatedHow similarthat two strains(Canchayathat invasive genetic

Materials

and methods

campestris, ATCC33913

a bacterial

are from the same species?

What is theet al 2004;

strategy

and annotation

al

and

comparative

studiesResults

lbi ic.unicamp.br/bacteria

have been theand support theand in a relational

and Xanthomonas campestrisfrom freshly

pv.

grown cultures

usingof Xac and Xcc are described

in da

were

mostlydatabase as described

carriedmanuallyatin Van Sluys et al (2002)

and Discussion

Genomic island

distribution

Xac genome was composed of circular(pXAC33

and pXAC64)

while

Xcc contains

chromosome (5, 175,554
one circular

Considering

the main chromosome, there are 99,367

distributed

as discrete

encompassing
LR1 (Large

regions

Rearrangementbp) and two plasmids

chromosome of 5,076,187

bp present(tableaccounts for 30% of this difference

1)

The

regionand was named

1) This region was composed in the two genomes of three specificand some rearrangements in gene order The interesting

aspect in these islands

is the presence in Xac of the syrE locus that is responsible

for the synthesis

Pseudomonas syryngaeae, this nonapeptide

with a long unbranchedacid chain that has an antifimgal

INDEL in Xac corresponds

bp.

in Xac and absent in Xcc

around the chromosome backbone

the terminus of replication

genomic islands

fatty

can

Nakagawa et al 2004;

are coming to light306

genomic DNA was prepared(2002)

2003,

These questions

elements are tools for genome diversification.

Sequencing

http://www

two strains?

Casjens

procedures
et

into

shouldXanthomonas axonopodis pv. atri, strain

Silva

and conserved

Genome sequences of plant pathogens

observation

the impact ofand strains bringing

level and strain level and human pathogenic

main focus of these questions

standard

speciesof invasive DNA that would differentiate

at both species

Perna et al 2001)

of closelyinto a 61,169

is associatedactivity(Menestrina

bp insertionand Semjen

of syringomycin.
1999)

In

3-hydroxy
The largest

where RTX toxin and most of the non-

Table 1 : Xac genomic island distribution

and content

gammabacteria genes are found. Also in the LR1, there was a region corresponding
froma discrete region also found in Xylellafastidiosafastidiosa

PD (A^-Temecula)a DNA methyltransferase
2000;

da Silva nal

strain (Van Sluys et al 2003)

regionswith a particularof OLf filamentous

campestris strains was identified.

is associatedthe Rhizobiaceae(Agrobacterium,

familylocus present reduced virulenceinteraction.

in some bacteria such as the members of

Rhizobium and Brucella)and Miller

1994;

for which, mutants in thisBriones et al 2001,

Thomashow et

has two main features probably

to overcome growth inhibition

The third region harbors three genes similarof Streptomyces (macrolides

protein,

fromX campestris pv.

and a tannase encoding gene. The latter is considered
inducedto those involved

involvedinto be an importantby tannins synthesizedin antibiotic

bybiosynthesis

and streptomycin)

the terminus region, other genomic islands

chromossome backbone.

region in

These are the presence of the avrBsl gene not found in Xac flanked

enzyme for microorganisms

Outsideet alregion, a cyclic beta 1,2 glucan synthetasewith virulence(Breedveld

also compose the equivalent

phage, which is specific

The second genomic island identified

by two transposases

resistantinterest

Inside the delimited

was present This protein

plant-pathogen

In Xf-9a5c, this region encompasses

that harbors an group II intron not present in Xac (Simpson

Xcc.First of all, an insertion

plantsCVC (Xf-9z5c) strain and not present in X

2002)

Three specifical 1987)

to 19 genes

In Xac, genes encoding

hly activator

proteinpennicilin

and most important

could be found scattered
modifyinganotherenzymes, acriflavinavirulence

avrPphE were found and were not present in Xcc. In Xac the most similar

along thegene known asgene to avrPphE is atthe side of lSXac3. Also, it is interesting
specificallypresent

that 1030 genes coming from non-gammabacteria arein Xac genome most of them locatedwithin the genomic islands

described

here.
In Xcc a full length
version was present

copy of P2/CTX phage was found while in Xac only a truncated

In Xcc, along with this full length

phage copy was located the umuC andumuDgenes which are absent in Xac. This would suggest that, probably,in Xac genome involving
involved

the loss of the phage region also resulted

a major rearrangementin the loss of these proteinsin repair

Transposon and IS elements
Table2 Transposonsand IS elements found inXac
Xac and Xcc genomes were invaded andXccgenomes
by several IS elements and each genome has
an element that is most represented
IS1478belongs

to IS5 familyof transposases

and was present in 16 copies
belongs

to the IS3 family(table2)

in Xcc. \SXac3

of transposases

andwas present in 21 copies

in Xac and has notbeen previously

detectedin Xanthomonas. The

impactelementsof thesecan be glimpsed

on these genomes

by the fact that only 4 IS

elements are shared by Xac and Xcc (IS1478,
IS1479,ISXcdl13 elements

and ISXacS)

are particularand all the otherelements to either of

the two genomes Table 2 presents
the 17 full-length
importantelements identified,(Tu

but it is

to mention that Xac genome carries

1 0 extra transposases
already

the list of

not fully characterized.

Tn5045is a composite

described

in X campestris as ISXC5

et al

1989)

This

originallydescribed

plasmidfrom Xcampestristransposontransposonwasto be born in pXW45J
pv citnstrain

XW45 In X axonopodis pv citn a full lenght
copy of theelement

was present

in pXAC64

thein thepXAC33

while

IS

unit

(ISXacl)

was missing. In both plasmidstheelement is in closely

associatedpositionsa sequence (gccatcgccagca)

5223-5235,

with the pthA gene. The transposon

region could have an impact on the variability

was present,

betweenthat was also found in pthA repeats
in the numbers of repeats

This

We also found thesame sequence as part of genes of unknown function

in Xylella(71%

AA identity)

andXanthomonas campestris (68% AA identity)

this coding

regionis present

in thepXF51

plasmid

In Xylella,

and in Xanthomonas is associatedcomponent (ISXacl)of the composite Tn5045

present

copiesin 8 identicalto Tn5053

has invaded the circularin Xac. \SXac2 is similar

to thispreviously

sequence are translated

described

insertion

ISXCD1

Most of the elements

by programmed frameshiftingas has been describedvulgahs Hildenborough

ISD1\SXac3 is closely

Erwinia amylovora transposon born in the ubiquitous

plasmid

pEA29 and to an IS-likepresentfor Desulfovibrio
in the Agrobacterium

The IS

chromosome and was

to a previously

sequence from Xanthomonas axonopodis pv. dieffenbachiae
relatedfamily transposons

tumefaciens PTI plasmidfrom the AB2/73

relatedlimitedto anelement

host range

strain. lSXac2 and lSXac3 were present in Xac generally

associatedwith genomic islands

listedin table

represented

being identified

only ashighly1 In Xcc, ISXacl
degenerated

and \SXac2 are very poorly

copies or truncated elements while \SXac3 was identified

them harbouring 26 bp internaldeletion.

be driversof the differentiationof the Xanthomonas genomes probablytransduction

or plasmid

conjugation

It is temptimg

would like to thank Dr Carlos

the helpful

and for providing

mention all the technicians,that these IS elements may
as a result of phage

events

Acknowledgements
The authors
discussions

to speculate

in 7 copies all ofstudents

the bioinformaticand researchers

made the Xanthomonas proj ect a reality

F M Menck and Dr Joao P Kitajima
support(JPK)

Also it is importantof the ONSA/FAPESP

consortium

for
to
that

(http ://watson.fapesp.br/onsa/Genoma3.htm)

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Mol

Gen.

Species
fusaria:recognition
and identificationof agriculturally
current status and future prospects

Kerry O'Donnell1.
1 Microbial
61604,David M. Geiser2 and Takayuki Aoki3

Genomics and BioprocessingUSA,

important

Research Unit, NCAUR-ARS-USDA, Peoria, Illinois

2 Fusanum Research Center, Department

State University,

University

Nationalof Agrobiological

InstitutePark, Pennsylvania

16802,

Sciences,of Plant Pathology,
USA, 3 Genetic

2-1-2 Kannondai,

The Pennsylvania

Diversity

Department,

Tsukuba, Ibaraki

305-8602,

Japan
Email

kodonnell@ncaur.usda.gov

Fusarium is a large genus of filamentous fungi that represents the single most important groupof mycotoxigenic plant pathogens
Fusaria have also emerged within the past two decades asopportunistic and often fatal pathogens of severely immuno-compromisedpatients
Members of thisgenus produce an amazing diversity of toxic secondary metabolites such as trichothecenes, fumonisinsand estrogenic compounds which pose a serious threat to human and plant health and food safety. In
order to develop molecular epidemiologicaltools for the rapid detection and identificationof the mostimportant fusarial pathogens, multilocus sequence typing is being used to investigate species boundaries
Knowledge of species limits is essential for understanding each pathogen's geographic distribution,
hostrange and toxin potential, and to establish successful molecular surveillance programs for economicallydevastating plant diseases such as Fusanum Head Blight of small grain cereals Because globalizationof
trade in agricultural commodities will continue to result in the inadvertent movementof foreign fusarial
pathogens worldwide, a global network of plant disease specialists is needed to meet this continuing
threatIntroduction
Fusarium collectively
phytopathogens
artificially
there(Marasas

represents

et al 1984)

immuno-suppressedof humans (O'Donnell

recognition(MSR)

Reinking

1935,
120

speciesestimates

using genealogical2000),

descriptions

2003,

which

multilocus

over 300 phylogeneticallyimportant(Fisher

et al 2002;

and agriculturally

over the past twenty years,

Althoughmorphological& Nirenberg

speciestaxonomy (Wollenweber

andNelson1983),

only

acceptedin the taxonomies

cited

above,

up to July, 2004.

By way of contrast,

preliminary

sequence
distinct

speciestypingrecognition(GCPSR)(Tayloret(MLST),

suggestthat

Unfortunately,

relatively

few medicallyKoufapanou et al

1997;

speciesfungi (Carbone

1982;

threatening

et al

Kasuga et al 2003,

importantin the number ofand lifeof Fusarium

concordance phylogenetic

employsincrease

as opportunistic

2004a)are currently

publishedcomprises
2003)

et al
Gerlachmorphospecies

includingalof fusaria

has formed the foundation
1971,

genus of mycotoxigenic

Moreover, due to the dramatic

recognition

pathogens

Booth

most importantand immuno-compromised patientshas been an increased

approximately

the single

et al

1999;

Litvintseva

Couch & Kohn 2002;

Fusarium
et al
Craven etal 2001,

Geiser1998a,

1998b,

et al

1998a,

2000, 2004b;

1998b,

2000,

2001,

Steenkamp et al 2002)

O'Donnell

1997,

have been subjected

A recent study of the Fusarium graminearum species
number of economicallyworldwide(Windels

diversity(O'Donnell

devastating
2000),

potential

institutional

provideset al 2004b)

alone may be insufficient
the toxin

potentialof Plant

Pathology,of all fiisaria

head blightfiisaria

identification

Because this study conclusively
and plant pathologistsof Fusarium

species,

speciesto accurately

report on

we have developed

a multi-boundaries

using a combinationof

we have teamed with mycotoxicologist

Dr Gretchen Kuldau,

The Pennsylvania

to reassess

State

University

framework and molecular
testing

markers for long-term epidemiologicalscientificashows that morphology

and mycotoxin analyses
studies,

community via the Internet

the toxin

plant pathologist

within the F graminearum complex. Results of these studies,to the global

includesspecies

USDA, St Paul, MN for pathogenicity

of molecularavailable

complex, whicha clear example of how MSR underestimates

within a robust phylogenetic

Dr H. Corby Kistler,

to GCPSR.
and scab

network to investigate

In addition,

et al

(FHB)

and pathogenicity

MSR and GCPSR.

O'Donnell

agents of Fusarium head blightfor mycotoxicologists

collaborative

Department

etiological

2000;(Geiserofwhich include

will be made freely

et al 2004;

Kang et al

2002)
Materials

and methods
Researchers

CultureCollection

from around the world send fiisaria(NRRL),

Peoria, IL, the National

to Agricultural

Institute

Researchof Agrobiological

Service
Sciences(MAFF), Tsukuba, Japan and the Fusarium Research Center (FRC), University Park, PA to be
characterized phylogeneticallyusing DNA sequence data from one or more loci and/or
phenotypically.

Each strain accessioned

in liquid

vapor at -175°C

nitrogenthe scientificor by lyophyhzation

for future reference and distribution

tocommunity upon request

Proceduresfor the polymerase

and DNA sequencing
Subsequentis given a unique accession number and they are stored

are describedto the publication

chain reaction (PCR)-basedin O'Donnell

et alamplification(2004b)and referencesof the first set of PCR primers and protocols

of fungal nuclear and mitochondrial

ribosomal genes (White

singlecodingand/ortranslation

elongationlevel studies(Geisergenes interrupted

by large

have been shown to be more informative

et al 2004;

et al 2004b)

EF-3> 5'-GTAAGGAGGASAAGACTCACC-3',
amphcon, makes it possible

to generate

fiisaria with just one sequencing primer

et al 1990),

numerous introns,

factor ( la), generally
O'Donnell

therein.

for the amplificationand direct sequencing
copy proteinof target locisuch asfor species

Use of the internalsequencing

pnmer

is nested

within

EF-la

which

a nearly complete

justsequence of this locus

thefor mostSequencing
3730

capillaryreactionssequencers

sequences are edited
exportedare currently

which yield highversion 4.1.2(Gene Codes, Ann Arbor, MI) and thenphylogenetic

analysisusing

PAUP* version

4.0bl0unknown strainsto species

or species

complex, partial

EF-lasequences are used as a query to BLAST the FUSARIUM-ID v.1.0 databaseat http //fusarium.cbio.psu.edu

3 100 orsequences of 800 base pairs or more. Allwith Sequencher
To identify

Biosystems-Hitachi

qualityfor maximum parsimony(Swofford 2002)

being run on Appliedhoused at The Pennsylvania(GeiserState University,

et al 2004)

University

Park,& O'Donnell

1998,

PA.

Species
1999a,b;limitsare assessed

Aoki ef tf/ 2001,

using a combination

2003, Himta et al 2001)

of MSR (Aokiand GCPSR (Taylor

et al 2000;

O'Donnell

et al 2004b)
Results

and Discussion
Substantial

evolution,

progressbiogeography,

has been made over the past decade at elucidating

host range and mycotoxin potential

withina phylogenetic

framework.

studies,which may be generallyof agriculturally

Results of these morphologicalapplicablethe systematics,

to other agronomicallyimportantand molecularimportantfusaria

systematics

fungi, are outlined

below.
1) Progressin Fusarium systematics

among scientists
Netherlands)
includinghas been greatly

facilitatedat the FRC, MAFF, NRRL, BBA (Berlin,
culturecollections,allmany ex-type strainsof whichand strains

house

importantby intensive

collaborations

Germany) and CBS (Utrecht,
extensivecollections

Theof Fusarium,from a mycotoxicologicalperspectiveof novel germplasm in publicallyaccessible(Marasas etf a/ 1984)
2) Supportof and the continued

international

culture collections3) Coordinatedresearch

help bridge

accessioning

is criticalinvolving

to continued

scientists

progress in Fusarium systematics

using MSR and/or GCPSR is essential

the enormous gap between the number of morphospecies

number of phylogenetic

species(-300;O'Donnell,

unpubl )in a study of the F graminearum species complex (O'Donnell
assume that many phylogeneticallyusing MSR.

distinct

species

resolved(-120)and the estimatedHowever, as recently
et al 2004b),

in order todiscovered

it is reasonable

by GCPSR may not be diagnosabletofujikuroi

4) The terms macro- and microconidia

obscure the morphologicalshould be replaced

descriptors

with more informative

as discussed

5) Discrete

DNA sequence data possesses

considerably

speciesthan other types of molecularmarkers

limitsnuclear ribosomalinternal

subunitdomains Dlspanning

transcribedspacer (ITS)

diversityof conidia

andin Aoki et al (2003)

more information

for investigating

However, DNA sequence data from theregionand the 5' end of the nuclear large

and D2, the two most widelyused loci for species

levelsystematics
in the Fungi, are too conserved to resolve most Fusarium species
However,
nuclear genes such as EF-la and P-tubulin,which are interrupted
by phylogeneticallyinformative
2004b

introns,have enormous utility

and referencesin GCPSR

et al 2004;

O'Donnell

et al

therein)

6) DNA sequence-based

phylogenetics

has shown that the subgeneric,

sectional

classification

molecular

phylogenetic

includingGibberella(O'Donnell

1998b),

Fusariumgraminearum or the dominant(O'Donnell(Geiserof Fusarium is mostly non-monophyletic.
studies

et al 2000,

have identified

2004b;

several

et al

1998a;

Ward et al 2002),

morphological-based

Fortunately,monophyletic2000),

results

speciesof thecomplexes,

F.oxysporum (O'Donnell

B-trichothecene

mycotoxin

and F solam (O'Donnell

2000;

et al

producers
Aoki et al

2004)7) Molecular

evolutionary

genetic studies

2003)

have shown that the evolution

species

complex is discordantphylogenetic
selectionevidenceof the trichotheceneof these 12 clusteredwith the species

suggests

phylogeny

monophyletic

15ADON+DON and NIV) have been maintained
a phylogenetic

chemotype in plant pathogenesis
gene cluster
withinmake it possible(Ward et al 2002)

Furthermore,is under a novel form of balancing

chemotypecladesover multiple

speciationto accurately

multiplex
predict

the(i.e.,

3ADON+DON,

eventsResultsofthe role of each trichothecene

PCR assays targetingthe trichothecene

the ends of thechemotypeof each strain

the F graminearum species complex (Ward et al 2002)

8) While

genealogicaldiscordances

concordance

have been detected

appears

distributed

where their evolution

has been homoplastic

2000),b) highly

divergent

to be the norm (O'Donnell

within all of the major species complexes

xenologs are uniformly
1998a,

the F graminearum

framework for investigating
In addition,(Kimura et al

genes withinthat the toxin gene clustersuch that three reciprocallythis study provide

toxin gene cluster

across the G fujikuroi
P-tubulin(O'Donnell

et al2004b),a) ITS2 paralogs

orand F oxysporum species complexes
and Cigelnik

1997;

O'Donnellet alsequences within the F solani complex confoundintergenic

F.meridionaleinphylogenetic

reconstruction,and c) a nuclear ribosomal

spacer region phylogeny

the E graminearum species

complex does not track with the multi-locus

et al 2004b)

through the power of GCPSR one natural hybrid was detected

Fortunately,

the E graminearum complex which possesses

some alleles

phylogeny

of(O'Donnell
withinof E asiaticum (Asian parent)

in amostly(South American parent) background(O'Donnell

et al 2000)

9) The Internet-based

tool FUSARIUM-IDet al 2004)

for the identification

correlating

host

range, geographic

sequenceforE

fusaria represents

a significant

v.1.0 (Geiser

step towards

distribution

and toxin potential

with species

10)

The

recent(http//www.broad.mit.edu/annotation/fungi/fusarium/),

completionofaccuratelyoflimitsthewholegenomecoupled

with

plans

graminearum
to sequencearepresentativeof the G Jujikurot(i.e., E vertiallioides),E oxysporum (Di Pietro

et al 2003),

and E solani

species(H

persresultin thehigh resolution

MLST

epidemiology

via thecomplexCorby

identificationof a plethoraof phylogeneticallygenotypingof agriculturally

important

Internetll)

Kistler,

informative

fusaria, thereby

commun.) shouldloci for use

facilitating

global(Geiser

et al 2004;

Kang et al 2002; Taylor and Fischer 2003)

Molecular

surveillanceof the FHB fusaria and their toxin potential

researchpriorities,

giventhat hybridization

pathogensmay give rise to super pathogens

following(Brasier

the global
2001)

is one of our top

transpositionof foreign

Because of the globalizationworld agriculture,

we are collaboratingwith Dr Maarten van Ginkel

Mexico to develop

of a global network of plant disease specialists

ofof CIMMYT, Juarez,

to meet the continuing

threatsuppliedofFHB
Acknowledgements
The authors thank all of the individuals

and institutions

who generously

strains

Kaku, National

Institutefor study.

KO gratefully

Research and the Organizing

thanks Dr Hisatoshi

Committee for the generous invitation

10 symposium. This research was partiallygrant 0304-OD-037

to participate

in the ICCC-

by a US Wheat and Barley Scab Initiative

to KO. Names are necessary to report factually

the USDA neither guarantees
by USDA implies

supportedof Agrobiologicalon available

nor warrants the standard of the product,

no approval of the product to the exclusion

data; however,and the use of the nameof others that may also be suitable.

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Acad. Sci USA 99:9278-9283

White, T. J., T. Bruns, S. Lee & J. Taylor (1990)
ribosomal& K. O'Donnell

impacts

Great Plains(1935)
355 pp.

and Applications,

Academic Press, Inc., Sanof Fusarium head blight
Phytopathol

Die Fusarien,changing

farms

90: 1 7-2 1

lhre Beschreibung,Schadwirkung

Functional
analysis for pathogenicity-relatedusing mutagenesis approachgenes of xanthomonads

Chaozu He and Wei Qian
National

Key Laboratoryof Plant

Sciences,

Beijing

P. R. China

Email100080,

Genomics, Instituteof Microbiology,

ChineseAcademy ofhecz@im.ac.cn

Gram-negative bacteria Xanthomonascampestris pv. campestris {Xcc) and X. oryzae pv. oryzae(Xoo) are casual agents of black rot of crucifers and bacterial blight of rice, respectively.
They cause
severe yield loss in each crop every year Understanding the genetic basis of essential steps duringpathogenesis may lead to innovative approaches to control these two diseases Here we analysed two
random transposon insertional
libraries, which contain 16,5 12 QCcc) and 16,712 (Xoo) transformants,
respectively, for their ability to cause disease in susceptible
host plants Seventy-five non-redundant,
single-copy mutants defective in pathogenicityof Xcc and 73 mutants of Xoo were identified. The
mutated genes belong to multiple
functional
categories,
includingundetermined
pathways,fiinction-unknown genes and three genes specific to Xcc 8004 strain that were not found in published Xcc
ATCC 339 13 genome database. Our results suggests that Xcc and Xooevolved with similar strategies toattack their hosts, but adopted different pathway regarding their host specificity. Our work represents oneof the largest scales in screening for bacterial pathogenicity-related
genes and provides new insight into
the global view of pathogenesis of xanthomonads
Introduction
Almost all species

belong to the genus Xanthomonas are plant-associatedmost of them are plant pathogens

with definite

two typicalplant-bacterium

interaction

relationshipwith rice cultivars

where genetic

between a bacterial(Shenavirulenceand Ronald 2002)

Xcc has the ability
three strains
citn

ofXanthomonas

306 (da Silva

genetic

phytopathogens
knowledge.
addition,

For example, thoughannotations

it has predictedbeing involvedin pathogenicityof the some 4, 100 protein-codingof genes(< 320)approximately
category

predictions

has

one third(da Silva

been

identified(> 1,300

et al 2002)

genes)

Therefore,

was developed

resistancegene (R-gene)has not been fully(Alvarezsequences"gene-for-gene"of matched specificity

2000)

Recently,

evolvedso thatthe completionof[Xcc ATCC 339 13, X. axonopodis pv.

and Xcc 8004 (our unpublishedand valuableso-called

and a plant disease

plantswhole-genomic

However, genomic

in the the genome)

Xoo evolved

relationshipgene (avr gene)

et al 2002)

information

relationships

most cruciferous

andhost range. Among them, Xcc and Xoo represent

As for Xcc, this kind of relationshipto infect

bacteria,

to explore

data)]

providedpathogenesis

have reflecteda profileof these

inadequacyofimportantin our current

that more than 250 genes (about

6 % of genes

ofXcc, many of them remain undefined.

In

genes annotatedin each genome, only a small groupexperimentallyin xanthomonads,

and there

been assigned

to any functional

genomic techniques

are requiredthateven haven't

high-throughput

aretoprove the functionsof these genes, which are applicablegenerated by in silico studies,but also to gain further

discoveringnovel genes or biochemical

Constructionof two mutant libraries

The mutant librarycultivar

Jingfengand screening

under optimized

conditions

Large-scale(temperature

verification

flanking

insertionusing EZ: TN <KAN-2>

cultivarIR24)

was conducted clone-by-clone

relative

humidity

analysed by searching
Preliminary

were repeated to exclude these possibilities

One hundred seventy

mutants were obtained.
mutants were detected

insertion.

1995)

The identified

insertion

by Southern blotting

As for Xoo, though its whole-genomic

intergenic

there are five insertions

Additionally,

However, since these75

non-redundantsequence has not been published,

genes indicates

genes

asymmetric interlaced

disrupted

Each of these mutants contains a single-copy

search and alignment with similartransposaseandagainst local or public databases

regions were identified.

regions

Copy numbers ofsites and flanking sequences were thenanalysis of pathogenicity-related
genes
From 172 Xcc pathogenicity-deficientmutants,

ORFs/intergenic> 95 %) To avoid

factors, at least four rounds

sequences of transposons were obtained by TAIL-PCR (thermal

PCR) (Liu and Whittieroleracae

were set up to screen

or other unpredictablein pathogenicity-deficientbymutants/ e. Xcc-cabbage (Brassicaof 28~35°C,

two Xcc and 200 Xoo pathogenicity-deficienttransposonpreviously

plant inoculation

result caused by phase variationof independentof pathgenicity-deficient

Two stable pathosystems,

mutants

into Xanthomonas pathogenesis
in infection.

as described

1) and Xoo-nce (Oryza sativa

pathogenicity-deficient
false-positive

insight

pathways involved

was constructedtransposome (Sun et al 2003)

not only to confirm the predictions

that at least 73 are insertedof Tn5
database

within ORFs or in(one in Xcc and four in Xoo) located ingenes maintainas multi-copies

in the genomes,

accurate insertion sites of them are uncertain.
Rapid development of genomics brings a paradigm

shift in studiesof plant-pathogen

interactions

components encoded in bacterial

To elucidate

all of the pathogenicity-related

genome has become a prevailing
available

genomic information

systematicalunderstanding

subject

for pathologists

in the near future.

on xanthomonads, our study representsof the essential

Based on theone of these efforts

in

aspects during pathogenesis

Acknowledgements
This work was supported
and Technology

by Chinese

Academy of Sciences,of China and National Natural Science Foundation

the Ministryof China.

of ScienceReferences
Alvarez, A.M. (2000)

in Mechanisms

of Resistanceto Plant

Diseases

Fraser, R. S. S., van Loon, L. C.) 21-52 (Kluwer Academic Publications,da Silva,

A.C. et al (2002)

differing
host specificities
Liu, Y.G. & Whittier,amplification

Comparison

Dordrecht)of the genomes of two Xanthomonas pathogens

Nature 4 17, 459-463
R.F. (1995)
Thermaland sequencing(ed. A.J. Slusarenko,

of insert

asymmetricend fragments

chromosome walking. Genomics 25, 674-68 1
Shen, Y. & Ronald, P. (2002) Molecular determinantsfrom PIof disease

Xanthomonas oryzae pv. oryzae and rice. Microbes Infectinterlaced

PCR:

automatable

and YAC clones

and resistancewithin interactions

forof4, 1361-1367.

Sun, Q. et al (2003) High-quality
mutant libraries
of Xanthomonas oryzae pv. oryzae and X.
campestris pv. campestris generated by an efficient transposon mutagenesis system. FEMS
Microbiol

Lett. 226, 145-50.

Genetic diversity of Ralstonia solanacearum strains
the Southeast Asian countries
KenichiTsuchiya1*.

Mitsuo Horita2,

Wong Boonsuebsakul3,

in Japan andJosehto,E. Villa1'4

and Karden

Mulya5
1 National

Institutefor Agro-Environmental

Ibaraki

305-8604,

Japan.

2 National

Sapporo062-8555,

Japan. 3 Department

Thailand.

4 Gifii University,

Agricultural(NIAES),

3-1-3,

Kannondai,

Tsukuba,

ResearchCenterfor Hokkaido

Region,of Agriculture

1-1 Yanagido,(DOA),

Bang Khaen, Bangkok

Gifu, Japan. 5 Indonesia

and Genetic Resources Research Institute,
EmailSciences

Agricultural

10900,

Biotechnology

Bogor 16 1 1 1, Indonesia

kentsuch@aflfrc.go.jp

Bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases
distributed
in worldwide. Japanese strains were divided into four pathogenic groups Groups I to III were
pathogenic to mainly solanaceous crops, and corresponded to race 1 Group IV was pathogenic to potato,
and corresponded to race 3 A dendrogram constructed based on the rep-PCR analysis of Japanese strains
defined 35 fingerprint types Each strain that differed by race or biovar represented a distinct fingerprint
type. The strains were separated into two main groups, one with all race 1, and the other with only race 3
Race 1 strains were further subdivided. The rep-PCR fingerprints of strains from Japan and various
countries revealed two mamclusters Cluster 1 comprised all biovar 3, 4, and 5 from Asia and Australia as
well as biovar N2 race 1 from Japan Cluster 2 included most of the biovar 1, 2, and N2. The 16S rDNA
sequence of Japanese race 3 biovar N2 strains was homologous to that of a certain strain from Indonesia.
Twonewly identified race 4 biovar 4 strains responsible for bacterial wilt of Zingiberaceae plants might
presumably invade fromeither Thailand or China
Introduction
Ralstonia
speciessolanacearum

and 50 families

bacteriumof plants

is a heterogeneousis the causal
in tropical,

species,organism

subtropical

R.solanacearumrace and biovars,

based on host range and utilization
bacterial

wilt

strains

more than 40 species

and 20 families

systematic

among strains

relationshiphave brought

unexpected

Improvementof detection

and identification

methods

and indigenous

R. solanacearumof foreign

strains

only taxonomic problems

but also plant quarantine

systems,onbackgroundand thetrade in productionof seeds

investigated.

systems as well as global
occurrences

to binaryhas been reported

However, the genetic

have been poorly

For more

respectively.

caused by R. solanacearum

and saplings
diversity

accordingof carbohydrates,of plants

Recent changes in agriculturaland genetic variability.

have been classified

diseasewilt of more than 200

and warm regions in the world. This

which shows phenotypicthan four decades,
In Japan,of bacterialof new host plants

matteris importantstrains

or invasive

strains

by elucidating

genetic

from the viewpoints

of notRecent advanced molecular
genetic diversity

or genetic

techniques

relationshiphave been effectively

in certain plant pathogens

appliedto analyze

as well as plant-associatedbacteria.
Under these circumstances
fromJapan and diverse geographical
Phenotypiccharacteristics

studiesof genetic

origins

were investigated.

and pathogenicityof Japanese

R. solanacearum is heterogeneouson host range and utilization

diversityand dividedof carbohydrates,

strains

into five races and five biovars

respectively.

so far, which were pathogenic

tobacco and eggplant

for the former, and potato for the latter

pathogenic

groupsto mainly solanaceous

tests, the Japanesestrains

Three groups (I to III) were pathogenic

tomato and eggplant,

which corresponded

based

In Japan, races 1 and 3, biovars N2,

3 and 4 have existed

Based on pathogenicityof R. solanacearum strains

crops such as tomato,

examined were divided

into fourto many solanaceous plants

such asto race 1 Group IV was pathogenic

to potato, weakly

pathogenic or nonpathogenic to tomato, and corresponded to race 3 Race 1 strains were isolated
fromvarious plants in most parts of Japan, whereas race 3 strains were found only in cultivatedpotato fields

in limited

regions including

In biovar determination

Nagasaki Prefecture.

tests, strains

were divided

into three biovars

Biovars 3 and 4 were most common.Japanese N2 strains were distinct
and N2 strains in several phenotypic
Genetic diversityof Japaneseand foreign

strains

group 1 with strains of biovars N2, 3 and 4, which belonged
N2 that corresponded

representing

strains

into two groups,

to race 1, and group 2 with strains

to race 3 Group 1 strains all had identicalsequences, and strains

the three biovars within the group did not differ from each other Group 2 strains

had characteristic

nucleotides

thatdifferedat seven positions

comparative analysis

of Japanese and foreign strains

group 1 was closely

related to Asian and Australian

from group 1 strains

biovars 3, 4 and 5, and belonged

group 2 was homogeneous with Indonesiansuggestinga close relationshipbiovars

2 and N2 in subdivision

based on rep-PCR genomic fingerprints

The three primer sets, REP, ERIC and BOX, defined 35 fingerprintlevel

The strains

were separated

to division
2b,

between them.

A dendrogram was constructedsimilarity

In abased on 16S rDNA sequences, Japanese

1 Japanesestrainsfrom foreign biovars 2

traits

A comparison of 16S rDNA sequences separated the Japanese
ofbiovar(N2, 3 and 4)

Each strain that differed by race or biovar represented
into two main groups

race 3 Race 1 strains were further

subdivided

a distinctof Japanesetypes at the 95%
fingerprint

type.

one with all race 1 and the other with only

into six groups at 80% similarity.

Withinrace 1,

each biovar (N2 and 4) separated

into a single

group, with high similarity

On the other hand, biovar 3 strains

were divided

within each biovar

into five groups, with low average similarity

amongthe strains
Comparative
rep-PCR
strains,

analysis

fingerprints
includingCluster
Australia,

from Japan andrevealed two main clusters

1 comprised

and 5 (races

theof R. solanacearum

six biovars

various countries,

ofall strainsof biovars

3, 4,

1, 4 and 5) from Asia andand strainsof biovars

1 and N2

(race 1) from Reunion and Japan. Cluster
includedmost strains ofbiovars(races1, 2 and 3) from 13 countries

In cluster

2, the averagestrainsfrom Brazil

respectively,

1, 2, and N2(Fig.

similarity

biovar 2 from eight countries

2
1)

withinand biovar N2

was 94 and 65%,

whereasaverageFig. 1

similarity

between biovar N2 strains from Brazil

Dendrogram showing genetic diversityof R. solanacearum isolated

andfrom Japan andforeign countries based on rep-PCR

Japan was 21%

NewR. solanacearum strains
Ginger (Zingiber
medicinalspp.),

parasitic

to Zingiberaceae

introduced

gingerfields in 1997,

neighboring

citiesmainly for food in Japan. Curcuma {Curcuma
to the same Zingiberaceae

to Japan. In 1995, a bacterial

wilt disease

by R solanacearum occurred in the cultivatedproduction

the same Prefecture.

successively

Bacterial

wilt of zingiberaceous

disease29 species

containing

in theplants are caused

in several countries,

among 14 families

thehas spread toto mioga plantations

by R. solanacearum race 4 strain and have been reported
had been recorded

causedin Kochi Prefecture,

the outbreak of this disease

and since 1999 it has expandedfamily, wasof C. alismatifolia

fields of a few localities

center in the country. Subsequently,
withinsources of spice orfor cut flower also belonging

from Thailand

leadingin Japanofficinale) and mioga (Z mioga) are important

crops, which have long been cultivatedornamental plants

plantsbut no suchof host plants

reported

before 1995 in Japan.
pathological,
strains

Thus new bacterial

strains

physiologicaland molecular

as well as those obtained
The disease

above mentioned
biologicalwas examined to characterize

viewpoints

in comparison

from geographicallydifferent countries

symptoms of three plants

were almost similar,

fromof indigenousof which yellowing

andwilting

startedgoldenbrown and wilted.
Allfrom lower leaves,isolates

quickly

spread upwards until the whole plant became entirely

from the diseased

plantswere identified

as R. solanacearum based on

physiologicaland biochemical

tests and were proved to be biovar 4. On the other hand, thosefrom Thailand

and Indonesia

consistedof both biovars

3 and 4, and those from Australia

andChina were biovar 4.
besides

Strainsfrom zingiberaceous

plantscaused wiltingof potato

and mangold severely

ginger,while

and sweet pepper

did not wilt

but showed vascular

discoloration.

tomato, tobacco

HR was induced in tobacco.

races (1 and 3) nor indigenous
wiltedgingerpreviously

strains isolated

Based on the pathogenicity

unknown in Japan (Table
Genetic diversity

ERIC as primersfrom Japan, Thailand,

from eggplanttests these isolatesof representative

Japaneseand tomato in Kochi Prefecture
were designatedas race 4 -a race1)of the strains

AnalysisOn the other hand, neither

was tested by rep-PCR analysis

was carried out using selected

Indonesia,

Australia

Table 1 Comparison of pathogenicity
Zinglberaceae
strains and indigenous
race 3 strains in Japanbetweenrace 1 andusing BOX, REP andcurcuma, ginger

and mioga strains

and China as well as representative

Japaneseraces

strains

isolated

and biovars

Fig.

2.

BOX-PCR

from ginger

profilesofand representative strainsof Japaneseraces and biovars
Lanes 2-7 ginger

strain,2, 12, 13,race

3, 14,15

1/bv

8, 9, race 1/bv4,

10,ll,

race 1/bv

race 1/bv

4, 16-18,race3/bv

2, 1, 19, DNAmarker

The DNA profiles

obtainedstrains,

two types (Typeneitherthe DNA patterns of indigenous

Kochi Prefecture

were highly

reproducible.

I and Type II) of DNA fingerprint

In rep-PCR

patterns were obtained.

R solanacearum strains isolated

nor those of representative

analysis

Japanese races nor biovarsof theHowever,from vaeous plants
were identicalinto any of

the two types (Fig.

2) The DNA patternof Type 1 strains was identicaland Curcuma spp. strains from Thailand,
which were originatedfrom Australia

A dendrogram
diversity

based

as well as their relationships

all Curcuma strains,
Type 2) contained

mostly ginger strains

2 pathogenic

strains

contaminatedseed materials

outbreakstrains

obtained,from either

Group A (containing

it was consideredthat a possibility

imported around 1995,
and subsequently

in ginger in 1997.

these

two exotic K solanacearum
proportions

strains

through

genetic

Type 1) consistedofthat Type 1 and Typeindependently

and remained undetected

were found in mioga after 1999. It was therefore
in epidemic

revealed

also

whereas group B (containing

curcuma or ginger were introducedin Curcuma sp. In 1995

strains,4)

strains
spreadinganalysisand all mioga strains,(Fig.

to ginger3)

on the rep-PCRamong strains

some ginger

Based on the results

and that of Type 2 was identicaland China (Fig.

constructedto that of several ginger

concluded

until the first

Furthermore, Type 1that the disease

has started from different

throughoriginscaused by

and has been

separate routes

Fig. 3 BGX-PCR profiles of Zingiberaceous
strains fromdifferent countries

Fig 4. Dendrogram showing genetic diversity ofK solanacearum isolated from Zingiberaceae
plants on the basis of rep-PCR

References
Hayward, A. C. (1991)

Biologyand epidemiology

solanacearum. Annu. Rev. Phytopathol
Horita, M. & K. Tsuchiya
Ralstonia

solanacearumof bacterial

wilt caused by Pseudomonas

29:65-87.

(2000)

Comparative

analysisof Japanese

based

on 16Sand foreignrRNA genesequencesdiversityof JapaneseJ

strains

of

Gen.

Plant

Patholstrainsof Ralstonia

66:132-137.
Horita,M. & K. Tsuchiya(2001)

solanacearum. Phytopathology
Tsuchiya,

Wilt

Disease:Springer-Verlag,

p6 1-73

Tsuchiya,

9 1 :399-407.

K. & M. Horita (1998)

Bacterial

Genetic
Genetic

Molecular

andK., M. Horita, W. Boonsuebsakul

diversityof Ralstonia

Ecological

Aspects& K. Mulya (2002)

solanacearumin Japan.

(Ph.

al

Prior

Occurrence

adaptationof new strains of R. solanacearum associatedwith Zingiberaceae
Japanese agro-ecosystem. Abstracts 3rd Int Bact Wilt Symp. P89.
Tsuchiya,K., H. Sawada, T. Yoshida

adaptation
Zingiberaceae& M. Takahashi

of new race strains of Ralstonia
plantsin Japan. Phytopathology(2004)

Invasion,

solanacearum, causal pathogen
94 (6):S

104.

et

eds)

and epidemic
plant

in thetransmission

andof bacterial

wilt of

Taxonomic analyses of soybean SDS and dry bean root-rot
based on a MAFF - NRRL Collaboration

pathogens

Takayuki Aoki1 and Kerry O'Donnell2
National

Instituteof Agrobiological305-8602
2Microbial

Japan.
Genomics

and Bioprocessing

Utilization

Research,

UnitedPeoria,Illinois,Emailtaoki@nias.aflfrc.go.jp

Sciences,States6 1604-3999,

Research

Department

Genetic

Diversity

Department,

Unit,

National

Centerof Agriculture,

Tsukuba,for Agricultural

Agricultural

Research

Service,

USA.

Taxonomic and phylogenetic analyses of soybean sudden death syndrome (SDS) pathogens anddry bean root-rot pathogens were performed based on a MAFF - NRRLcollaboration.
Isolates of thesepathogens from the United States, Argentina, Brazil and Japan were accessioned in both culturecollections
Detailed phenotypic comparisons of macro- and microscopic features and phylogenetic
analyses of multilocus DNA sequence data, including those on the nuclear ribosomal intergenic spacer
region and the single copy nuclear gene translation elongation factor 1-ot, indicated that they comprised
five distinct species
Soybean SDS in North and South America was found to be caused by four distinct
species Fusarium virguliforme, E tucumaniae and two undescribed species of Fusarium. Further, drybean root-rot in North America and in Japan was found to be caused by R phaseoh and an undescribed
species of Fusarium. Amongthem, an undescribed species was a commonpathogen to both soybean
SDS in Brazil and dry bean or mungbean root-rot in the United States, Canada and Japan.
Introduction
regions

Soybean sudden death syndrome (SDS) has been reported from all major growing
in North and South America (Nakajima et al 1993, Ploper 1993, Anderson and Tenuta

1998,

Rupe et al2001)

commercial production
agent of thisglycinesdiseaseand this diseaseof this crop.

has been reported

Roy (Roy et al 1997)

and closely(Burkh)relatedpart of a collaboration
et al

2003)

of the SDS pathogen
pathogentypicalsymptoms

phylogenetic

analysesof Phaseolus

vulgarisof Fusarium

disease.

These

f. sp.

2000,

in Argentina

were recovered
isolateshomogeneous

L, E solam f. sp. phaseoh

Li et al 2000,

Rupe et al 2001)

for the soybean SDS pathogen

between INTA-EEA, Marcos Juarez, Argentinaof thethe etiologicalof DNA sequences,have been shown to be geneticallyfield surveys were conducted

Twenty strains

in 1972,

to thecomplex or section Martiella comprises at least 26
2000), many of which have not been described.

W. C. Snyder & H. N. Hansen (O'Donnell

In 2000 and 2001,

in Arkansas

constraint

as F. solam (Mart ) Sacc. or its forma speciahs,

Recent molecular

to a root-rot

become a serious

Since its discovery

however, revealed that the E solam species
phylogeneticallydistinct
species (O'Donnell
North American isolates

has recently

and JIRCAS,

from soybean plants

were depositedasJapan (Aoki
exhibiting

to the Microorganisms

Divisionof the MAFF Genebank System (MAFF)

Sciences(NIAS,

Collection

Tsukuba,(NRRL)

Ibaraki,Japan)

at the National

and in the Agriculturalat the National

Center for Agricultural

Peoria,Illinois,USA)

A systematic

pathogensand related

fusaria

was initiated.

Argentina,

strains

pathogen,E solam f. sp. phaseohPennsylvania

State University,

USDA/ARS,

Instituteof soybean SDS pathogens

Utilization

Research

StatesStrains

pathogen(O'Donnell

2000)

pathogensfrom Brazil

and Argentina,

pathogensfrom Canada were made at MAFF and/or NRRL and included

objectiveof this researchin 2002and 2003,

concordance phylogenetic

mung bean (Vigna

radiataalliesmorphologicallyand(Taylordiversityand species limits

Materials

and methods
In total, 24 Argentinean,

14 Brazilian

and 8 USA isolates

USA and 2 Japanese isolatesof dry bean root-rot pathogens,and 1 isolate

and compared systematicallyCollection

Wilczek

and phylogenetically.
nitrogen

National

SDS) root rot

in the study.

Theof the soybean

using

genealogicalto understandtheir

genetic

of soybean SDS pathogens,2 Canadian isolatesof mung bean

All strains

includedvapor at the Agricultural

Center for Agricultural

Utilizationin the study are stored

ResearchResearch

Service

Cultureand at the MAFF

Genebank System (Microorganisms

Division),

National

Institute

Typicalbiological

specieswithin the E solam species complex

strains

comprising

et alof E solam, including

mating populations

comparison.
Detailed(2003)

procedures
Fusarium

I-VII, preservedfor investigatingstrains

1990)

in 9-cm plastic

fluorescentlightor underdaylight

Average and standard deviation(S.D.)

MolecularPetridishesof Agrobiological

Sciencesat NRRL and MAFF were also examined formorphologicalwere incubated(Nirenberg

50 randomly

characterson potato dextroseat 20°C in complete

to study

morphologicalin the size of conidia

were describedin Aokiagar (PDA)and SNA

darkness,

under continuousand culturalcharacteristics

were based on the measurement of

selected

conidia,based

phylogenetic

analysesof DNA sequences fromthe strains followed

O'Donnell(2000)

DNA sequences from the entire nuclear ribosomalintergenic

spacerand Aoki et al (2003)

3from an unknownhost from the United States were examinedor in liquid(NRRL),

et al 2000),

to the SDS

of soybean(L.)

molecularly

recognition

by lyophilization

relationshipnew accessions

speciesroot-rot pathogens,of E solari f. sp.

was to compare North and South American isolates

and their

fromStates and Japan stored at NRRL, FRC (Thein the study because of their close phylogenetic

pathogens(NCAUR,and a dry bean root-rot

phaseoh were included

SDS

Culturestudy on soybean SDSUSA) and MAFF were examined.
Also

Serviceto the new soybean SDS isolates

in the United

in the United

Researchand phylogenetic

In additionof Agrobiologicalon the number of septa

and cultural

conditions

a

(IGS)

region, the nuclear ribosomaltranscribedspacer(ITS)internal

regionanddomains Dl and D2 at the 5' end of thenuclearlarge

portionof the translation

(1-a)

gene

Applied

Biosystems-Hitachi

3730 genetic
editedsubunitwere

rDNA (28S),

and aelongation

factorobtained

using

an

Prism 3 100 or

analyzer

Sequence data werewith Sequencher

version 4. 1.2 (GeneCodes, Ann Arbor, MI) and then the aligned
sequenceswere analyzed

in PAUP* version 4.0bl0

phylogenetically(Swofford

2002),

using maximumparsimony.

Fig. 2. Microscopic
morphologyof Fusariun
tucumaniae isolated from soybean plants exhibiting
typical
SDS symptoms in Argentina. A: Aeria
falcate conidia
with a foot-cellformed on tal
conidiophores
B: Aerial minute conidia formed on
short conidiophore
C-H: Sporodochialfalcafeconidia with a foot-cell and a branched conidiophore
I-J ChlamydosporesA, H-J on SNA; B-G on PDA
A-C, H-J NRRL 34546 = MAFF 239252; D: NRRI
34547 = MAFF 239253, E: NRRL 34548 = MAFI
239254;
F: NRRL
34549 = MAFF 239255,
G
NRRL 34550 = MAFF 239256.

Fig 1 One of 2 most-parsimonious
phylograms
showing evolutionary
relationships
among thesoybean SDS and Phaseolus root rot pathogens
The combined data set of 4560 aligned base pairs
was rooted with sequences of Fusarium spp. NRRL
22387 and NRRL 22574. Strain numbers are those of
NRRL

Fig. 3 Microscopic
morphologyof Fusarium
virguliforme isolated from soybean plants exhibiting
typical
SDS symptoms in Argentina
A: Aerial
minute conidia formed on a short conidiophore
B:
Aerial falcate conidia with a foot-cell formed on tallconidiophores
C-F: Sporodochialfalcate conidia
with a foot-cell and a branched conidiophore
G-I
Comma-shaped
sporodochialconidia
J-K:
Chlamydospores
A-C5 J on SNA; D-I, K on PDA.
A-Q J, K: NRRL 34551 = MAFF 239257; H: NRRL
34552 = MAFF 239258, I NRRL 34553 = MAFF
239259Results
Parsimony analysisof the combinedof soybean is caused by fourbean root-rot in the United
species(Fig

1)

DNA sequence data (4560phylogeneticallydistinct

speciesStates and in Japan were resolved

One of these latterbp) indicatesthat SDS

strains

causing dry

while

as two phylogeneticallytwo species also contains

distinct

strams that are responsibleforsoybean SDS m Brazil and for mung bean root-rot in Canada.
Detailed

comparisonsof conidial

morphology

bean root-rot pathogens

share unique, aerial

conidia

on tall and mostly simple

with a foot-cell

nonseptate(Figs

minute ellipsoidal

2B & 3A)

complex represented
soybean(so-calledin that they

aerialconidiophores

microconidia)

by mating populations

strains)

produce

I-VII

Most (21diagnostic

septateStates and Brazil and those of the
The Argentinean

dry beanconidiophoresof the E solam speciesofsporodochial

the Argentinean

conidia(so-called

pathogen was described

described

as E virguliforme

as E tucumaniae T. Aoki, O'Donnell,Yoscontains

3 strainsforms uniquesporodochial

Fig. 2), which

O'Donnellcomma-shaped sporodochialconidia(macroconidia;

causes soybean(macroconidia)

with roundedin the Unitedet alspeciesis diagnosedby the production

rostrate

apicalbean root-rot

3G-I)

One of the other species,(6 strains),

produces
this

septatespeciesFig. 3) andwith

septateFusarium sp. 3,

sporodochialconidia

The other soybeanof septate

sporodochialconidia
basal

This

(macroconidia)

foot cellwith aIn addition,a dry

known as E solam f. sp. phaseoh,wasphaseoh (Burkh ) T. Aoki & O'Donnell
septateSDS

as well as dry bean root-rot inand mung bean root-rot in Canada (2 strains)previously

by shorter

also

togetherpathogen

It is characterizedThis species(Fig.

beak and a distinct

as E

was

conidiacell like a wedge-shaped(2 strains),

2003)

causing(8 strains)

et al 2004;

Fusarium sp. 4, causes soybean SDS in Brazil (1 strain),and Japan (3 strains)

States(Scandianiends that differentiateStatesin Brazil

in Argentina

Fig. 3D-F))

SDS in Brazil

was also discovered& T. Aoki (Aoki

of soybean SDS pathogens

in theStates

The soybean SDS pathogen

2003)

aerialin the United

soybean SDS (7 strains)

recombined

2A & 3B) androot-rot pathogens

et al 2003;

the United

all form septate(Figs

on short
strains

Homma& Lattanzi (Aoki

species,featuresFig. 2C-H) that are longer than those of the other soybean SDS pathogens

and Japan.

whichthat the soybean SDS and dry

These features were not observed in typical

SDS pathogens

macroconidia;
Unitedconidiaconidial

revealed

at the rank of species

sporodochialconidia(macroconidia)

basal foot cell that is typicallyhookedventrally.

acuate apicalcell and a distinct

undescribed

Fusarium spp., i.e. spp. 3 and 4, formal descriptions

are in preparation.

(Aoki

et al

with an
For the twoTable 1 Diagnostic
bean root-rot

morphology

Discussion
For differentiating
speciesthatof Fusarium species responsible

the four Fusarium species responsiblegroup is as follows

distributionof each of

E virguliforme

SDS, dry bean and mungfor soybean SDS and the twocause dry bean or mung bean root-rot, diagnostic

summarized in Table 1 The known
monophyletic

for soybean

morphologicalfeaturesarethe five species

withinthis- United States and Argentina,

E tucumaniae

Argentina and Brazil, E phaseoh - United States, Fusarium sp. 3 - United States and Brazil,
andFusarium sp. 4- United States,

soybean SDS pathogen
et al(2004)

pathogenin Argentina,

Canada, Japan and Brazil
E virguliforme,

Fusarium virguliforme

The discovery

of a second

recently

by Scandiani

was reported

also appears to be present in Canada as a soybean SDS(Terry Anderson, pers commun.)
The causal pathogens

described

of soybean SDS and dry bean root-rot have been called

as E solam f. sp. glycines and E

solani f. sp. phaseoh, respectively,on host pathogenicity(Roy 1997,

Roy et al 1997)

furtherin the present

study, neither

demonstrated

dry bean root-rot pathogens
pathogenicity

form exclusive

in Aoki et al (2003),

evolutionarygroups, strongly
originstaxonomic and phylogenetic

andnor the twosuggestingthatThis finding challenges

naming system because it obscures the fact that geneticallyare being referred to by the samsforma specialis

based primarily

the four soybean SDS pathogens

or monophyletic

to each host may have independent

theformae speciales

As discussedanddiversespeciesname. Results of the present study provide aframework for understanding

the genetic

diversityof pathogenic

Fusarium species that have had strong negative economic impact on soybean and green or dry

bean production

within North and South America.

Results
scientificof the present

collaboration

complementary
agriculture.

among scientists

scientific

tools

study

serve to illustrate

the collective

international

collections

who bringof importanceto world

at different

to bear on plant pathologicalcultureproblems

Moreover, it is throughthecontinued

depositand phenotypicof novel germplasm

withinpublicallyaccessible

microbial

characterization
appliedsystematic

agricultural

benefitofand genetic
collectionsthat

science will continue to progress

Ackowledgments
Specialresearch

thanksare due to Dr Y. Homma,JIRCAS,

Japan and members of the soybean

group at INTA-EEA, Marcos Juarez and at INTA-EEA, Famailla,

Phytopathology
isolates

Section,

EEAOC, Tucuman, Argentina

of soybean SDS pathogens

Embrapa Soja, Londrina,
Pedro, Argentina

Brazil,for depositingin Argentina.

for their assistanceCordial

Dr M.M. Scandiani,
BrazilianthanksLaboratorio

and Argentinean

the mung bean root-rot pathogens

in obtaining

fresh

are due to Dr J. T. Yonnori,isolates

Agricola

Rio Parana, Sanof soybean SDS pathogens

NRRL and MAFF. Thanks are due to Dr Terry Anderson, Agriculture
for depositing

and members of theinand Agri-food

Canadaof Fusarium solam f. sp. glycines

causingfrom Canada.

References
Anderson, T. R. & A. Tenuta (1998)

First

reportsudden death syndrome of soybean in Canada. PI Dis 82:448.
Aoki, T, K. O'Donnell,

Y. Homma& A. R. Lattanzi (2003)

is caused by two morphologicallysolam

species

complex,

America. Mycologia

and phylogenetically

F virguliforme

in Northfrom other F solam based

Phytopathol
Nakajima,

death syndrome of soybean

distinct

species

differentiation

on mitochondrialof Fusarium solam f. sp.

small

subunitrDNA sequences

90:49 1-497.
T, T. Mitsueda

Pennsylvania& M. J. D. Charchar(1993)

State University,

University

Occurrence
Abstract,

Recent advances in the taxonomy ofFusarium.

O'Donnell,

K. (2000)

Molecular

D. (1993)

Sindrome

Fusariumsudden deathWorkshop,

Park, p. 79.

H. I (1990)

species complex. Mycologia

of soybean

7th Int

Nirenberg,

argentino.

the Fusarium

America and F tucumaniae in South

Molecular

syndrome caused by Fusarium solam in Brazil

Ploper,within95 :660-684.

Li, S., Y. K. Tarn& G. L. Hartman (2000)
glycines

Suddenphylogenyof the Nectria

Stud. Mycol 32:91-101

haematococca-Fusarium

solam

92:9 19-938.
de la muerte siibita:

Avance Agroindustrial

Nueva enfermedad de la soja en el noroeste

Ano 13 N° 54:5-9.

solaniRoy, K. W. (1997)

Fusariumsolani on soybean roots

sudden death syndrome and identity

and relevance

NomenclatureofF solani form B. PI Dis

Roy, K. W., J. C. Rupe, D. E. Hershman & T. S.Abneysoybean. PI Dis

solaniJ. C. Guerber,

Differentiationf. sp. glycines,

pathogenicity,(1997)

Suddendeath

agent of81:259-266.
syndromeof

81 1100-1111

Rupe, J. C, J. C. Correll,

Yount (2001)of the causal

C. M. Becton, E. E. Gbur, M. S. Cummings & P. A.

of the sudden death syndrome pathogen
from otherand mitochondrial

isolatesof F

DNA restriction

basedof soybean,

on cultural

fragment length

Fusariummorphology,

polymorphisms

Can. J.

Bot 79:829-835
Scandiani,(2004)

M., D. Ruberti,
Recentvirguliforme
Swofford,
VersionK. O'Donnell,

Outbreak

31:21-31

of Soybean

SuddenDeathand Fusarium tucumaniae in Argentina.

D. L. (2002)

PAUP* Phylogenetic

4.0b 1 0. Sunderland,

Taylor, J W., D. J Jacobson,(2000)

T. Aoki, R. Pioh, L. Giorda,

Phylogenetic

species

Massachusetts

Analysis

A. Luque & M. Biasoh

Syndrome

PI Discaused88: {inpress)

Using Parsimony(*and other methods)

Sinauer Associates

S. Kroken, T. Kasuga, D. M. Geiser, D. S. Hibbett
recognition

by Fusariumand species

concepts

in Fungal& M. C. Fisher
Genet

Biol

Systems biology Initiatives
Magnaporthe grisea

in the rice blast fungus,

Yong-HwanLee
Schoolof Agricultural

Biotechnology,

Emailyonglee@snu.ac.kr

Seoul National

University,

Seoul 1 5 1-742,

Korea

Magnaporthe grisea, the causal agent of rice blast, is an economically devastating fungal plant
pathogen. Whole genome sequence data has been released and over 8,800 unique ESTs of this fungus areavailablein public databases
Recently we generated 20,000 mutants using Agrobacterium
turne/adens-mediatedtransformation (ATMT) and developed a high-throughput phenotype assay system.
Thus far, over 1,000 loss of virulence and several hundred transformants, including auxotrophs,
developmentally-defective
and oleate-nonutilizing
mutants, were obtained from an ATMTmutant library
The T-DNAtagged sequences from the mutants are being rescued by TABL-PCR technology. As the firststep to establish a system for this pathogen, we developed databases to manage all of the phenomics andgenomics data on these transformants
Furthermore, a web-based portal system has been established for
efficient data acquisition and analyses, utilizing BLAST and INTERPRO searches, gene ontology (GO)classification,
expression profiling, phylogenetic
and comparative analyses, and tools for additional
functional genomics research. Fungal systems biology initiatives
provide a platform for a system-level
understanding of the biology ofM grisea, and they are also applicable to other fungal pathogens
Introduction
Rice blast,
study plant-fungi
worldwideclassical

caused by Magnaporthe grisea, was developed

interactions

This is due not only to the economic significancebut to the genetic
genetic

crossingand development

and molecularbeen conducted to understandseriesinfectionof the host plantbeen identified(restrictionof studies,of this pathogen.

and gene knock-out technologies

Extensive

researchmechanisms of the pathogen
biologicallevelscues and related

determinants
integration)mechanisms required to complete

throughthe diseasesignaling

decade.

Throughansystems

involvedinmutagenesis

strategies

and moleculargrisea were completed
accurate annotation

cycle are not fully understood.

Recent advances inand made publicallylevelsIn 2002,

available.

of pathogenesis

whole genome drafts of rice and M

Currently

much effort is being focusedonof genes in both organisms

Agrobacteriumtransfer

biologicalusing REMI

However, the precise

genomics research are making great progress towards an ultimate understandingat the biochemical

hasMore than a dozen genes haveinsertionalor reverse geneticmaps,

and defense mechanisms ofdunng the pastby the fungus have been elucidated.

enzyme mediatedincludegenetic

the environmental

as pathogenicity

These features
extensive

infection

toof this diseasemating types,

and molecular

elegant

tractabihty

using strams of two differentof transformation

the host at the cellular

as a model plant disease

tumefaciens-mQdiatQd

genes to a wide vanety of plants

transformation(ATMT)

has long been used toand has also been used extensively

as a tool forinsertionalmutagenesis

in Arabidopsis

transformed using A. tumefaciens.
potential

as an alternative

conventional

thaliana.

For insertional

tool to REMI

transformation

More recently,

techniques

severalfungi

have beenmutagenesis,

this techniqueoffers enormous

One of the principal

advantagesof ATMT overis the versatility

in choosing

which starting

material

totransform Applicable to several fungi, A. tumefaciens can transform protoplasts, hyphae, spores,
and blocks of mushroom mycelial tissue. Furthermore, ATMT generates a high percentage oftransformantswith a single

subsequentisolation

insertof taggedof T-DNA in the fungal genomes, which will facilitate
genes especially

Recently we developed ATMTtechnology
To dissect
in the rice blast

the function

ATMT 2) development
throughput

technology

mutants of interestof a highlackinga sexual stage.

in M grisea.

of potential

fungus, we initiatedfrom those fungi

thefungal pathogenicity

a projectthat includes

throughput

screeningfor DNA extraction,and finallyMoreover, we are establishinggenes throughout
1) generation
technology4) rescuing

an infrastructure

the genomeof mutants using

for mutants 3) highflanking

sequencesof bioinformatics

fromtools forsystems biology.
Fungal strain

used in this study

We assessed the genetic

make-up ofM grisea KJ201,

different

transposons,genes, growth rate, and mating ability.

different

from strain 70-15,

avirulence

number of transposons
the genetic diversity

and avirulence

genes These elements are generallyof a high throughput

technologyof HTS have been developed

useful for comparing

screening(HTS)(mutants)

system is required.

The conceptinOne bottleneck

and are widely used in the pharmaceuticalandfor new drugs However, HTS systems are not well developed

the plantis the lack of specificsites we can target

systems have been developed by companies that develop pesticides
All single-sporedmutants were grown in a 24-well tissue
medium to obtain mycelia.

at the same time,tocandidates

Using these plates,in liquid

we measured growth rate, pigmentation,and theof each mutant By changing

the composition

cultureHowever, HTS
plate

to measure the ability of each strain to conidiate.
In addition to HTS for phenotypes, we developed

DNAextraction.

is quiteindustryevaluate potential

technology

KJ201

of strainsdevelopment

colony morphology

Strainusing

for which the whole genome was sequenced, based on the copy

Development of high throughput
screening technology
To characterizephenotypesof many transformantssciences

a Korean field strain,of media, we can use thisa high throughput

technology

forHigh throughput

pathogenicity

Traditionallyassay

plantsare grown in soil in pots to test fungal pathogenicity.

is not always easy to replicate
are all aware, disease
pathogen,the same environmental conditions

development

provideseveralgermination

advantages

Further,a protocol

As weof host plant,

for growing rice plants

on a tissue

we removed nee seed coats Removing seed coats

includingand accelerated

in every experimentis the outcome of a complex combinationand the environment We developed

culture medium in the laboratory.

However, it

efficientsurfacesterilization,

plant growth. It is importantto pre-treat

more homogeneous
dehulled

rice seeds m

100% ethanol before surface sterilization.

This pretreatment

seed surface, rendering

more thoroughsterilizationof the seed surface.

control the compositionand concentrationof ingredientsin the tissue culture medium as well It

is well known that most rice cultivars
are supplied

reduces surface tension

become more susceptible

on the rice

Further,when higher

levelswe canof nitrogenin the culture medium (soil)

Mutants selected for further study
Weobtained more than two thousand transformants
HTS procedure. These mutants were further tested
Once defects

were verified

on rice seedlings,

genomic DNA was isolated.

showing defects

for pathogenicity

defects

from the primaryon rice seedlings

these mutants were grown in liquid

By Southern blot analysis,

T-DNA insertion

medium andinto the chromosome

and its copy number was verified for each mutant
Identificationof flanking

Pathogenicity

sequences
defectiveof T-DNA insertion

mutants, verified

subjected

to TAIL-PCR to rescue flanking

available,

we were able to identify

far, we have identified
changessequences

as a single

integration

Since the genome draft ofM grisea is

ORFs based on sequences flanking

the insertion

more than 200 loci where T-DNA was inserted.

by T-DNA insertion,

we knockedof T-DNA, wereout the same locisite. Thus

To confirm phenotypicusing

a gene replacement

technique.
Developmentof infrastructure

In addition

for systems biology

to our genomics research, we are constructing

enormous amount of genomics data. Presently,
for efficient
ontology

data acquisition(GO)

classification,

tools for additional

functional

and analyses,
expression

databases

to manage thea web-based portal system has been establishedutilizing

BLAST and INTERPRO searches,pro filing, phylogenetic

genomics research.

and comparative analyses,

gene
and

Acknowledgements
This work was supported

by BioGreen2

1 project

Referencesde Groot, M.J.A., P. Bundock, P.JJ
tumefaciens-mediatedDean, R.A. (1997)

Signal

Hooykass& A.G.M. Beyersbergentransformationof filamentous

pathwaysand appressorium

fungi(1998)Nature Biotech.

morphogenesis

Agrobacterium
16: 839-842.

Annu. Rev. Phytopathol

35 211-234.
Krysan,P.J , J.C. Young & M.R. Sussman (1999)

Arabidopsis.

Plant Cell

Liu,

CAMP regulates

fungus Magnaporthe

Y.G. & R.F. Whittier

amplification

andMullins,

E.D.,

infection

structure

Thermalof insert

Agrobacterium-msdiatedasymmetricinterlacedend fragmentsfrom PItransformation

R. Raina,

Phytopathology

Ou, S.H. (1985)

Rice Diseases,

2nd ed. Commonwealth Mycological

3 9-16.

and YAC clonesfor

Exploring

Institute,

tumefaciens-mediatedfungus, Magnaporthe grisea. Mol Cells
Having a blast& S. Kang (2001)
tool insertional

91 173-180.

Rho, H.S., S. Kang & Y.-H. Lee (200 1) Agrobacterium

Microbiol

automatableof Fusarium oxysporum : an efficientand gene transfer

N.J. ( 1995)

PCR:

D. M. Geiser

mutagenesis

Talbot,in the plant

Genomics 25 674-68 1

X. Chen, P. Romaine,

the plant pathogenic

formation

grisea. Plant Cell 5 693-700.
(1995)

sequencing

chromosome walking.

mutagen in

1 1 2283-2290.

Lee, Y.-H. & R.A. Dean (1993)
pathogenic

T-DNA as an insertional

the pathogenicity

12: 407-41

Kew, England.
transformationof1of Magnaporthe

grisea. Trends

2. NEW PARADIGMS OF
BIOLOGICAL RESOURCECENTERSPreface
WFCC-MIRCEN World Data Centre for Microorganisms(WDCM)

organized

thest

WDCMsymposium entitled "Microbial Resources Centers in the 2 1 Century: New Paradigms"
on February 16th, 1999. The symposium was immediately followed by "OECD Workshop
Tokyo '99 on Scientificand Technological

Infrastructure- Support for Biological

Resource

Centres (BRCs)" on 17-18 February, 1999.
Since these events, the term of BRCs has beard a new meaning, especially
microbial

culture collections

Based on the efforts

Professor
published

Hideaki Sugawara (Japan)
an official report entitledlife sciences

and biotechnology"

for themade by the Task Force on BRCs chaired byin the Working Party for Biotechnologyof OECD, OECD
"Biological
Resource Centres - underpinning the future of

in 200 1

In 2002 and 2003, the Task Force on BRCs led by Dr Louis Rechaussat(France)hasdeveloped standards to be called BRCs and also a strategy to realize the Global System for
BRCs In the meantime, NITE BRC was established in Japan; the EBRCN project was activatedin Europe; ATCC trademarked
Thailandorganized

international"The Global
meetings

out in many countries
Therefore, it is the opportune
concept of BRCs and also implementation
BRCs in the special

Bioresource

Center", African countries,

on BRCs, and other relevanttime for us at ICCC-10

activities

China andwere carried

to reach consensus about theof the concept

We will revisit the new paradigm ofsymposium II and the panel discussion

to have concrete plans toward BRCs

st

inthe

21

century.
HideakiSugawara WDCM,National Institute

Makoto M. Watanabe National Instituteof Genetics,

for Environmental

Studies,

Jean Swings BCCM/LMG Culture Collection,

Japan
Japan
Belgium

The new paradigmof the Biological
Resource Centres
challenges
and opportunitiesof culture collections
or BRCs(BRCs):

J. Swings and V. Storms
BCCM/LMG Culture Collection
Email

K.L.Ledeganckstraat

35 Gent B9000,

Belgium

Jean. Swings@UGent.be

Different collections or BRCs are in different financial and environmental conditions and do
not necessarily have the same challenges and opportunities
In general however, three important
cornerstones can be distinguished.
As a first cornerstone, BRCs organize the professional conservationand distribution of biological materials and the data related to them. Key values are authenticity, genetic
integrity and validity of the information provided. There is a need for global standards to assure top
quality of biologicalmaterials and data through appropriate Total Quality Management The second
cornerstone is the linking of BRCs to scientific centres in taxonomy, molecular genetics, biochemistry,cellular biology, biochemistry or genomics The third cornerstone, commonfor all BRCs is the valuationand thus demonstrable utilizationof its holdings and its data. The bioeconomic logic of industry,healthcare and the research related to it is here clearly the driving force of a mainly economic reality.
Biological

Resource

underpinning

life sciences

Operation

and Development)

thought

centres(BRCs)

and biotechnology.
thinkof the actual

culturevisionarysciencesandstorefront) )Different
actualsituationdifferent
theirfinancialinspiredcollections

The effort,

Resource Centres,

which

underpinningoffor the future
has taken so

by many WFCC members, has resulted

can be distinguishedcollections

in an

the futureof lifein any vision developed

startingfrom theIt is obviousthat the actual

collections

are inat different

stages of cutting

Perfect situations

do not occur We can think of a grading

system for BRCs

however, the challenges

definedand opportunities

criteria.

A tiered structureof culture collectionsin the areas discussed below.
BRCs organize the professionalpreservationand the data related

to them. The biologicalviruses,animal cells

fungi, tissues,and consequently

cultureconditions

to a number of clearlyand generated

has done a great effort

and environmental

cornerstones

accordingfor Economic Co-(http //oecdpublications.gfi-nb.com/cgi-bin/oecdbookshop.

cornerstonesof Culture

The OECD (Organization

and reference

document ('Biologicalbiotechnology'of the infrastructure

Resource Centre and forms the basis

manyyears and was from the beginning
importantparttank on BRCs (1999-2004),

to define the new Biologicaldevelopment

are an essentialthat have to be conserved together

BRCs are repositories

of high quality

biologicalof biologicalmaterials

concerned are of all kinds

bacteria,

fields of the bioeconomy,materials

with the data and the literaturematerial

In generalfor the next 5 years are situatedand distribution

materials

In different

can be envisaged.

from industry,

research,

are usedreferencesfrom clinicaltraceability)

centres and also from the great biodiversity

study efforts

materials

Therefore,

need to be maximallyand genetic integrity
achieve global

guaranteed.

of the material

standards

and validityof the informationof biologicalAppropriate

QualityTotal

important cornerstone. Concerning the Information
to a data model integrating
decades,

bioterronsm

Biologicalall scientific,

affected

Resource Centres therefore

provided.

materialsof theThere is a need toand data related

Management therefore
Technology,

administrative,

microbiology

and the stability

key values of BRC's are authenticity

to assure top quality(including

The quality

on a high degree

face continuallyforms a first

culture collections

literature

to themare moving

and strain data. In the last

(cfr. Antrax, The Butler

increasing

Biosafety

Case)

and biosecurity

demands
As a second cornerstone,
taxonomy, preferably

linkedBRCs are, and will continue

to university

departments

to be, centres of excellence

In the future,especiallygenomic

will be of importance.

Indeed,there

taxonomy and taxonomy of unculturable

organisms

traditional

and taxonomy of bacteria

link between culture collections

needs fortification.

The present

developments

in phylogeny,

require from the taxonomist that he adapts himself
taxonomy will synthesize

microevolutionary,inand fungi

This basic link

genetics

and genomics

population

and rethinks

phylogenetic

is a

his science.

The future genomic

and genomic data in a new synthesis

Since today only a very small part of the microbial

diversity

has been studied,

we canexpect that in the future, there will be massive incorporation of biodiversity
items The needed
absorption capacity of BRC's will be huge. New Technologies as automatization, robotisationand miniaturisation
Besidesneed to be increasingly
their preservation

applied.

role, culturecollectionsof biodiversity.

At the Earth Summit in Rio de Janeiro

comprehensive

strategyfor "sustainable

Rio was the Convention on BiologicalAn important
benefits

arising

pharmaceuticalout of the commercial

intellectual

propertyrights,without

importanceof Property

RightsrelatedFurthermore,culturecollections

world leadersagreed on a

One of the key agreements adopted

debateinvolves

ataccess to and sharingof theof genetic

material,such aswould be sold and protectedby patents

or otherand other utilization

Often, the products

for the conservation(www.biodiv.org)

part of the biodiversity

products

in 1992,

development"
Diversity

are important

fair benefits

to the source countries

to the biodiversity

This stresses

thedata, as well as data and databaseprotection.
biodiversity-rich
importantpublicguidelines

countries

to betterresource for public

and the policy

also

understandinformationplay

a capacity-building

and utilize

and policy

formulation.

makers can call upon for objective

for the safe and ethicaluse of biologicaltheir

resourcesrole,

to help

diversity,and anThey are bodies

that themicrobial

help in developing

regulationsandFinally,
utilization

anotherof its holdings

research related
valorisation

importantcornerstone

commonfor all BRCs is the demonstrable

and its data. The bioeconomic

to it is here clearly

the driving

healthcare

and theforce of a mainly economic reality.

Thus, theof BRCs will be the use of its treasureslogic of industry,

for the biodiscovery

in the developing

bio-

economy, taking into account the IPR and CBD. It is clear that WFCC has a role to play in thefurtherdevelopmentof a BRC concept

and in the standardizations

document forms the basis for further

developments

linkage

BRCs to internationalof the WFCC and individual

will be of major importance to provide
policydevelopment,

an effective

e.g. the GBIF initiative,

WIPO,

-42-

needed.

because of its overarchingorganisations

voice in international

The OECD

vision. Electronic

and Global networking
initiatives

and science

The criticalrole of Biological

Resource Centers

Raymond H. Cypess and Shung-Chang
American Type Culture

Collection

in public

healthJong(ATCC),

1080 1 University

Blvd. Manassas, VA 201 10-2209,

USA
Email sjong@atcc.org
Designing appropriate mechanisms for the security of infectious agents and biodefense-related
pathogens requires the availabilityof centralized Biological Resource Centers (BRCs) and collaboration
between government agencies, academia, industry and the public health community. Of particular
relevance to meeting these requirements is the American Type Culture Collection (ATCC), which has
garnered a reputation for neutrality, integrity, quality, service, and personnel with comprehensive
bioresource expertise since its establishment in 1925. In recognition of its success in global surveillance
for public health, The National Institute of Allergy and Infectious Diseases (NIAID) of the National
Institutesof Health (NIH) has recently established the Biodefense and Emerging Infections Research
Resources Repository (£e/resources)
and the Malaria Research and Reference Reagent Resource Center(MR4 Center) at ATCC, where qualified, registered scientists can obtain quality-assured
materials andinformation they need to study organisms or reagents that might be used as agents of bioterrorism or thatcause diseases such as malaria, SARS, West Nile virus, and hepatitis C. Also, in 2002 the Center ofVeterinary Medicine (CVM) at the U. S. Food and Drug Administration (FDA) awarded the ATCC a threeyear contract to study the mechanisms and speed of development of microbial resistance to veterinary
antibiotics once they are in commonuse for domestic animals The goal is to test susceptibility
patterns of
5,000 strains in total, which include Escherichia coli, Salmonella, and Campylobacter, identified
in thelast five decades
Introductiondiseases,
centralAs a consequence of recent bioterrorism

events and the threatof emerging

infectious

the U.S. government and the scientificcommunity recognizeda specificneed for a

resourceof these threatening

their

qualitylikelyones require
control,

referencestandards

qualityassurance,

inspection

to assure compliancesecurity,

access, and distribution

meeting

thesecoordinating

activities,

whileof all these agents and training

to identify

the most

that are based on the complexityof analytical

testing,

and access to them. Possession

with the more complex tests requiring
qualityagentsrequirements
the shared

government agencies,

to enhance research

the most stringent
proficiency

standards

testing,and documentation,with all U.S. and internationalof the materials

Resourceuse of quality-assured

biologicalacademia, and the public

One of the established
AmericanType Culture

conjunction

with other public

BRCs

Collection
healthsupporting

regardingdata. Of particularCenters(BRCs)

materials
healthresearch

tools,

as well as regular

regulations

and associatedare Biologicalindustry,

for personnel,

controllingthatsafety,relevancetospecializeinand information

among

infrastructureis thecommunity.

the public(ATCC)

Since

its founding

partners,

has been involved

healthin 1925,in global

the

ATCC,

in

healthservicestocontrol,improve, and protect

assuring

the security

community health.

of infectious

reagents

Recognizing

and standards,

awarded the ATCC more than 130 million

dollars

programs
1) The Biodefense

Research

establishedand Emerging

by NIAID/NM

Infections

inthe organization's

success

the U.S. government recently

contracts

to carry out the following

Resources Repository(beiresources),in 20032) The Malaria Research and Reference Reagent Resource Center (MR4 Center),
1998

NIAID/NIH
3)

inestablished

byin

The Use of Existing

Microbiological

Pathogens to Antimicrobial

Collections

to Examine Historical

Agents (Drug ResistanceSusceptibilityofProgram), establishedby CVM/FDA in

Resources Repository(/^/resources)

2002
The Biodefense

and Emerging

Infections

The use of biologicalResearch

agents in acts of terrorism

or war dates to ancient timesLong

before the germ theory of disease

was understood,

potentially

toxic material

from ill peopleordead bodies

armies, catapulted

into cities,

or placedin water supplies

inwere used against

attempts to demoralize and kill perceived enemies When microbes were discovered to cause
infectious diseases, research into the potential use of microbes as weapons began. In the UnitedStates,for example, President

Franklin

Roosevelt"aninhuman form of warfare." Privately,
biological

warfare capability.

publicly

denounced in 1942 germ warfare as

he approved a top-secret plan for the U.S. to develop

A year later the U.S. had a four-pound anthrax bomb. In 1950 the

U.S. Army tested

the spreadand survivalof "simulants,"

marcescens bacteria,

by spraying

them over San Francisco.

which were actually
WithinSerratia

days one San Franciscan

was dead and many others were ill with unusual Serratia infections, but the Army called this"apparently coincidental" Similar tests were conducted in the New York City subway system,at the Washington
fall of 2001

National

Airport,and elsewhere.

are a reminder that other potential

targets

and water supplies

At least 2 1 attacks

against100 years Federal

health agencies are evaluating

The anthrax attacks in the U.S. mail in theof bioterrorism

food/agriculture

exist, including

the food

have been documented in the last

and accelerating

measures to protect the publicfromhealth consequences of such an attack.
Homeland security
component The National
Institutesof Healthis a multifaceted

Institute

includingagentsestablished

theof Allergy

conducts and supports

tools to detect and prevent infectious
that

could

Biodefense

and Infectiousof which biodefense
Diseases(NIAID)is a criticalof the Nationalmuch of the research aimed at developing

diseasesintentionallyandendeavor,

essential

caused by a wide variety of emerging pathogens,
be introduced.

Emerging

Infections

In September
Research

2003

the NIAID

Resources

Repository

(ieiresources)at ATCC, where qualified,materials

and information

registeredscientists

they need to study organisms

can obtainthat might

quality-assured

be used as agents ofbioterrorism

or that cause emerging diseases

such as SARS, West Nile virus, and hepatitis

The targeted

agents have been categorized

and prioritizedby the U.S. Public

HealthServicedisease,panic and social disruption

and thebased on their severity

in causing widespreadneed for special

healthpublicpreparedness

publicand response. NIAID has also set research priorities

and goals for each organism in the broad area of biodefense
center named "ieiresources"
Category

helps

facilitate

A, B, and C Priority Pathogens

development

and evaluation

It offers

an alternative

distributeselect agents
In order to facilitate

information
throughof diagnostics,

for universities

development

electronic
and publicationand emerging
therapeutics,

proactive

and workshops,

communication

additionto securing acquisition,

generates

new reagents as scientificreagents,

on theseand informationmaterials can be monitored under strict biosurety
prevention

available

nationalof infectious

this informationtransferthroughof reagentscompanies

In

agents, fteiresources

also

that, in the long-term,forreflectingbythis function,practicescollects

resource and clearinghousea concerted

effortaccess to and use of theseand proceduresmaterials that are essential

and treatment

accessed at the ie/resources

technologyof biologicalorganisms,

CDC, USDA, and ATCC. By centralizing

Aeiresourcesand disseminates

advances are made. It is anticipatedspecimens,

and aid in thethat no longer want to store or

and pharmaceuticalstorage, and distribution
government's

detection,

agents,commercial developmentwith biotechnology

will become the Federal

A list of currently

infectious

enhances

Aeiresources
NIAID,

of NIAID

and vaccines against these organismsof methods, and facilitates

throughof the pathogenesis

the understanding

reagents and standards

media,The new

research and product development,

about biodefense-related
print,and emerging infections

and other institutions

relevantC.

diseases

for studying

may be obtained

the mechanisms,
from ATCC or

website, www.beiresources.org.

The Malaria Research and Reference Reagent Resource Center (MR4 Center)
Malaria, the most deadly of all tropicalparasitic diseases, has been undergoing
dramatic

resurgence. The World Health Organization

500 million

estimatesthat between 300 and

new cases of malaria occur each year, and that annual deaths

number between 2 and 3 million.
States(WHO)were reported

In 1997, approximately

to the Centers

for Disease

Control

resistanceof the malaria parasite to effective drugs presents

infections

At the same time, increasing

control of transmissiondifficultresistancefrom the disease

1,800 cases of malaria in the Unitedand Prevention(CDC)

Increasingproblems for the treatment of activeof mosquitoes to standard insecticides

to achieve. At the International

a

makes

Conference on Malaria in Africa

heldin Dakar in 1997,

malaria experts

from 35 countnes and representatives

malaria research funding

agencies identified

a specific need for a new biologicalfor standardized

reagents

and methods to ensure the comparability

from major
resource centerof results

for malaria

and Reference

Reagents

research and control
In responseto thisstatedneed, the Malaria

Resource Center, known as MR4, was established

Research

at ATCC in 1998

components of the Multilateral

Initiativeon Malaria (MIM),

involved

in malaria research,

controland development

National

Institutefor Allergy

and Infectious

central source of quality-controlled

Diseasesmalaria-related

mosquito vectors, molecularantibodies,

antigens,

molecular

biologya cooperative

assistanceof agencies

sponsored

reagents and informationby the U.S.

registeredusers The reagents include
synthetic

and human reagentswith a 3-part material transfer agreement between a depositor

the second party, as well as a statement of the terms and conditions

peptides,A process to help

and a recipient,of registrationato the international

items, recombinant proteins,probes and constructs,

used, is in place to meet the legal requirements

effort

The mission of the MR4 is to provide

malaria research community, free of charge, to qualified,parasites,

as one of the four

through

MR4 asunder which reagents are

and ownershipof the material thatis in the MR4 Center
In order to promote technology

transfer and interaction

between malaria investigators,

between field-based

and laboratory-based

as well as to encourage better

linkageprograms, MR4 also provides

an electronic

programs,
reagents

comprehensive
An International

center in prioritizing

onlineboard for users, workshops

and printed

catalogs

Advisory Committee has been establishedreagent acquisition

The Noguchi Memorial Institutebeen selected

databases

bulletinand serving

for Medical

as the first MR4 satellite

as a liaison

describing

researchand training

the available

to provide guidance

to theto the malaria community.

Research at the Universityof Ghana has recently

in Africa, which will increase its ability

to communicate

and serve the African malaria community.
As of this date, MR4 has more than 400 registeredalmost 320 new reagents, distributedand conductedantigens,

two workshops

plasmids,

approximately

850 items to scientists

Over 800 reagentsare now available,from the MR4 at ATCC or accessed

www.malaria.mr4.org. The updated
document" of malaria protocols,
file.

In 2003,

EST clones, and genomic DNA and CDNA libraries

current reagents may be obtained

PDF (*.pdf)

users (Pis)

MR4 acquired

all over the world,
includingantibodies,

A complete list of
at the MR4 website,"Methods in Malaria Research Manual", a "working-is available

The manual is intended

for downloading

as a Microsoft word (*.doc)

to give enough detailin the methodology

or
that

someone who is not an expert in the field of malaria will still be able to make use of these
protocols

The Use of Existing
Pathogens

MicrobiologicalCollections

to Examine Historicalto Antimicrobial
Agents (Drug Resistance Program)
Few issues evoke as much discussion and disagreement

antibiotics

in feed/animal

antimicrobial

resistanceproduction(AR)systems

Increasedin human microbial

pathogenshealthsubtherapeutic

from feed animal production.

animal production

specialists

thus adversely
the practiceand increased

impact human health.
of adding

traces of antibiotics
Controlconcerns

Infections

antibiotics

and Prevention(DCD)

more than 30,000

However, many veterinarians

andtoo many antimicrobials

producingloads in feed animals andgreater

resistanceone of its top

antimicrobial

for AR and MAR. MAR has emerged in foodborne

factors,

including(FDA)

resistanceper year An(MAR)

Since 1996

environment have been
Many

to compounds other than antimicrobials,

andresistanceelements,

influence the developmentof Veterinary

andand commensal bacteria.

species,the Center

more serious

States may be as high as $4.5 billion

from animals or their production

In 2002,(AR)

are becoming

isolates

movementof mobile genetic

might leave

The Centers forof multiple

serotype,

yields,that

among bacteria.

antimicrobial

analyzed

Administrationofis the development
originatingby feedAcademy of Sciences reported

microorganismsofto advocate the elimination

pathogendrug resistancecalls

caused by resistantproblemmicrobial

to animal food, while

difficult to treat, and the cost in the United
additionalthat are also carriedprofessionals

In 1967 the Nationalin meat, thus increasing

Diseaseas the use or misuse of

have been concerned that eliminating

would lead to poor animal healthofconcern over the development

animals has led many public
antimicrobials

and medical

Susceptibility

Medicineawarded the ATCC a three-yearof MAR(CVM)

at the U.S. Food and Drug

contract to study the mechanisms andspeed at which microbes develop them after antibiotics

are introducedon the farm The goal is to test the susceptibilityof 5,000 strains in total, which include

isolatesof Escherichia

Antimicrobial

panelscob, Salmonella,comprisedpatterns

and Campylobacter,of several

antibiotics

and become widely used

identified

in different

in the last five decades
concentrations

specified

the FDA are custom-manufactured. ATCC acquires and characterizes

resistantthe mechanisms and degrees of resistance,and makes cultures

to medical

researchers

around the world. The CDC is a valuable

source for this work; they provide isolates

dating backto before

1950.

diagnostic

services,tested,In addition,and public

ATCC has also obtained
healthlaboratories

and a database has been constructed

References
Cypess, R.H. (ed.)(2003)

Biologicalavailableisolatesstrains,byfrom universities,

To date, more than 3,000

studiesveterinary

strains have been

for analysis

Resource

Centers

Their

ImpactCommunity and the Global Economy. American Type Culture Collection,on the Scientific
Manassas, Virginia.

pp.163
American Type Culture
The Biodefense

Collection:

http //www.atcc.org/

and Emerging Infections

http //www. beiresources.

Research Resources Repository:org/

The Malaria Research and Reference Reagent Resource Center http //www.malaria.mr4.org/
The National

Instituteof Allergy

and Infectious

Diseases

Controland Prevention:

http://www.bt.cdc.gov/agent/

Organization:

http ://www.who.int/

The Centers for Disease
The World Healthhttp ://www. niaid.nih.gov/biodefense/

National
Biological
Resource Center to establish
infrastructure
for life sciences and biotechnology
Ken-ichiro

theintellectual

SuzukiNITE BiologicalTechnology

Resource Center (NBRC),

and Evaluation,

Department

2-5-8, Kazusakamatari,

of Biotechnology,

National

InstituteofKisarazu, Chiba 292-08 1 8, Japan

Email : suzuki-ken-ichiro@nite.go.jp
Recent progresses in microbiology have revealed that the extent of diversity of microorganisms
will be much greater than previously thought and most of them are as yet unknown. The roles of
biological resource centers (BRCs) are expanding into various fields: one of its most important roles willbe, of course, a reference center of living microorganisms described in literature and standard organisms
for specific quality control including taxonomic type strains, as culture collections have been serving till
now.However,BRCs are expected to go several steps further to catch up with the technical advancement
to maintain and characterize novel fastidious microorganisms such as extremophiles and others as muchas possible. Needless to say, most of the reference organisms commonto world-wide culture collections
have to be preserved with the quality high enough to meet the global standard. In addition, newapproaches to bioresources are envisaged by emphasizing their possible applicationsin industries basedon the government's policy to stimulate industrial use of microorganisms. Screening of new drugs
including lead compounds will be supported by supplying a large number of newly isolated diverse
microorganisms. Also, international collaborations
amongst researchers will create bioresources of novel
characteristics.
At the same time, technology transfer will make it possible to isolate desired novel
microorganisms and supply of specific gene clones derived from genome analysis accompanied bysequence information will be of value for seeking methods of application of enzymes and other proteinsencoded. Biosafety and intellectual
property right are also the key issues for BRCs. The roles of BRCs
will thus become diversified by the expansion of biotechnology.
Introduction
As defined in the OECD report issued in 2001,
an essential

partof the infrastructuresciencesand biotechnology,

diversefieldsmaterials,

extendingincludingagriculture,

those of the scientists

at various

research

institutes.

government's

in biosafety

and intellectual

bioresources
properties

Considering

Ministryof Economy, Trade and Industryof Japanreport on Biologicalpointsof view.

was inaugurated

Resource Centers.

I shall

discuss

and biotechnology.

Lifedrug discovery,life.

In addition
fieldsetc., coverto supplying

and the knowledge

are required

to be as high as

BRCs may sometimes be affected by theand agreement among industries.
which

are

food production,consensus

Resource Center (NBRC)

resource centers (BRCs)

sciencesto play various other roles in the relatedof BRC staff members in handlingpolicylife

from the advanced research to our daily

BRCs are expected

and technology

underpinning

biologicaland concernedsuch significant

decidedin 2002

to establish

with the social

roles
NITE

of BRC, theBiologicalas the first output after the OECD

below on the roles of BRCs from various

Essential functions of a culture collection
1. As a supplier of reference organisms to maintain the standards
Examinations
antibiotics

such as sterilization

as well as quality

which the microbial
national

control"strains"

laws or regulations

performance,

activitiesof media are described

to be used are specified.

by the numbers in the domestic

authorized data are desirably

reagents

orin each of the official guidanceinStrains

valid under the internationalof aseptic

are generally

cultureregulations

designatedcollection.

These locallyor those of other countries.

This is in accordance with the certification

system of BRCs that has been discussed

Task Force of OECD that the certificated

BRCs to be the international

globalstandard for smooth internationalbyin the BRC

official supplierfortrading.

2. Depository oftaxonomic type strains
Typestrains

speciesof prokaryotes

culturable

have to be depositedcollectionsofin culture

to show the availability.

Prior to the proposalof a newspecies,

the type

strainspeciesisdeposited

to beappropriate
collections.

of the"public"incultureThis rule is employedin BacteriologicalCode(1992)

and

in

other

spreadingmicroorganisms

suchFig. 1. The numbers of genera and species

as yeasts

published

and fungi. The number of validlyisvalidly.

of prokaryotes(Data based on J. P. Euzeby)

publishedspeciesincreasing

as shown in Fig 1. According

to the data of Dr. J. P. Euzeby, the number ofprokaryotic

speciesis almost 6,000(including

rapidlysubspecies)(as of July 2004)

and this is threetimes the number in January 1980.
In addition
are becoming difficultof studies

to the increase in number, cultivationand preservationin both techniques

One of the reasons is the recent trend

on extremophiles.

may lose the status.

and equipments.

If a problem be found in the culture

Furthermore,culturecollections

maintenance and availabilityof a new species.

loaded with the responsibility

for preservation.

preserved,

are requested

Consequently,of these organisms
the proposedtaxon

to issue a certificateofculture collections

are heavily

3. Considerationsof the intellectual

property rightspecimens

have been regarded

Biologicalaccessible

to allbioresources,
deposit.

who are interested

BRCs have to clearly

CBD and relatedmaterials.

as internationalin. However, considering
indicatethe limitof theirlaws of the country of origin

The access procedure and conditions

heritagesthat are madethe potential

benefituse as the conditionsoffor theirsometimes control the use of biologicalshouldbe indicatedin the catalogue

with thenameof country of origin.
4. Biosafety
Microorganisms

are classified

into four biosafetyof laboratories

The containment

levelsand choicemicroorganisms.

Severallists

However, BRCs are requested
speciesor unidentified

Packaginglevel

by the regulations

specifiednational
illegalbased on the biosafety

to be able to classify

microorganisms

This

is closely

for shipment

relatedis an international

international

attentionof biologicaleasy access with correct information

attached.

are available.

to accept newly proposed

matter dependent

upon the biosafety

Plant quarantine
information

be paid to protect

materials

level

of

of identification

Local BRCs must have a function
transfer

5. Identification

shouldupon thoseto the abilityof IATA and postal services.

to say, highest

use of bioterrorism.

unnecessaryofis controlled

on biosafety

byto users'

microorganisms

fromto reduce the number ofby ignorantusers and support

theirfor BRCs and is indispensableforby biosafetytoservices and quality control

Correct identificationof microorganisms

the evaluationof depositors'

givingnames to them. Microbial

scientific

Whenever possible,

identification

servicecharge of each group of microorganismsof preserved

is essential

data and for their classification
culturesevery time ampoules are renewed by referencing

qualityin BRCs are dependentlaws. BRCs are expected to show the appropriate

community. Needlesson the basis of their infectivity.

of microorganisms

materials.

microorganisms.

levelsbioresources

level,

in addition

are examined and their identity

is confirmed

to the data obtained

will be made available

at the time of deposit.

to customers.

are expected to enhance their capability

by performing

their

Scientists

into improve theown research in taxonomy and related

fields of science.
NewApproachesof BiologicalThe bioresources
standard

strains

Resource Centerspreserved

for various tests,

at BRCs are mostly reference

organisms

taxonomic types, organisms used in scientificseems that the time ripe for BRCs to develop bioresources

such as thepapers,etc. Itof their own and add values to them.

Following

approaches

are ongoing at NBRC.

1. Development and supply of biological
Restrictionin acquisition

resources difficult to accessof bioresources

has recently

biomaterials

from resource-rich countries is controlled

Convention

on Biologicalvarious applications

Diversity.

by laws stipulatedThose who are interested

seek the way to obtain desired

Exporton the basis

in microorganisms

ofof theusefulinresources. The barrier is, however, generally

difficultto break for those in private sectors. Therefore,

to legallytransport

theirbecome stricter.

BRCs are expected to establish

a systemand supply materials to users with clear guidance with respect to the range ofuse. Sometimes BRCs are involvedin the benefit-sharing
collaborations

and further

contract

industrialuses. NITE-DOB is promoting

researchers

in South East Asian countries under the memorandumof understanding

the institutes

in individual

taxonomicallycharacterized,

to explore novel microorganisms

countries in accordance with CBD. The isolated
providedfor the assay of production

evaluation.

The resultant

microorganisms

utilization

with a certain material transfer

for their

are deposited(MOU) withmicroorganismsof bioactivein the culture

with
are

compounds forcollections

for further

agreement (MTA). These organisms are potentiallygood resources for further investigations.
2 Clones derived from genome analysis
The genome of some two hundred microorganisms
are valuablewith a large amount of information.

made available
especiallyto basic and applied

The clones constructed

for genome analysis

fields of research and NBRC is preparedin the case of those organisms that are difficult in cultivation

as hyperthermophiles
3. Collaborations

or eukaryotes
with private

such as the Koji mold (Aspergillus

scarcely used. To stimulate
for screening

from universities

use of such sleeping

are preservedbioresources,areclonesor DNA extraction suchoryzae).
resources

at BRCs, most of them are

the resources of NBRC will beof usefulfunctionsto the cooperative

research teams including

scientists

and private

sectors.

The results

be exploited

industrial

applications

and the data obtained

our planningof bioresource

open to the public.

to supply

sectors to explore research seeds by using biologicaland the associated information
While a large number of microorganisms
offeredhas been analyzed to date. The data

will

for their

will be added to the NBRC database as they are valuable

collection.

Applications

for cooperative

researches

forof this type areEstablishment

and strengtheningof a local BRC network

Formation of a global BRC Network will be one of the subjects of the second term
BRC Taskforce of OECD. A regional network will, therefore, be important to discuss in thisconnection by raisingsome specificRoles of the scientificsociety, JSCC

Japan Society
culture collections

for Culture Collectionsin Japan and the related

etc. One of the most importantof a national

problems.

biologicalby the Sciencestudies

activities

Councilsuch as taxonomy, preservation,of Japan through the Liaisoncollections.
individual

Practical
collectionsof

informatics,of the establishment
1999,

and approved

Committee on Microbiology.

The annual

on the matters about the management of culture

matters, such as CBD, plant quarantine, biosafety,
were also discussed in business meetings of JSCC.

etc., commontoof a combined catalogue

JSCC has been publishingsince 1962. The latest
1 998 that contains

editioncombined catalogues

was the sixth edition

8,470 speciesof bacteria,

virus. By exploiting

the opportunity

establish

databasea combinedof culturesof JSCC Catalogue

to revise the Catalogue,of microorganisms

a catalogue will be published.

3. Establishment

ofa consortium to supply microbial

JSCC

preservedof Cultures
is currently

in the JSCC

publishedinprotozoa, andplanning

tomember collections

resources with research-quality

The diversityof microorganismsthat a single

BRC is not able to cover the entire

affiliated with JSCC have theirof the member collections

archaea, fungi & yeasts, microalgae,

and consequently

collections

to promote the activities

resource center. This was drafted by JSCC in Julyof JSCC was the place to discuss

diversesocietyof JSCC was the recommendation

meeting

2. Publication

is a scientificand theirscientificbackgroundare so wide andfield even. Twenty-five member

own specialty

and characteristics

The total number of strains maintainedin these 25 collections

is 229,840

as of the end of March

2004 (Anon. 2004).

by these collections

were 22,554.

Affiliated collections

are classified
shipped

Cultures

distributedin the collection.

into five groups as shown in Fig. 2. Almost 50 percent of the total distribution

by theapproximately

top

three

collections,

17% of the total.

accessionand distribution.

Therefore,

collections,they will contribute

although

These

the

collectionsorganisms
have personnel

if they would function

to the efficient distribution

as virtualpreserved

by themof administration

is
are
for

centers of the memberof microorganisms.

Capacity

building
Not only the techniqueshandlingofofmaterials^ but the knowledge

operating

BRCs

has

becomeimportant in laws and regulations.

Toperform a cooperative

research is agood occasion to transfer

technology

and knowledge, which can be realized
throughetc.

workshops,

to be

held

in

countries. Essential
isolation

andmicroorganisms
in theirtraining

courses,resource-richtechniquesin thecharacterization

of

Fig.

as well as knowledge

taxonomy and biosafetyareimportant to control the management
and transfer
progressesof biomaterials.
in bioinformatics

for the identification

2. Numbersof holdings(areaof bars)of cultures(lengthAffiliatedcollections.

corresponds

to AHU, ATU5HUT, NMC, OUT, and RIFY

"Applied

Microbiology"

have made it possible

to analyze a large amount of sequence dataof various genes and theirprotein products.

Data management is thus
construction.

standardizationthat may be

as serving for an essentialfunctionand biotechnology.

Industries

BRCs to take the leadershipexpect nationalof code of conduct for handling
modifiedbe to coordinate

industriesof bioresources.

Supplythe activities

of users.

However, it is quiteorganisms requires

socialof qualified

materials

The biosafety

BRCs have to provide
Materials,

not used for a long time. The existencelevel for hazardous

information

will surely

supportused in popularresearch

subjects.

is not used for several

decades

until it ismaterials

therefore,

have to be maintainedof a stable financialof an infrastructure
are required

even if they are

backup system makes it possible

tofor the community, as mentioned in the OECDeach BRC is a component of the global

and perspectives

in theconsensus. One of the roles of BRCs will

and appropriate

commonthat a microorganism

work for the establishment

the community of lifeand consumers. An equally important role will be to expand the users

re-focused for its certain function.

Report. In addition,of bioresources.

withinIt should

BRCs are envisaged

and/or geneticallystrategy

from JSCC

Recentalso be stressed that technology transfer will be important for global
regarded as a non-monetary benefit for resource-rich countries.

establishment

anddistribution

important to join in the BRC network as well as for in-house databasesciencesof ovals)

BRC network. The long term

for the management of national

BRCs.

References
Anon. (2004)

Annual reportfrom affiliatedculturecollections(In Japanese).

Microbiol.

Cult.

of cultures

sixth edition

1998.

standing

inColl.20,17.

Editorial

Committee

Japan Society
Euzeby,J.

for JSCC Catalogue

for CultureP.

(2004)(1998)

JSCC

Collections,

Tsukuba.

List

bacterialofcatalogue

names

with

nomenclature.

(http ://www. bacterio. cict. fr/).
Lapage, S. P., P. H. A. Sneath, E. F. Lessel,(1992)

International

code of nomenclature

Microbiology

Washington

D.C.

OECD (2001)

Biological

Resource

biotechnology.

V B. D. Skerman, H. P. R. Seeliger,

Organisation

Centresof bacteria(1990

underpinningfor Economic Co-operation& W. A. Clark

revision).

Americantheof lifefutureand Development,

Paris.

SocietyforsciencesandMicrobial
Dwightdiversityand pharmaceuticalindustry

culturecollections

Baker

Albany Molecular

Research, Inc., Bothell

Research Center, 1 8804 North Creek Parkway,

Bothell, WA 9801 1-8012 USA[Current address: Cubist Pharmaceuticals,

Inc, 65 Hayden Avenue, Lexington,

MA 0242 1 USA]

Email : dwight.baker@cubist.com
Pharmaceutical industry culture collections constitute extensive archives of microorganisms ofa limited number of phylogenetic
groups. Although industrial collections
may lack taxonomic or
phylogenetic
breadth, they often benefit from taxonomic depth. Major public collections
typically
provide broad phylogenetic diversity, but may limit the depth of the collection to a few representative
strains for each taxa. These differences in collection strategy reflect the functions which the collections
are to serve. Pharmaceutical industry collections
are biased to provide chemical diversity of small
molecular weight compounds, and more comprehensive taxonomic breadth may not effectively support
pharmaceutical discovery efforts. For this reason, industrial and public culture collections should not beinterpreted as redundant of each other. The pharmaceutical industry cannot realisticallydepend on thestrains found in public culture collections to serve their needs, nor can the public collections realisticallyunder-represent industrially
important taxa in their collections because industry overrepresents these.
Industrial culture collections will continue to have value to the pharmaceutical, biotech and animal healthindustries, but the provision of collection services is likely to be by smaller more focused researchorganizationsin the future. These smaller organizations
can provide the specialized
expertise ofmaintaining and utilizing the collections more efficiently than the pharmaceutical companies themselves.
Introduction
Duringthe

1940's,pharmaceuticalresearch in earnest to exploit
began to collectbioactive
studiessecondary

Qualities

culturescompanies

metabolites

or as assay targets

generally

to support

have abandoned(Baker2004).

would not be includedof industrial
culture collections
Industrial
culture collections
developed

industrial
culture collections
are not extensively
even physiology
or metabolism.
The majority
from diverse

sources,companies,

e.g. Kosan Biosciences,modifiedstrainsbearingalthoughdiscovery.

A few companies

Since that time, many of

theirused for generationcollectionsof microorganismsofused for genetic

in those lost from the industry.

for drug discovery

purposesdifferfromin common among them. In general,thesecharacterized
to taxonomy, phylogenyor toof strains held in such collections
are initial

a number of the smaller

combinatorial

Maxygen, Diversa, etc., will have collections

metagenomic

microbiologicalthis research.

Collections

company to company, but there are some features

isolatesbegan to conduct

the newfound successes in antibiotic

and hold microbial

the major pharmaceuticalcompanies

DNA within

a common host.

biologyof genetically

For the most part,pharmaceuticaldominatedcompany collections

will

be

strains

to

the

by

belonging

actinomycetes

and filamentous

fungi,

withmuch

representatives

from

other

fewer

phylogenetic

groupsresulttheof(Table1). This

predominancewithinbias

ofTable 1. Representationof microbial
phylogenetic
groups within industrial
collections.

is abioactive

secondary

metabolites

these two majorgroups.

The Chapman and Hall Dictionary

NaturalProductsof(Buckingham

2004)

providesa compendium of identified

naturalproduct

compounds, and if one reviews the number ofcompounds by selected

phylogenetic

becomes very clear why industrial
fungi.

Because the industrial

the number of closely

relatedgroups, itcollections

collections
strains

will be rich in the actinomycetes

are strong in a limited
withina single

number of phylogenetic

group will be greater

repositories.

For example, if one looks at the data available

for two major

repositories,

theCentraalbureau

fur Schimmelcultures(CBS),Type

Culturethe representationgroups,than in most publicaccess microbial

Americanand filamentous

Collection

on the internet(ATCC)

of industriallyor therelevant

fungalstrains by taxonomic name is very different

than that represented

Similarfor comparisons among taxa of the actinomycetes,

differences

would be observedin Chapman and Hall (Fig.

1.).

eubacteria, etc.
Industrial

culture collections

can be better

centers for pro-active

and systematic

curating

the collectionof microorganisms

microbial

resource

curation.

center.

Unlesscultures will not be regularly

refreshed.

Risks to industrial

culturecollections

Industrial

culturecollections

golden years of antibiotic
withinthe industry

characterized
Typicallya strainis actively

cultureand natural product discovery(Bakerresources has also been lost. Thirdly,
uncertainty

in the use of microbial

Other risks to the holding

rather thansetting
being

than there

utilizedcollections

or studied,preserved2004).

programs are over. Secondly,
redundant

pharmaceuticalfinancialcollections

liabilities

industry

continue to arise.

toora great wealth ofTreaty (Rio Convention)

resources and this has biased

themergers

companies have discarded

With the loss of these collections,

the BioDiversity

or use of industrial

and/orfor

would be at a

are under siege for a number of reasons. Firstly,

have made culturecollections

operations

there are less rigorous standards

in an industrial

successor companies. The majority of the largest
sold theiras safe-keeping

has also addedaway fromtheir use.

Fig. 1. Taxonomic breadthof publiccollections

represented

by numbers of strains

for selectedfungal genera

Bio-terrorism regulations
Since

2001,

greatercollections

both in the public

collections

are required

scrutiny

has been placed

and private sector.

to hold regulatory

on access to and use of microbial

Withinthe United

permits for many of their strains.

prior to 2001 for certain plant and animal pathogens,but the stringency

increasedpathogenic

greatly.

The regulatory

and Plant Healthjointly

Inspection

administeredagency overseeing

Servicewithpathogenic

species,holdinga diverse

furthermicrobialcollection,

repositories

of secondarypurchasedfrom publicaccess repositories-U.S.$150-200

collectionof approximately

metabolites

the Animal
is now

The added regulatoryburdenmost likely

someis cost prohibitive.

per individualstrain,

5000 strains by acquisition

contains
their use.

purposesfrom

The current rate forwould mean that creatinga strainwould cost close to U.S.$ 1 million,
after acquisition.

the costs associatedwith creatinga new microbial

strainsfrom Rio-compliant

environmental

samplesacquisition

costs identified

above, but much more expensive than retaining

collections.

Therefore,

option for utilizing

the least expensive

hasof Agriculture,for drug discovery

counting the cost of use, storage and maintenance of the strains
estimatedof the regulations

or creating new collections

a new librarycultures,This was requiredcompanies from continuing

Building
microbialthatmost industrial

microorganisms,of the U.S. Department

biases pharmaceutical

Cost of strain purchases from public
strains(APHIS)

with the Department of Homeland Security.

associatedStates,collection

to be lessnot

Baker (2004)

by de novo isolation

costlymicrobialof

than the commercial
existing

resources

microbial
for industrial

purposes, and also the most opportunistic
and flexible
for future uses, would be to retain
existing
industrial
strain collections
and provide access to these for scientificresearchsupporting

human, animal and plant health.

How this might be accomplishedlogisticallyis theprimary question.
Intellectual

property limitations
Creating

within,publicindustrial

repositories

with perceived
strainsor microbial

strains

separate

from, or distinct

resource centers may impose additionalrisks associatedpharmaceuticalcollections

companies desire to have ownershipbecause it allows them to preserve their intellectual

were owned by a contract services

would typically

only be able to license

creative

development,

businessfor microbial

lower value. In general,

patent or producing
microbial

repositories

the strains

the liabilities

difficultyis that having

a relatively

large

providermay not be seen as self-sustaining

provider,the pharmaceuticalfor patent purposes.

of this approachmicrobial

property.

in the long term.

held

If thecompany

However, with a bit ofcould probably

collectionofbe managed. The

by a contract

Subsidies

servicesor semi-exclusiveaccess privileges
collection

for interested

industrial

partnersin the short term, but the collection

may be able to maintainmust yieldwould need to receive a continuing

stream of royalties.

Having industrial

acquired by public

culture collections

it might seem to be an ideal mutuallyindustrial

collections

as well as providing

industrial

collections

into microbial

increasingly

characterizedcollection

efficient

immediate

scrutiny,

for protection

The historicalcollections

for their

exclusive

for selectingof industrial

corporations

services

providermaintainsfee, is the current businessfrom industry,a collection

will be forced to prune

culturemaintaining

use in pharmaceuticalappear to be the most likely

seems to be obsolete,maintenance and utilization

for success. The first,

Albany Molecularand provides

access to organizations

semi-exclusive

common,yet has been promoted as a mechanism for building
financial support dependingof industrial

for academic purposes.
of diversity

collections

is probably

of most microbial
resource

centers

diversity

which

replicatedin an academic

might

maintains

a collection

memberships,

is not at all

naturalscenario is that it is subject

and industrial

collections

However, in reality

microbial

arising

product

discovery

to fluctuations

inon membership participation.

between public

One criticism
industrialof this lattercollections

for a

Research, Inc., and Cerylid

The second scenario, in which a not for profit organization

programs. One of the difficulties

of industrial

in which a contract

model for a number of natural product discovery

via continuing

or ataccess to materials in the collection

Biosciences.

Cross-communicationcollections

and using their own microbial

development

and provides

e.g., MerLion Pharmaceuticals,

will rarely

strains for disposal.

least on the wane. Two scenarios for the continued
culture collections

poorlyeven the mostculture collection

repositoryofresource centers

use would seriously jeopardize

and utilization

model of public

for protecting

Microbial

The majority of strains in an industrial

with no adequate rationale

Recommendations

opportunity

and acquiring a relatively

be accessed over a decade of maintenance, and the microbial
the collection

product for which it

greater access to those resources, the incorporation

coming under financial

culture collection.

beneficial

resource centers is unrealistic.

with limitedof therepositories

Althoughare themselves

some tangible

viability

collections

is that they limit access to importantthe breadthof diversityof taxa represented

not of that great importance to basic science.
phylogenetic

groups will more likely

than

in industrial

collections.

be better

represented

withinsettingwitha limitedindustrial
effort.

Also,

the depth
uniqueinGreater breadth

be represented

collections
Truly

taxa, even

withinof taxonomic

can probably

beor metabolicallyinterestingstrains

patent purposes,

from industrial

collections

routinely

and in this manner can precipitate

protocolsfor patentpublicculture collections

References
Baker, D. D. (2004)

depositswill

get deposited
new interestin public

repositories

in that taxon.

ensure that cross-communicationfor

The Budapest

between industrial

andwill continue to occur into the future.

Industrially-held

microbial

diversity:

Are culturecollections

reallythnecessary?
In: Kurtboke,
Melbourne, Australia.
Buckingham,

J. (ed.)(2004)

I. and Swings,
Dictionary

J. (eds),of Natural

Proceedings
Products,of the 13

ISBA

conference,

Chapman and Hall, London.

Biosafety
demands
challenges
Christine

and the self-image

Rohde

DSMZ-German Collection
D-38 124 Braunschweig,
Email:

of modern BRCs - Globalof Microorganisms

and Cell

Cultures,

Mascheroder

Germany, Tel 0049-(0)53

1-26 16-220,

Fax 0049-(0)53

1-2616-41

Weg lb,8.

chr@dsmz.de

If we were asked to identify the most prominent demands for a modern vital Culture
Collection
or to fix the general outline for a new paradigm of BRCs, we would certainly
mention profound long-term expertise of staff, high technical standards and high standards ofqualitycontrol measurements, well-functioning
management systems, reliablelong-term
fimding and, among further characteristics,
staff having a constant look at all the complex
bio-legislation
aspects, many of which are more or less affecting the heart of a BRC in thataccess to and handling and despatch of biologicalresources are governed by a number ofdifferent and often confusing national and international
regulations.
Whenever we talk about
bio-legislation,
we have in mind the "destiny" of an isolate/culture/strain:
it is accompaniedfrom the very first process of isolation/sampling(see CBD) through all the lab procedures(health and safety requirements)
until its long-term storage and finally its despatch to serve
science, applicationor teaching. The transportation
chain with all export and shipping
regulations are at the final point of this "destiny" when the strain is supplied in order to function
as a valuable tool in the recipient'slab, maybe after a long and highly-paidjourney, maybe inanother continent.
Introduction
The complex framework of bio-legislation
receive such special
and problems

emphasisin the past. An overall pictureof Culture Collections

However, alreadyin 1990on the situation,

drawn by Sigler

the WFCC had establishedand publishedmechanisms to be adheredchanged globala system of concepts which did nothas been nicely

Establishment
and Operation of Collections
Committee 1990). These guidelines
alreadyin regulatory

requiresaims, opearation(2004)and Smith (2003).

the Guidelines

for theof Cultures of Microorganisms (WFCC Standards
emphasized that Culture Collections
are embedded

to. Issues the PQSR Committee dealt with reflectdemands which also receive

constant

attentiontheby the WHO of course (seeWHO1993). Over the last decades and especially over the last few years, BRCs had and have tofamiliarise themselves with this progressively
branching out system of concepts. However, theuser of the BRC, this means the bio-scientificconsequently

surprisedcommunity, is often not aware of all this and isabout administrationaldeliveryof the orderedcultures.

personnel

and theirpromotingthese requirements.

harmonised

standards

training

Therefore,

expenditure,
all these

It is the understandable
levelscosts and delay

in

BRCs need adequate support in monetary terms forin order to fulfil

on different

paperwork,

includingaims, in keeping
spiritbiosafety.

with the times andof the times to develop
In that respect,common

BRCs are ideal

examples of scientificinstitutions

because of the large versatile

they hold and because of their attitude
The OECD Global

to be up-to-date

spectrum of biologicalwith the regulations

Forum on Knowledge Economy: Biotechnology

document for the operation(Smithresources
et al. 1999).

focuses in its guidanceof all BRCs on all the above mentioned premises and key features(See OECD 2001).
An example of action in Europe
In order to harmonise
European EBRCN project

standards

and practicehas focused on the development

be used as working documents with the attitude
Whereever a BRC is located,

independent

documents cover the most relevantCollections.

of implementationof helpfulof regulations,basic

to make BRC-internalthedocuments that can

implementationeasier.

of the size and whatever its main domains are: thelegal

aspects

havinga high relevancefor all Culture

A BRC should comply with:•EClassificationof microorganisms

on the basis of hazard•EQuarantine regulations•EOwnership of intellectual

propertyrights•EConvention on Biological

Diversity(CBD)•ESafety information•ERegulations

provided

governing•EControl of distribution

to the recipientof microorganisms

packagingand shippingof possibly

dangerous microorganisms•EHealth and safety requirements
This listis certainly

not complete

remain working documents as they
BRC-specific resources and activities.
Prevention

but offers

a basishave to be adaptedof malafide use of microorganisms:

and the respective

to national

bioterrorism

documents

legislationor to theand biologicalweapons

discussions
Unfortunately,
contrast

BRCs also have to focus theirto all other biosafetymeasures against

misusebio-weapons. Biosecurity
and reliable
authorised

despatch/accessaspects,of certain

involves

to work with the material.

used reference

or researchstrainsemphasisthe term biosecurity

evolvedbiologicalwithprotection

control

specialresourcesof facilities,

dependent

on biosecurity:inand means precautionary
a potential

to be used ason the resources they holdin order to make sure the recipient

is registered

However, certain pathogens

and frequently

are valuable

on the one hand but fall under dual-use regulationsoror theBiologicaland Toxin Weapons Convention

reportedin one or more cases of misuse. Among such species,

microorganisms
Escherichia

like

Staphylococcus

coli or several fungi

mass destruction

had sadly

abstract

but unexpectedly

thinking,

collections.
2002,

allocatedor Clostridium

invitation,

known whereas bioterrorism

became an incision-like"A neutral platform

for discussing

NDA meeting in Brussels,

had only existed

to this issue (Swings

on bioterrorism

How would we define scientifictracking

and traceability

recipientsof pathogens

NATO and EU Security

pathogenic

containing

is required

to produce designed
by Culture

and it is very importantthat thesecommonline is highly

desiredorganisms?

based
needed for

Who are theWhich control

Of course, all these questions
several similar

projects

do not justwork in parallel

without

to the export

offices, ministries

of export

elsewherea feedback

to thecontrol

etc. Aand at last the reflux to the United Nations where international

for disarmament regularly

beyondmaterial

bio-weapons?

Collections?

"black market". There are probably

authorities:

Upon

And how do we deal with transfer of

do not cover the existing(national)

Policies".

a scientificallybiologicaldangerousordered from Culture Collections?
verified

Bioterrorism

freedom and how far can this go? How do we ensureof consignments

mechanisms can be realisticallyet al.

and bio-weaponry

and to add to the discussion

research?

What knowledgeinJune 2004, the chance was taken to represent

how do we deal with exchange of highly

BRC services

in the context ofa project funded by the European Union. NDA

ductus:experts delegations

verotoxigenic

issue for WFCC and the member

Since a while, new projects

the voices ofWFCC and the EBRCN project

respective

perfringens,

One of the new ones is the New Defence Agenda (NDA)at the initialknowledge?

we find toxin producing

to Risk Group 1. Bio-weaponryGroup (www.newdefenceagenda.org),

has the subtitle

on the other hand because they were

no. 34, January 2002, was dedicated

Smith & Rohde 2002).

Reportingaureusbeen already

WFCC Newsletter

defence are evolving.

(BTWC)

meet.

"usual"curatoractivities:

intermediaryconsulting

requiressufficient control mechanisms, biosafety

serviceon thelaws
High quality
on many levels
forum for Cultureof human action including
Collections,organ for communicationtransferof knowledge.

of theirproblems

for detection

between the individual

requiresresponsibility

The WFCC is a wonderfuland so WFCC functions

members and the world-wide

as anscientificcommunity. As a result of the communication

with the user/customer,

the BRCs function

mirrors and can articulate

are. BRCs ideally

their self-image

they also offer intermediarytime-consuming:

where the problems
consultation

due to the complexity,that BRCs are recognised

to be institutions

follow

service in the bio-legislationasin thatarea which can be quite

this goes far beyond the FAQ level and demonstrates
carried by true expertise.

Such activities

are moving

away from scientificwork but are terribly

conferences and meetings dealing
seminars or individual

needed.

Furthermore,with matters of bio-legislation

training

on these issues

BRCs send experts

toand on the other hand offer

so that they are centres of information

andadvice.
The complexityof transport

regulations

and new shipping

highlights

The ever-changing postal, packaging and transport regulations
for microorganisms
have been in the limelightsince years and our efforts towards certain changes in the UN Model
regulations("OrangeBook")

only recently

culminatedrewarding our input. This happens in times when shipping
when costs of dangerous

in a successful
regulations

tighter,

climbing
irradiated.

extraordinarily
and when letter post (containing
harmless
This success of WFCC made clear that a constant and positive
regulatory

the developmentof new pathways

information

bodiescould even be defined

worlds leading

scientists

contributing

with profound special

A very outstandingknowledge

publishes

the recommendations

An overview on all questions

that WFCC goals were highlighted
infectious

substances

broad basis
nationalof implementation

co-operative

andto the distributionofthis co-operation

withof bio-legislation

partners for regulatory

andauthorities.

on packaging
1999).

goods shippingand international

and traceability

and with member scientists
specialist

for all modes ofshippingof biological

Another example of WFCC activitiesof packagings

was a topic among others. So, WFCC as a globalof expertiseThis UNisduring the UN BTWC Meeting of Experts for disarmament

UN, Geneva, where tracking

or internationalof Dangerous Goods (UNSCETDG).

for dangerous

material is given by a DSMZ brochure (Rohde
2003,

co-operation

example for this aspect is that WFCC has been granted observer status to the

UN Sub-Committee of Experts for the Transport

in Augustorganisms)
is being
contact with national

knowledge are working with BRCs. Due to theiraddress the problems so that they are trustworthytransport.

considerablyareas one of the missions of modern BRCs since some of thework, they have hands on practical

Committee

Group 2 organisms)

and raised awareness. With regard to our paradigm question,

the authorities
daily

(Risk

is of high relevance and can lead to fruitfulwhile

processare becoming stricter andcontrols

and international

goods transportdecisionsocieties

Summary: consequences for designingwith a very

who have good contacts to the large

like IUMS, FEMS, ASM, SGM or VAAMhas best

chances to be heard and we have experienced how deeply recognised
however, is that we have active individuals

initiative

containing

this input is. A prerequisite,

who keep going.
a new paradigmof BRCs

Because a new paradigm of BRCs cannot be seen without the international
key recommendations for modern BRCs should no doubt include global, co-operative

context,
thinkingtogether,

not thinking

alike,

towards harmonisationof standards,

proceduresand good practices

and positive thinking towards careful but active development of models for shaping the future.
BRCs are expected to be exemplary prototype institutions
being concerned about being on "thesafe side"and not outside

the law. Due to growing

professional

staffing including

the bio-legislative

Culture Collections/BRCs,
necessary as funding
Cultureresponsibility

and the necessityofarea and in order to maintain the credibilityof

their status within the individualis the outstanding

Collections/BRCs

most crucial

cannot be overestimatedcountries

problemshould be upgradedin many cases. The role of thewhether we look at the precious

resources or at the precious

expertise

Abbreviations
used
ASM

American Society for Microbiology

biologicalof staff.

BTWC

Biological

CBD

Convention on Biological

EBRCN

European Biological

FEMS

Federationof European MicrobiologicalSocieties

IUMS

International

Union of MicrobiologicalSocieties

PQSR

Postal, Quarantine

SGM

SocietyV

Vereinigung

fur Allgemeine

Associationof General and Applied

AAM

where

and Toxin Weapons Convention
Diversity

Resource Centres Network

and Safety Regulations

Committee ofWFCC

for General Microbiology
und Angewandte Mikrobiologie,
Microbiology

References
Atlas, R.M. (2002) Bioterrorism:
from threat to reality. Ann. Rev. Microbiol. 56: 167-185.
OECD (2001)
Biological
Resource Centres, underpinningthe future of life sciences
biotechnology.
Brochure, ISBN 92-64-1 8690-5.
Rohde, C. (1999)
Shippingof infectious,
non-infectious
materials,
Sigler,international

L. (2004)

Cultureregulations.

Brochure available

collections

in Canada:

and geneticallymodifiedandbiologicalfrom DSMZ (sales@dsmz.de).

perspectives

and problems.

Can. J. Plant Pathol.

26:39-47.
Smith,

D., Rohde,

C. & Holmes, B. (1999)

the law. Microbiology
Smith,

D. & Rohde,

Convention
Smith,

D. (2003)

and distributionof micro-organisms

andToday 26: 14-16.
C. (2002)

and other related
CultureHandlingThe

initiatives

collections

implicationof theBiologicalandfor WFCC members. WFCC Newsletter
over the world. Int. Microbiol.

6: 95-100.

Toxin

Weapons

34: 4- 1 1.

Swings,J., Mergaert,

J., Smith,

D. & Rohde, C. (2002)

WFCC's

WFCC Newsletter 34: 1-3.
United Nations (2003)
UN recommendations

on the transport

NewYork, ISBN 92-1-139057-5.
World Health Organization(2003)

Biosafety

edition,

WHO, Geneva.

Laboratory

handling

bioterrorismof dangerous

goods.

13th edition,

guidelines.

2nd revised

Manual. Interim

issues.

3. PROGRESS

IN THE RESEARCHON AGRICULTURALMICROORGANISMS

Overview of the MAFF Genebank project
Toyozo Sato. Toshirou Nagai, Keisuke Tomioka, Kasumi Takeuchi,

Motoko Iida

and Masae Kawada
Genebank, National
Ibaraki

305-8602,

Instituteof Agrobiological

Sciences(MAS),

Kannondai 2-1-2, Tsukuba,

Japan

Email: s l 043@affrc.go.jp
Keywords: culture collection,

database,

genetic resource, microorganism,

workshop

Introduction
The MAFF (Ministryof Agriculture,

Forestry

Microorganisms

Section,establishedin 1985,12

sub-banks(NARC,NIFTS

and10 institutes).

The

microorganisms

includingfungi,

bacteria,

yeasts,phages,insect cells.

These microorganism

1.

in Japan)of a central bank (Genebank,viruses,resources are distributed

the world. Here we describe the activities
Activities

consistsand Fisheries
Genebank

Genebank
NIAS)

containsandvariousnematodes, protozoa, andnot only within

Japan but all overof the MAFF Genebankof the project
Preservation

and Distributionof the MAFF strains:

MAFF Genebank preserves

20,472 microbial
strains that are preserved by cryopreservation
for fungi, by
freeze-drying for bacteria and yeasts and by L-drying for plant viruses (Fig. 1). The
MAFF Genebank is characterized
Pyriculalia

griseadepositedand distributed

2. Exploration
explorations

by a wide collection

and Xanthomonas oryzae pv. oryzae.
amount to 500-800

bacteria

phytopathogenice.g.,

The numbers of strains(Fig. 2) and 700-800

pathogenic

fungi.

diseasepathogento Hokkaido

a year, respectively.

fiingi,

Shikoku

Fourteenand Hokkaido

nematodes,

and 25 fungalof the MAFF strains:

isolatesof a chrysanthemum wiltof some herbaceousand characterized.

MAFF Genebank and will be available

in fruit trees and so on.

areas were searched for nematodes and

17 fungal

isolatesresources

enzymes, fungicide-resistantmushrooms, viruses causing diseases

were obtained

3. Characterization

for useful microorganismthat produce polysaccharide-degrading

In 2003 the Hokuriku,

pathogens

pathogens,for Collectingof Various Microorganisms
within Japan: Three to four
within Japan are done every year. We have explored many areas in Japan

from Okinawa and the Bonin Islands
includingof plant

These isolatesleaf blightdiseases

were deposited

into thedata on characterization

were

to the public.
In 2003,

12,738

collected.

Items of characterization

region), gene (toxin
race, production

and antibiotic

pathogenicity,

production),sequencing

host range, microscopic(rDNA and ITS
morphologies,

of enzymes, and others. These data are stored in the MAFF database,and some data were presented
4.

includedin papers.

Publications:
To promote the use of our strains, MAFF Microorganism Genetic
Resources Manuals are publishedevery year and the latest is No. 16, which describes
enzymes producedby filamentous

site as PDF files. We also publish
MAFF

Genebank;

thefungi.

The manuals can be retrieved

annual reports on exploration
formerareavailableonfrom our web

and on activitiesof the

our

sites

web

(http ://www. gene. affrc.go.j p/micro/).
So Construction
storedof Web Sites

in a database,and Databases:

All of the data about deposited

6.

your strainsor to get the strains

youfrom our web pages.

Pro¥isioiiof Financial
Support to SeYeral Microorganism
Studies:
The Genebank
provides domestic institutes
studying microorganisms with financial support. In 2003, 4
projects

7.

areand users can access with them via the Internet. Informationneeded to access the Genebank (how to depositwant) is also obtained

strains

were supported.

Organizationinternationalpresentedof the international
workshops are held,and discussed

Agricultural

Microorganisms'5,

Korea, Philippines,

Fig.1

Details

actively.

Thailand,of MAFF strains

MAFF Genebank Workshops: Every 3 or 4 years,
where studies of agricultural
microorganisms
areUnder the latest

19 topics

theme "Diversity

were presented

by researchers

and Use offrom China,

USA and Japan.

in 2003

Fig. 2 The number of preserved strainsin the central bank and total number inthe MAFF Genebank

ACCC.

Current

statusresourcesinand future

Ruibo Jiang. Jingang
and Zhlyong

Ruan

Agricultural

CulturePlanning,

prospectsof collection

and use of microblal

Gu, BIngquan Fan, Xiaoxia Zhang, Xiaotong
Collectionof China,

CAAS, No. 12 Zhongguancun

Institute

Ma, Shigui

of Agricultural

South Street,

Beijing,LI

Resources

China, Postcode:

and Regional
10008

1

E-mail: accc@caas.ac.cn or jggu@caas.ac.cn
Key Words : biodegradation,
In China,

biodiversity,

research

and application

focused on mushrooms rhizobium
microorganisms

degradepesticides,

and straws,

resources

biofertilizers,

biocontrol,

microbialof microbial

inoculation,
rapid

inoculation

microbial

the collection

after microbial
faecesmicroorganismsof agricultural

Inoculation,

marsh gas. ACCC's mission includes
of soilbioferlllizers,

resource diversity,

Isolationand screening

identification

were mostly
foods,

the zoology

of strains

1:

Straw Biodegradatlon

Table Is Content of humic add and humic substance, WSC/organlc
T re atm en t

H u m ic A cid

H m h iic

N, T value

W S C /O r gN

te rm in al:o rig in a l)

lOd

15d

S u b sta n ce
lO d

lO d

15 d

lOd

15d

1 C K

0 .3 3 7 a

1 .1 7 3 b

1 .8 3 5 d

2 .2 9 3 c d

2 .1 13 a

0 .9 5 a

0 .6 3 a

2 A

0 .3 4 3 a

1 .3 7 6 ab

2 .6 1 3 b c

2 .5 3 2 b cd

1 .4 8 6 a b

G .7 4 ab c

0 .4 8a b

3 B

0 .3 7 5 a

1 .2 3 l a b

2 .2 5 2 c d

1 .8 5 1d

1 .5 2 9 a b

0 .6 1c

0 .4 1b c

4 H

0 .3 4 7 a

1 .2 6 6 ab

2 .3 0 2 c d

1 .8 9 0 d

1 .2 3 2 b

0 .6 9b c

0 .3 7b c

5 A +B

0 .2 9 4 a

1 .3 6 7 ab

2 .0 9 4 d

3 .7 8 3 a

2 .0 0 8 a b

0 .8 7 ab

0 .4 4 b c

6 B +H

0 .3 3 2 a

1 .3 7 2 ab

2 .8 3 9 a b

3 .1 3 9 a b c

1 .9 19 a b

0 .7 7 a b c

0 .3 9 b c

7 A +H

0 .3 6 4 a

1 .2 7 4 ab

2 .8 4 3 a b

3 .2 6 0 a b

1 .8 2 4 a b

0 .7 3 a b c

0 .4 1b c

0 .3 7 4 a

1 .6 2 8 a

3 .3 1 3 a

2 .6 10 b c d

1 .8 4 5 a b

0 .5 8 c

0 .3 1c

A + B +H

whichof mushrooms, and biofertilizer

manufacture.
Part

and

-3 highly

effective

straw-degradingstrains

Trichoderma viride, T. harzianum and Paenibacillus-Straw Inoculated
contentof humic

value(Terminalacid

and humic substanceand the valueof WSC/organic

N and T

C/N).
Diseasesfungus and three Actinomycesin Vegetable

Production

and two Actinomyces

after pot experiments,

Phytophthora

like thepabuli.
with the parametersof Soilbornein which the protect

to be

has been tested

In our research, two bacteria
were selected

and identified

with these 3 strains

C/N: Original

Part 2: Biocontrol

have been isolated

in which the protect

againstRalstonia

solanacearum

efficiency was 70% or 50%. One

against Fusarium oxysporum were selected

after pot experiments,

efficiency was 50%. Twobacteria, two Actinomyces and three fungi against

capsici

were selected

after pot experiments,

in which the protect

efficiency was

90% or 70%.
Part 3: Isolation

and Applicationof Rhizobium

In the past 50 years, ACCC have obtained
portionsof indigenous

legume-plantrhizobiumcountry. In 2002,

more than 15 institutes

ACCC to isolate

and screen legume rhizobium

more than 1500 strains

samples collected

to soybean cultivars,

Yuejin-5,

from 20 provinces

and 60 persons of microbiology

across thewere organizedby

all over China. In our study, we have used 26

strains of /?, fredii and 25 soybean cultivars for symbiotic
nitrogenase activity
is usually 3-6nmolL"1g"1h 1(CZ142).
frediifrom almost 5000

Shanning-2,

association formative efficiency. The
The nitrogen fixation ability of /?.

Ludou-4, Minquan round bean, Shanghai

earlybean is similar or superior to that of B. japonicum 15006. Compatibilityof /?. fredii towards
soybean cultivars is stricter than that ofB. japonicum. The average nodulation rate of 26 strains
against203 soybean cultivars

nodulation.
4 strains

is 49%, among which strain

Summer soybean cultivars,
of/?, fredii.

15067(USDA

191)

gives

Yuejin 5, Ludou 4 and Shaming 2 were inoculated

The seed production

Part 4: Rapid IdentificationofLentinus
Antagonism, isozyme, intergenic

89% inwith

was increased by 9.4%-3 1.4%.
edodes and Pleurotus spp.
spacer (IGS) and random amplified

polymorphic

DNA (RAPD) have been chosen for quickly identificationof L. edodes. In the present
investigation,
43 L. edodes strains including 4 1 cultivatedstrains cultivatedon a large scale and2 wild strains were collected
On the basis
analysisfrom China.

of the fruit-bodywere used in the study of genetic

sapidus, P. ostreatus,

P. florida,

morphology,
diversity

antagonisticof 79 strains

P. sajor-caju, P pulmonarius

test,

isozyme and RAPD

which were classified

into P.

and P. cornucopiae, respectively.

It makes possible

to identify

species,and breedingof oyster mushrooms.

Part 5: Isolationand Evaluation

cycling,

clear synonyms of strainsof Phosphate-dissolving

Phosphate-dissolving

microorganisms

converting

phosphate

insoluble

were obtained.

Two of them, P8 and Pn1 were identified

one Aspergillus

sp. In plate

dissolve

Ca3(PO4)2,

ATCC14581.
dissolveplay an important

forms. Three strains
as Penicillium

However, in broth

Morocco rock phosphateof P-dissolving

fungi

oxalicum, and the othercapability

to

5H2O, CaHPO4, FePO4 and bone meal than ATCC2085 1 andassay, P8 consistently

demonstrated(MRP) than ATCC2085 1. Results

P8 and Pnl, using NO3-N and NH4-N as N sources, indicatedbroth enhanced the releaserole in phosphorous

assay, the strain of P. oxalicum P8 showed higher

Ca8H2(PO4)6for genetics

Microorganisms

in soils

into soluble

and give the basis

higherof incubation

efficiency

toof MRP withthat both NO3-N and NRrN inof P in MRP, and that NO3-N form was more efficientthan NH4-N.

Collection
Apirusht

and identification

Somrith. Pattana Sontirat,

Plant Pathologyof plant

Fusarium in Thailand

Niyom Khaimook and Tharntip Bhasabutra

Group, Crop Protection

Department of Agriculture,

pathogenic

Research and Development Office,Chatuchak,Bangkok 10900, Thailand.

E-mail: apirush009@yahoo.com
Keywords : collection,

Fusarium, identification

Introduction
Fusarium is one of the most devastating

plant pathogen

ever causes economic crops

diseases in countries. In Thailand, this pathogenic genus also causes major plant diseases. Many
crops severely destroyed are field crops, tuber crops, vegetables, ornamental plants, and fruit
crops. Having many kinds of plant hosts,

survival

in soilhabitatvariation, and toxic substances production,

Fusarium collection

for years, naturaland identification

genetic

would ideallybe the useful and important task to reach the knowledge of its origin and epidemic as well as
distribution
resources. Each identified
Fusarium species culture has been preserved in the
CultureCollectionof Department of Agriculture

viabilityand variation have been checked every 6 months, is very useful for further

researches

such as the natural

identification,

morphological

Materials

and Methods
Plant pathogenictransplanting

morphological

identification(DOA)

for 3 years. The culture,

variation,

molecular

and also toxic substances

Fusarium was isolated

valuable

study,

DNA

production

from plant diseasemethod on WA (Water Agar) and PDA (Potato

structure

which its

specimensby tissue

Dextrose Agar). Each specimen

cut into 3x3 mmpieces were immersed in 10% Chlorox and placed on WA. After incubated
26-28°Cfor 24-26 hrs. mycelial

tip was transferred

selectedby single

by transferringvial,

streakingspore techniquespore suspension

to PDA. Pure culture of Fusarium was

Fusarium spores into sterile distilled

on WA and incubating

germination

was checked and collectedunder lOx objective

transferredto PDA. Morphological

identification

followed

by Nelson et aVs method (1983).

at

at 26-28°C

water infor 16-24 hrs. Sporelens of a compound microscope andof Fusarium was based on pure culture,

Colony characteristic

as well as pigment,sclerotial

and sporodochialformation was investigated on PDA and CLA (Corn Leaf Agar). Slide culture
method was used to study sporogenous cells, phialides,
microconidia and macroconidia of each
Fusarium species. Morphology of macroconidium, conidiophoreand chlamydospore
were
investigatedafter placing culture on CLA under NUV (Near Ultraviolet)
light at 26-28°C for10-14 days. Four practical preservation
techniquesof Fusarium culture, lyophilization,10%
glycerin at -80°C, dried filter papers at 8-10°C and dried garden soil at 8-10C
were studied.
Viabilityand variation

Results

and Discussion
From a hundred and five of plant-diseased

areas of Thailandof isolates

duringwere checked on PDA every 6 months.

October

2000

were obtained

from tissue transplanting

culture7 species

revealed

scab diseasedisease- September

eighty-eight
Speciesformae speciales

from plantation

isolatesof Fusarium

identification

based on pureF. graminearum from ear

from stalk rot of corn, F. proliferatum

from Bakanaeof soybean and black spot of potato,

fromfusarium wilt of sugarcane, F. semitectum from dirty panicle

Most of F. oxysporum isolated

from plant disease

was classified

diseaseF.

of rice.

systemicallyinto 8(f. sp.). They were F. oxysporum f. sp. asparagi, F. oxysporum f. sp. cubense,

F. oxysporum f. sp. gladioli,

F. oxysporum f. sp. lycopersici,

oxysporum f. sp. tracheiphilum,
zingiberi.

were collected(f. sp.) as follows;of rice, F. solani from sudden death disease

subglutinans

2003,

method isolation.

and 8 formae specialesof wheat, F. moniliforme

specimens

F. oxysporum f. sp. phaseoli,

F. oxysporum f. sp. vasinfectum

Colony characteristic

and morphologyof species

derivedand F. oxysporum f. sp.
from Thailand

those from Fusarium keys of Nelson et al. but sometimes there was a difference.
of variationsof Fusarium occurring

identification

study on Fusarium.
The culture preservation

naturally.

ThisoccurrenceF.

resemble toThat's because

would be emphasized
for its maintenancefor

3 years. According to the long viability
and no variation after preservation,lyophilization
been the best preservation compared to 10% glycerine at -80°C which has been also better

has
than

preservationof Fusarium species has been studiedforon dried filter papers at 8-10°C

and dried garden soil at 8-10°C. Dried preservation

method, when preserved more than 2 years, sometimes have a lost of viability
andcontamination
so that lyophilizationand 10% glycerin at -80°C method would a goodalternatives

for long term Fusarium preservation.

References
Nelson, P. E., Toussoun, T. A. and Marasas, W. H. O. (1983)
manual for identification.

The Pennsylvania

London. 193 p.
Smith, D. and Onions, A. H. S. (1983)
Commonwealth Mycological

Institute,State

Fusarium Species:

University

The preservation

Press,

An illustrated

University

Park andand maintenance of living

Kew, Richmond, England. 5 1 p.

fungi.

Maintenance of microbial
freeze- drying

genetic

resources

by cryopreservationandToshirou NagaL Toyozo Sato, Keisuke Tomioka, Kasumi Takeuchi, Motoko Iida
and Masae Kawada
Genebank, National
Ibaraki

305-8602,

Instituteof Agrobiological

Sciences(NIAS),

Kannondai 2-1-2, Tsukuba,

Japan

Email : nag@affrc.go.jp
Keywords : culture collection,

viability

test

Introduction
MAFF (Ministry
establishedin 1985.

of Agriculture,

Forestry

The microorganisms

sectionsub-banks (NARC, NIFTS, NIFS and 9 institutes)of the section preserves
The preserved
viability

microbial

microorganisms

and Fisheriesconsistsof the central

mainly locatedstrains using cryopreservation
are tested

tests are useful for selecting

for viability

a preservation

are summarized to a table in an appropriateof Japan)

Genebank wasbank (NIAS)

in Tsukuba. The central bankand freeze-drying

at regularintervals.

format. In this study, we describedstrains after one-month and one-year of preservation.

Materials

and Methods

techniques.

The data fromprocedure for a new deposited

microbial

and 12strain if they
the viabilityofFreeze-drying:
Cells suspended in 10% skim milk with 1.5% monosodium glutamate
were dispensed
to ampoules, frozen at -40°C overnight and freeze-dried. To revive thefreeze-driedcells,

they were re-suspended in 100 |xl of water. The suspension was transferred

onto an appropriate plate medium and incubated.
successful preservation.
Cryopreservation:
mycelia

Mycelialdiscs (6 mm diam.) were cut out of an agar plate on which

and freezing (-70°C),

nitrogen(-165°C).

quickly

in a water bath at 30°C. The discs

Results

growth on the plate was assessed aswere growing. Five or ten discs were put into a vial containing

After cold-hardening

incubated.

VisibleVisiblethe vial was transferred

To revive the frozen mycelia,

lml of 10% glycerol.

to an atmosphere of liquid

a vial containing

frozen cells

were put on an appropriate

growth in at least 80% of discs was assessed as successful

was thawed

plate medium andpreservation

and Discussion
In 2003,the microorganisms

sectionand the central

bank maintained

20,472and14,836

microbial

viability
viabilitystrains,

respectively.

samples are testedforand the records of the tests are input to the databaseof the MAFF Genebank.

Thedata have been accumulated

bits of data were stored in 2003.

These data were processed
numbers of strainsthat

At regularsince 1985,

to give a tablesurvivedintervals

preserved

and 27,877

showing genus names, numbers of tested

one-month of preservation,strains,

and numbers of strains

thatsurvived one-year of preservation.
In the preservation
preserved

successfully;

bacterial

strains,of yeasts, 99.0% (100 strains

only a Candida strain failed

1,992 strains

survived one-year preservation;

could survive one-year of preservation.
In

Although

Table. Cryoprservation

MAFF

Genebank,

fungalandoomycetous

cells

are

pres ervedof strains could be

three Lactobadllus

As for actinomycetes,a few microorganisms

method, the method was found to be an excellent

thestrains)

to survive for one month. Of the 1,996

one Pseudomonas fluorescens could not be preserved.
by the freeze-drying/10 1 tested

curvatus and237 tested

strains

could not be preserved

one.

of fungi and oomycetes.

using

cryopreservation.

Of 6,68 1

fungal(99.3%)

strains,
and

6,631
6,578

(98.5%)

strainssurvived

one-monthpreservationand one-year preservation,
respectively(Table).

In thepreservationof oomycetes,

72.5%(300/414)

and

63.8%

(264/414)

strains

are

could

be

successfully

preserved

for one monthand one year, respectively(Table).

Oomycetous strains are difficult(Smith1982).

Our results

also

to preserveshow low viabilities

preservation

dependedon the strain.

maintainedby subculture

on slant.

by cryopreservationof oomycetes,

as known previously
and that

Cells that can not survive cryopreservation

are reluctantly

References
Smith, D. (1982)

Liquidnitrogenstorage offungi.

successful

Trans. Br. Mycol. Soc. 79: 415-421.

Long term preservationfor 20 years of entomogenous fungi

Sanae Wada. Ritsuko Murakami, Wataru Mitsuhashi,
and Kiyoshi
National

Kazuhisa Miyamoto

Kawakami

Instituteof Agrobiological

Sciences,

1 -2, Ohwashi, Tsukuba, Ibaraki

305-8634,

Japan

Email : sunny@aflfrc.go.jp
Keywords : Beauveria, freeze-drying, Metarhizium, pathogenicity
Introduction
The effective

long term preservation

study of microorganisms.

of the microbial

isolates

control

about preservation

importantas microbiologicalviabilityof the major entomogenous fungal isolates

agents of insectofof entomogenous fungi, which arepests. In this study,

we described

after 20 years preservationand isolates

after 8 years stored at 5°C on slants, and the pathogenicity

preservation

by freeze-drying.

theby freeze-drying

of survived isolates

afterof entomogenous fungi were preserved by freeze-drying

or on

and Methods
Fungi:

slants,for theMany efforts had been spent for the maintenance and preservation

them, however, there is few information

Materials

is indispensable

Seven species

Ascosphaera

apis, Beauveria

bassiana,

fumosoroseus, Metarhizium anisopliae,
Freeze-drying
sodium glutamate

Paedlomyces farinosus,

Nomuraea rileyi and Vertidllium

and reconstitution:

were dispensed

B. brongniartii,

Conidia

suspendedP

lecanii.

in 10% skimmed milk with 1%

into ampules in about 0.5ml portion.

And the ampules withconidial suspension were frozen below -50°C for 30 min before lyophilization.
The freeze-dried
ampules were sealed and reserved at 5°C. After 20 years, the conidia were reconstitutedbyre-suspendingin sterile

Storagedistilledwater.

at 5°C: The fungi were cultured

on the pupal-extract

sucrose agar slants at

25°C for two weeks and stored at 5°C for 8 years.
Viability

tests:

The conidia

from the ampules (freeze-dried)silkworm pupal-extract

sucrose liquidmedium. The cultureson the slants (storedof the pupal-extract

sucrose medium. Both transferredtransferred
isolatesto the fresh agar slants

were transferredto theat 5°C) were

were cultured at 25°C for two weeks.
Pathogenicity:

The fourth or fifth instar larvae of the silkworm,

J137),

and adults of the yellow-spotted

longicorn

conidiaof the revival

after freeze-drying.

cultured

isolates

beetle,Psacothea

Bombyx mori (C 146 X

hilaris,were applied

The insects treatedwithwere reared atroomtemperature for 14-20 days and the number of dead insects fromthe fungi were scored.

Results

and Discussion
The viabilityof total

isolates

freeze-dried

four species, A. apis, B. bassiana, B. brongniartii
the viabilityof N. rileyi

entomogenous fungi
viabilities

was 28.6%. Furthermore,

storedof the viability

Thus, the viabilities
testedand P farinosus

in thiswas similar

after long term preservation

study. Further

investigationof thewere all over 80%. However,except M. anisopliae

P farinosus,of each species

1). The viability

Table 2 showed that high viability

at 5°C on agar slantsofA. apis, B. bassiana, B. brongniartii,

The tendency

was 74.6% (Table

and N. rileyi.

Theand V lecanii were all over 90%.

in these two preservation

are consideredis necessaryof themethods.

to depend on the fungal

for the preservation

speciesof N. rileyi and M

anisopliae.
Weinvestigatedfreeze-drying

the pathogenicity

of survived(not shown). The tested isolates

isolates

after 20 years preservationofN. releyi, M. anisopliaebyand B. bassiana were

all kept their pathogenicity
against the silkworm larvae. And the isolates of B. brongniartii
exhibitedpathogenicity
against host insect, P hilaris. These results showed that the preserved
isolates

have kept the pathogenicity
even after 20 years.
This is the first case exhibited that major entomogenous fiingi

years by freeze-drying.
important

Maintenance of the properties

fungi, for example, microbiologicalto be a usefulof isolates

can be preserved

is essential

control agents. The freeze-drying

method for long term preservationand maintenance of fungal

for 20for economically
was consideredgenus Beauveria

and M.anisopliae.
Table 1Viabilityof the fungal isolatespreservation by freeze-drying

after 20 years

Table 2Viabilityof the fungal isolates

culture stored at 5°C for 8 years

on agar slant

Grouping of bacteria
rice plants
Hirosuke Shinohara1,

isolatedJun-ichirofrom leaf sheaths

Enya1' 2, Shigenobu

Yoshida1,

and panicles

Takao Tsukiboshi1'of intact

3and Seiya Tsushima1
National

Institutefor Agro-Environmental

Sciences(NIAES),

Kannondai

Ibaraki 305-8604,
Present address:

Japan
2Kyowa Seed CO., LTD., Ozawa 377, Chonan-machi,

297-0 142, Japan
Present address:

3National

Instituteof Floricultural

Sciences(NIFS),

3-1-3,

Tsukuba,

Chosei-gun,

Fujimoto

Chiba

2-1, Tsukuba,

Ibaraki 305-85 19, Japan
Email : hirosuke@affrc.go.jp
Keywords:

bacterial

flora, rice plants

Introduction
Bacterial

flora of rice plants(a microbe list)

includingspeciesmicroorganisms

and theirbioremediation.

However, little

in a field was investigatedname, sampling site, function,

diversefunction

information

is usefulis available

on bacteria

flora on a rice seed (Cottyn

sheaths

has been lacking.

rice plants

sheaths and panicles
and to classify
Materialsof cultivated,

epiphytic

bacteria

For five leaf sheaths
sampled at heading(cv. Koshihikari)cultivatedandcrops. Althoughbacterial

one LS per hill,

flora on leaf

bacteria

from leaf

in a paddy field

and other bacteria

includingat two sampling

time, 16S rDNA region of the isolates

and Discussion
The percentage

were sampled in a field 1 monthtime (B). Both ends of leaf sheaths

sampledand the PCR products (about 650bp)

bacteria(LS),time (A) and at heading

paraffin to separate

Resultsof crops

inhabiting

is to isolateon thesethem based on sequences of 16S rDNA for bacteria.

and Methods
Ten uppermost leaf sheaths

before heading

control

et al. 2001),

Our objective

intact rice plants

etc. The information

for disease

there is a report on bacterialof intact

to construct microbe inventory

times
(485

endophytic

were covered withbacteria.

(A and B) and four panicles
strains)

were amplified

by PCR,

were sequenced (Fig).

of Microbacteriumand Sphingomonas was about 30% each of total

in A samples, and that of genus Sphingomonas was about 70% in B samples. The genus

Pantoea was about 70% in panicles
isolatedat all. This

resultsuggests

sheathsof rice plant in a field.

(Table).

However, plant

pathogenicthat genus Sphingomonas inhabits

bacteria

dominantly

were notin the leaf

References
B. Cottyn,

B.5 Regalado,E., Lanoot, B., De Cleene, M, Mew,T. W.and Swings, J. (2001)

Bacterial

Populations

AssociatedPhytopathology

Table.

with

Rice

Seed

in

the

Tropical

9 1 : 282-292.

Bacterial
flora on leaf sheaths
intact rice plants

and panicles

Fig. The flow chart of an experiment.

ofEnvironment.

Pathogenic

diversityof Xanthomonas oryzae pv. oryzae in Myanmar

Them Lwin and Seint San Aye
Department of Plant Pathology,

Yezin Agricultural

University,

Yezin, Myanmar

Email : yau@cybertech.net.mm
Keywords: bacterial

blight,race, rice, Xanthomonas oryzae pv. oryzae,

Introduction
Rice is widelygrown throughout

Myanmar. Bacterial

blightXanthomonas oryzae pv. oryzae is one of the major constraintsof the diseasehas been informed

Myanmar.Growing resistantdisease(Ezukadetermination

on pathogenic

resistancein rice germplasms

may then be investigated.

is the most safe and feasible
To control

diversity

the disease

Occurrence

regionsby growing

resistantvarieties,

Evaluationofgenes into improved varieties
diversityofX.

oryzae pv. oryzae in major rice growing areas of Myanmar and to evaluate the resistanceof rice

germplasms to different
Materials

The presentof the resistant

all overcontrol measure against theof X oryzae pv. oryzae is a prerequisite.

and incorporation

caused byin rice production.

every year from various rice growing

cultivars

and Kaku 2000).

of rice (BB)study aims to determine

races of the pathogen.

and Methods
Forty-four isolatesofX. oryzae pv. oryzae collectedduring 1988 were evaluated for their virulence(TNI),

Kogyoku, IR 1545-339,

of the previous

IR 1545-339,

Yenet 7, Java 14 and Sinekari

representative

isolates

confirm the differential
resistantin the previous

collected

international

differential

3 were selected

experiment.

as Myanmar differentials
on selected

TN 1 and IET 8955 were used as a susceptible

from 6 locations

from 8 locations

and thevarieties

tocheck and aduring 2000, andduring 2003 were analyzed for their virulence

on proposedrice varieties for X oryzae pv. oryzae namely IRBB 1, IRBB 2, IRBB

3, IRBB4, IRBB 5, IRBB 7, IRBB 8, IRBB 10, IRBB ll,(Ogawa 1993).

to five representative

experiment, five varieties namely Bagolonethwe,cheek variety, respectively.
Fifty-two isolates ofX. oryzae pv. oryzae collected

1 1 isolates

namely Taichung Native 1

were tasted for resistanceof the five races were tested for their virulenceinteractions.

of Myanmar(Lead rice).

of five races of X. oryzae pv. oryzae differentiatedBased on the resultsfrom eight locations

on five rice varieties

Java 14 and Kheisaba

One hundred and twenty rice varieties
isolates

the pathogenic

TN 1 was applied

as a checked variety.

IRBB 13, IRBB 14 and IRBB 21

Results
Virulenceof X oryzae pv. oryzae Isolates
Isolates

virulence

Collected

on 5 rice varieties.

Accordingwas qualitatively

different

varieties

types, 44 isolates1). The virulence

could be accounted for screening and breedingof fortv-four

isolates

Twenty-nine varieties
to all races, respectively.
to others (Table

Varietiesof differences

belonged to five races on

could be placed

and 30 varieties

in other groups were resistantof 120 varieties

belongedin pathogenic

to 8

of 120 ricein 8 groups.

in Group-VI were resistantto some races and susceptible

2).

Table 2. Disease reactions

into 5rice

between the interactions

120 test varieties

in Group-I were susceptible

were classified

inprogram for BB resistance.

Resistance of Rice to Different Races
The distinct
disease reactions were observed
and 5 races. Based on these reactions,different

on each race on the 5 varieties

from that of other races. This kind

Table 1. Disease reactions

Varietal

1988

during 1988, were distinctly

to the reaction

as race 1, 2, 35 4 and 5 (Table

capacity

DuringofX. oryzae pv. oryzae, collected

races designatedfivevarieties

varietaland Discussion

groups to 5 races ofConfirmation

on tie Differential

Reactions

For the developmentof a differential

Myanmar,7 varieties were selectedtheirvirulence

interactionthat there was a distinct

differential

Isolates

Varieties

set to identify

based on the resultson the representative

between the varieties

of Selectedof previous-339,

Java 14 and Sinekari

experiment and retestedof the five races. The results

and the races was highlyinteraction

significant

between the selected

Among7 varieties tested In this study, 5 varieties
1545

races ofX oryzae pv. oryzae In

and a resistantvarieties(TableTable 3. Disease seYeritles (% leaf area infected)
races ofX oryzae pv8orwzmes

and the races.

namely Bago-lonethwe,

3 could be used as differentials

check, respectively

that theat P<0.0 1. This suggested

to identify

oryzae pv. Oryzae In Myanmar. TN 1 and 1ET 8955 should be Included
a susceptible

indicated

for

Yenet 7, IR

the races ofXin the differential

set as3).
between 7 rice ¥aiieties

and 5

VirulenceofX.oryzae pw. oryzae Isolates Collected during 2§Q§ and 20S3To in¥estigate the pathogenic
diversityof X oryzae pv. oryzae from Myanmar, 52

isolatescollected

from 8 locations

during 2000 and 1 1 isolates

2003 were analyzed for their virulencecollected

on proposed internationalfrom 6 locations

differentials

duringfor X oryzae pv.

oryzae (Ogawa 1993).
Based on the virulence
during 2000.
collected

Anotheranalysis,

12 races were detected

3 races could be identified

during 2003 on the differentials(Table4).

among 52 isolates

by the virulence

analysisof llcollected
isolates

Virulence

Table 4.

of 63 Isolatesof X oryzae pv*oryzae on 13 rice varieties

Conclusion
In the present

study,

15 races were identified

from 63 isolates

locationsof Myanmar during 2000 and 2003. These results

existing

races of X. oryzae pv. oryzae in Myanmar because a limited

isolatescollected

from some locations

only

collected

from 14

do not show a complete nature of

were tested.

number of bacterial

More races could

be found if

nationwide collectionof isolates were made. It is, therefore, necessary to carry out further
research on identificationand distributionsof races by using isolates collectednationwide.
Furthermore, it is also essential
Myanmarto understandlevel
nationalof pathogenic

the populationand genetic

level of screening

diversity

diversityof the pathogen.

of X oryzae pv. oryzae from
The awareness of the nationalofX. oryzae pv. oryzae will be a great help to theprogram for rice bacterial

A historicalreview of bacterial

blightresistance.

blightof rice. Bull. Natl. Jnst.

Resour. 15: 1-207.

Ogawa, T. (1993)
resistancethe genetic

structureand breeding

References
Ezuka, A. and Kaku, H. (2000)
Agrobiol.

to evaluate

Methods

and Strategy

genes to bacterial

Agri. Res. Q. (JARQ)

leaf blight

27: 71 - 80.

for monitoringrace distribution{Xanthomonas campesiris

and identification
pv. oryzae)ofin rice. Jpn.

Phylogenetic
analysis of Xanthomonas species based on the nucleotide
sequences of 23S rRNA gene and 16S-23S rDNA spacer region
Hirokazu Ochiai1.

Jean Swings2 and Hisatoshi

Genetic

Department, National

Diversity

Kannondai 2- 1-2, Tsukuba, Ibaraki
Universiteit

Kaku1

Institute305-8602,

of Agrobiological

Sciences(NIAS),

Japan, 2Laboratorium voor Microbiologie,

Gent, B-9000 Ghent, Belgium

Email : ochiaih@nias.

affrc.go.jp

Keywords: ITS, phylogeny,

23 S rDNA, Xanthomonas

Introduction
The genus Xanthomonas consists
importantof plant pathogenic

crops. To date, Xanthomonas was classified

DNA-DNAreassociationand 16S-23S(Vauterin

rDNA intergenic

Xanthomonas strains.
high conservation

Analysis

into 20 genomic species

Until recently,

analysesof 16S rDNA sequences has revealed

among xanthomonads (Hauben et al. 1997).
level

resolution

than

previous

study of 16S-23S

Materials

and Methods
Bacterial

rRNA intergenic

rDNA intergenic

strains.

maltophilia

ofon the basis ofof 16S rDNA sequence
analyses withina small divergence

andrDNA intergenic

spacerandofXanthomonas based on sequences ofspacer region (ITS),

and compared them withspacer sequences.

A total of 109 strains

strain of Stenotrophomonas

16S-23S

a variety

16S rDNA sequences (Goncalves

In this study, we analyzed the phylogeny

23S rRNA gene and 16S-23S

attackingspacer sequence were employed for comparative

sequences has showed a higher
Rosato 2002).

et al 1995).

bacteria

including(previously,

18 Xanthomonas species

designatedand two

as Xanthomonas maltophilia)

used in this study.
PCR amplificationof 23S rDNA and ITS.

The PCR primers for 23S rRNA and ITS

were designed from the conserved regions in the 16S rRNA and 23S rRNA genes. PCRs werecarried out with the following temperature profile: an initial denaturation at 94°C for 5 min; 30
cycles of denaturation

at 94°C for 1 min, annealing

2 min; and final extension

at 72°C for 7 min.

Direct sequencing.
using

at 55°C for 1 min, and extension at 72°C fora BigDye Terminator

The PCR products purified
Cycle

Sequencing

from agarose were directly

FS Ready Reaction

products were analyzed with a model ABI 3 100 genetic

analyzer.

sequenced

kit, and the sequencing

Data analysis.

Phylogenetic

program. Phylogenetic
Results

trees were constructedand Discussion
Based on partial

major clusters.

analysis

was performed by using

accordingto the neighbor-joining

Members of each cluster

were similar

to those of 16S rDNA analysis.

X. albilineans,

two clusters

X. hyacinthi,

whilecluster

98.8 %. Cluster I consisted
The obtained

results

rDNA (16S or 23S)of two subgroups,

were similar
analyses

II was consistedof 4

same group were more than

to those of 16S rDNA sequence analysis,
to delineateI

betweenwith each subgroup having heterogeneous

were not sufficient

into two
Cluster

X sacchari and X. translucens. The similarities

were ranged from 96.6 to 97.8 %, but those withinX

method.

sequences of 23S rDNA, Xanthomonas species were divided

was a major group which was composed of 14 species,
species,the CLUSTAL

species.

and suggestedthatxanthomonads at species level.

ITS was divided into three regions ITS 1, 2, and 3 by existence of two tRNAs,tRNA^ and tRNAIle. The length of each ITS region was 85-96 nt, 14-76 nt, and 223-228 nt,
respectively.

The lengthof ITS was very diverse

among Xanthomonas species.

tRNAs were highly conserved, they were classified
sequences. In tRNAAla, two nucleotides were different
were different
dividedin tRNAIle.

into two clusters.

there were slight

On the basis
in subgroup

I indicatedlevel.

Xanthomonas species

that subgroups

Comparative

were also

ITS

that

phylogenetic

species could be reflected by the topology

analysis

between subgroups

but
and

were defined by tRNA types. The tRNA typingof Xanthomonas species

suggested

into two types by slight differencesin
between two types, and three nucleotidesof ITS sequences,

was useful for clustering
analysistwoMembers of each cluster were the same as those of 23S clusters,

differencestRNA types in cluster

Althoughby ITS sequences.

relationships

The results

and lineages

obtainedbyamong Xanthomonasof the ITS tree.

References
Goncalves, E. R. and Rosato, Y. B (2002)
upon 16S-23S

rDNA intergenic

Phylogenetic

analysisofXanthomonas species based

spacer sequences. Inst. J. Syst. Evol. Microbiol.

Hauben, L., Vauterin, L., Swings, J. And Moore, E. R. B. (1997)

Comparison

DNA sequences of all Xanthomonas species. Inst. J. Syst. Bacteriol.
Vauterin,L., Hoste, B., Kersters,

Inst. J. Syst. Bacteriol.

45:472-489.

K. And Swings, J. (1995)

52:355-361.
of 16S ribosomal

47:328-335.

Reclassificationof Xanthomonas.

Development of genome database system for Xanthomonas oryzae pv.
oryzaeMasaru Takeya. Hirokazu Ochiai,
Genetic Diversity

Department,

Masahiro Satoh and Hisatoshi
National

Kannondai 2- 1 -2, Tsukuba, Ibaraki

Institute305-8602,

Kaku

of Agrobiological

Sciences(NIAS),

Japan

Email : katu@nias.affrc.go.jp
Keywords:

BLAST search form, ORF viewer

The genome database system has been developed for the complete genome sequenceofXanthomonas oryzae pv. oryzae (Xoo). The system provides sequence and gene information
obtainedfrom our genome sequencing project(MAFF311018).
designed

We analyzed

a relationalof Japanese

a data structure

database schema, thus eliminating

database will be accessed through the internet.
and viewer of gene information,

race I strain

to genome information
any inconsistence

T7174

on Xoo andof data mutuality.

The

Two kinds of search system, BLAST search form

have been prepared in the homepage of the database.

Sequence data is registered

to table"fragment". This table"contig", "genome", "orf', and "cron_microarray"of database

relative

representative

is linked

through reference relationships.

is shown in Fig. 1. The database provides

gene information

to table"cron",The schemawith ORF viewer and

BLAST search form. Moreover, the database system is used for microarray and gene knock-out
experiment.

Fig. 1. Schema of xoo databaseIn the BLAST search form, complete genome or ORF sequences can be chosen as atarget database,and the BLAST starts based on Inputted sequence of nucleotide

when the "Search" button Is pushed (Fig.

2).

In the viewer of gene Information,
product Is shown by a clicking
available
choiceresearchers

Interested

level.

gene informationsuch as gene name, length,andof target gene In the genome maps (Fig. 3). A keyword search isto the gene Information.
of magnificationor amino acid

The display

range of the genome map can be changed by a

The latest Information

on gene analysis

In this study by using the databasesystem.

Fig. 2. BLAST search form

Fie.3.

QRP viewer

ofXoo can be sent toPCR-based genomovar identificationand characterizationof
Burkholderia cepacia complex strains isolated from diverse origins
Kenichi Tsuchiya.

Sang-Tae Seo \ Chika Fujitani

2, Hiroyuki

Sawada, Takanobu Yoshida

and Mami Takahashi 3
Divisionof Microbiology,
National InstituteKannondai 3-1-3, Tsukuba, Ibaraki 305-8604,

for Agro-Environmental
Sciences (NIAES),
Japan. ] Present address: National Horticultural

Institute,Suwon, Korea. 2Tokyo Agricultural

University,

National

Agriculture

Atsugi,Japan. 3Present

address:

Research Center, Joetsu, Japan

Email : kentsuch@affrc.go.jp
Keywords: plant pathogen,opportunistic

human pathogen, genetic analysis,

environmental

risk

Introduction
Burkholderia

cepacia, first describedin 1950 as the pathogen

in recent years as an important human opportunistic
particularly

among cystic fibrosis(CF)antagonist

throughof various antibiotics,

consideredas biopesticides

the productionin the control

wide range of recalcitrant
distinct

species

complex (Bcc).

Despitethe acknowledged

their genomovar distribution
characterizationthat causes numerous outbreaks,

Burkholderia.

cepacia is also broad-spectrum
such as pyrrolnitrinof plant diseases

compounds. Taxonomic studies

of genetically

The objective

patients.

pathogenof onions, has emerged

and could be

as well as in bioremediationof ahave revealed that B. cepacia consists

or genomovars, which has become known as the B. cepacia
significanceof the Bcc strains,of natural strains, particularlyof thisstudyof the 1 19 Bcc strains

littleis known about

related to agro-ecosystem.

was to determine

the genotypic

recovered from clinicalidentificationand environmentalandsources inJapan and Thailand.
Methods and Results
One hundred nineteen strains of the Bcc and reference strains, namely, B. cepacia
genomovar I (ATCC 25416T and ATCC 17759), B. cenocepacia ffl-A(ATCC 25609, ATCC
17774 and ATCC 17460), B. cenocepacia HI-B (ATCC 17765), B. stabilis (ATCC 27515),
B.
vietnamiensis(ATCC BAA-2487), and B. pyrrocinia (LMG 14191 and K1112)
were used.
Genomovardeterminationof the Bcc strains was performed by a combination

the 16S rRNA and recA genes, and genomovar-specificthe epidemic

marker (BCESM)

prnC among strains

PCR. Furthermore,

encoded by emsR and pyrrolnitrin

were investigated.

All procedures

biosynthetic

in preparationof PCR-RFLP ofthe distributionoflocus encoded byof bacterial

DNA, PCRamplification,

digestionof PCR productswith endonucleases,

prnC, and others were carried out as described
Based on the results
Dde I , the Bcc strains
vietnamiensis)
All strainsof analysis(Seo and Tsuchiya

into two groups (Fig.

2004).
after digestion1): group

into eightpatterns

In the RFLP analysis
designatedwith

1 (including

B. cepacia genomovar I , B. cenocepacia

to group 2 except for one strain.

using HaeIII, strains were separatedof the BCESM andby 16S rDNA RFLP generated

were differentiatedand group 2 (including
belongedelsewhere

detectionB.

and B. stabilis).
of the recA geneas A, B, E, G, H, I, J and K?

of which pattern K was new(data not shown). When they were compared with those obtainedinprevious

were identified

B.

as B. stahilis(J)

studies,

where patterns designated

A-J were reported, 73 strains

cepacia genomovar I (D, E and K), 33 as 5. cenocepacia(G, H and I), threeand one as B. vietnamiensis

to pattern K were identified

strains

belongedcepacia genomovar I based on genomovar-specific

PCR.

Burkholderia.
pyrrolnitrin(74%)

biosynthetic

(A).

These

cepacia epidemicstrain marker (BCESM)

as B.

encoded by emsR and thelocus encoded by prnC were present in 22 strains(18%)

respectively.

to B. cenocepacia^

from all sources,

All emsR positive

strains

belongedand 88 strains

whereas most prnC positive strains belonged to B. cepacia genomovar I (Table 1). Comparing
the emsR and prnC strains, we observed a good match between emsR positiveand prnC
negative strains.

All of the emsR positive

group except for some cymbidium

strainsstrains

were found to belong(B. cenocepacia

to the prnC negative

III-B).

Table 1 prnC and emsRin different genomovars or species among
clinical and environmental isolates of the A cepocia complex

Fig. 1 EJeetrcptioresis patterns obtained after digestion of amplified
Burkholdria cepacia complex 16S rDNA with restriction enzyme
I. M, DNAsize standard (50-bp ladder). ITis genoiiMwa-stateof each strain is indicated by tie Romannumber

References
Seo3 S-T. and Tsuchiya,
Burkholderia
AppliedK. (2004)

cepacia complex bacteria

Microbiology,

39: 413-419.

PCR-basedfrom clinicalidentificationand characterizationand environmentalofsources. Letters inFlagellin

glycosylation

Kasumi Takeuchi1.

islandin Pseudomonas syringae

Fumiko Taguchi2, Chihiro

Etsuko Katoh1 , Yoshishige

Inagaki2,

Yasuda2, Hanae Kaku1, Katsuyoshi

Murata1,

Kazuhiro Toyoda2, Tomonori Shiraishi2

and Yuki Ichinose2
Genebank, National Institute of Agrobiological
Ibaraki 305-8602,
and 2Faculty of Agriculture,
Okayama 700-8530,

Sciences (NIAS), Kannondai 2- 1-2, Tsukuba,
Okayama University,
Tsushima-naka 1-1-1,

Japan

Email : kasumit@affrc.go.jp
Keywords:

glycosyltransferase,host specificity,

plant pathology

Introduction
The deduced
tabaciand P. syringae

hypersensitivetheamino acid sequences of the flagellins

reaction

posttranslational

posttranslational
isolated

modification
island).

are identical;

different.

modificationof theinvolved

We identified
and identified

however, theirflagellins.

To

investigate

to induceaseems to depend on
the

role

of thisbetween plants

and bacterial

in the posttranslational

modificationof flagellin

glycosylationand their

the genes involved

Using these materials,
glycosylated

abilities

The reason for the differencein the interactions

mutants were generated.

microbe interactions
Materials

are clearlygenes that are potentially(glycosylation
deletion

pv. glycineaof Pseudomonas syringae pv.

in flagellin

pathogens,we

we examined their influence on plant-amino acid residues.

and Methods
The bacterial

strains used in this study are Pseudomonas syringae pv. glycinea race 4and P. syringae pv. tabaci Isolate 6605. For the inoculation test, bacteria were cultured in LuriaBertani (LB) medium with 10 mM MgCU at 25°C. For purification of flagellin proteins, bacteriathat had been cultured overnight were incubated in minimal medium containing 10 mM
mannitol and fructose as carbon sources for 24 h at 23°C. Pseudomonas syringae strains witheach ORF in the glycosylation
island deleted
were generated based on homologous
recombination by using the mobilizablecloning vector pKl &mobsacB (5.7 kb, Km1) (Takeuchi
et al2003).
Results

and Discussion
The sequence data revealed three ORFs, designatedflagellum

structuralgenes flgL andfliC

orfN, which was found in the glycosylation(Fig.

orfl, orf2, and orJ3, between the1). orfl and orf2 showed significant
islandhomology toof P. aeruginosa strain PAK; at the amino

acid level there was 32% homology in an 860amino-acidregionandhomologyin

577-amino-acidrespectively.
producta
On theshowedglycosylation

other

38%
region,theorft

to orfC in theof P. aeruginosa(29%

which was homologous to putative

3-oxoacyl-(acylcarrier protein)theglycosyltransferases

are responsible

generateda defective

glycodetection

genes

that

exhibitedhomology

for flagellin

glycosylation

mutant with a mutation

revealed that the deletion

proteins,

Fig. I. Schematic representationof the ORFs in the region upstream of
the HiC (flagcllin)
gene from the genomic sequence of P s pv. glycinea
and pv. tabaci and their comparison with P, aeruginosa strain PAK.

synthase III. To

examine whether

flagellin

was

hand,

homologyislandhomology),

there

residues

genes

in each ORF. SDS-PAGE analysis

andeliminatedthe ability

remained in the flagellin

each mutant strain of P. s. pv. glycinea

putativewe

To evaluate the effect of a defect in the genes in the glycosylation
inoculated

encoding

in P. syringae pv. glycinea,

oi orfl completely

whereas some glycosyl

to theof the orf2 mutant.
islandonto soybean (host)to glycosylate
on plants, weand tobacco(nonhost)

leaves. When host soybean leaves were inoculated with each strain, the tSorfl and Aorf2 mutants
failed to cause prominent symptoms. In contrast, inoculation of the nonhost tobacco leaves withthe orfl and orf2 mutants resulted
inoculation

with the wild-type

in lesion-likeacid residues

the

10 days after inoculation,strain did not have this effect.

strain ofP. s. pv. tabaci onto tobacco (host).
to cause prominent symptoms.

To identify

changes

glycosylated

We also inoculated

These mutants, especiallywhereaseach mutant

the tsorfl mutant, failed

amino

on flagellinfrom P. s. pv. tabaci,

comparison of peptide

fragments produced by

the digestionof wild-type
and Aorfl mutant
flagellins with asparatic N-peptidase was carried out by the reverse-phasecolumn HPLC. The

amino acids sequencing

experiments revealed

analyses of the peptides

that all glycan linked

though serine residues

serines are locatedin the internal

flagella

MALDI-TOF MS analysis

filament.

Flagellin

glycosylation

specificity.

J Bacteriolof flagellin

193 and 201 (Fig.

2). These

to be surface-exposed

proteins

part infrom Ser/Ala-substitutedof molecular weight was about 540 Da in each serine.

F., Inagaki,
islandat 143, 164, 176, 183,

domain, which is expectedmutants revealed that the reduction
References
Takeuchi, K.. Taguchi,

and alanine substitution

T., Toyoda,K., Shiraishi,

in Pseudomonas syringae

185: 6658-6665.

T., and Ichinose,

pv. glycinea

Y. (2003)

and its rolein hostHuanglongbing
research
Siti Subandiyah1.

diseasemanagement in citrus in Indonesia-Current

Andi Trisyono1, Susamto Somowiyarjo1,

Andrew Beattie2,

Paul Holford2,

Zamir Hossain2 and Paul De Barro3
department of Plant Protection,

Faculty of Agriculture,

Yogyakarta 5528 1, Indonesia.
2Centre for Horticulture and Plant Science,

University

Keywords : Candidatus

Bulaksumur,

of Western Sydney, Locked Bag 1797,

Penrith South DC, NSW 1797, Australia.
3CSIRO Entomology, 120 Meiers Rd, Indooroopilly,
E-mail : ssubandiyah@yahoo.

Gadjah Mada University,

QLD 4068, Australia.

com

Liberibacter

asiaticus,

Diaphorina

citri, huanglongbing

management.

Introduction
Huanglongbing,
citruscaused by Candidatus

in Indonesia.

In the past,

approach was not sustainable,
fruit. Eradicationof diseased
disease-freematerial,

pesticides

for control

hampered attempts
Indonesia

Liberibacter

oxytetracycline

asiaticus,

is a severe disease

was used to control

the disease,of

but thisas it gave only temporary control and left antibiotic
residues ontrees has been practiced in Indonesia since 1980s, but a lack of

the high

cost of replacingof the vector, Diaphorina
to build

in collaboration

infected

citruswithfrom Australia,

improve the management of huanglongbing

and an ineffective

citri Kuwayama (Hemiptera:a sustainable
scientists

plantsindustry.

Researchuse ofPsyllidae),is being

have

conducted

inFrance, Japan and Viet Nam toand its vector and to understandtheir biology.

Research Project(1) Distribution
and occurrence ofD. citri and psyllid natural enemies. Surveys of
the incidence of D. citri and its natural enemies are being conducted in several areas of theIndonesianarchipelago

includingnatural enemies, including
are being collected
latitude,Java, Kalimantan,

entomopathogens,for identification

and altitude),climate,Sulawesi

predators,

their distributions

the nature and proximity

and Bali.

Psyllidfauna and theirand primary and secondary parasitoids
will be related

to geography(longitude,of other vegetation

and, where relevant,alternativehostsof D. citri

obtainedfrom: herbarium specimens

management practices.
(2)
asiaticus.

Distributionof known and potential

Primary data on rutaceous plants

are beingand L.

located at the Herbarium Bogoriense;

the living

collection

at Indonesian

Botanic Gardens; andrecent surveys conducted by staff from the Bogor Botanic Gardens. Secondary data are also
being obtained from published studies.
These data will be related to geography (longitude,
latitude,and altitude),ecosystems

surrounding vegetation)
infection by L. asiaticus

type(including

climate,and, where relevant, human activities.
is also being recorded.

(3) The impact

of temperature,and disease

severity.

1000-1200

m above sea level)

altitudesoil

Six experimental fields at three different

on the incidence

altitudes

development

climateand management practices.

Locationsat the lowest altitude

speciesof Citrus

to determine

theiralliesandofD. dtri(50- 100, 500-600,

with Siem mandarin {Citrus

determine the seasonal abundance of D. dtri and disease
and their

topography,

The presence of D. dtri and theand genotype

are being planted

type,

and

reticulata)

in relationtoto altitude,

will also be planted

withsuitability

as hosts for D. dtri and L.

behaviourof D. dtri and host plant

asiaticus.
(4)
phenology

The relationshipbetween the feedingon disease transmission.

in 3-4-year-old

orchards

psyllids

theirincludingduring flushes.

will

Vector populations,be monitoring to determine

movement, feeding

Glasshouse

experiments

observations to describe adult behaviour
different environmental conditions.
(5)
ovipositionThe

impactbehaviour

tree phenologyof mineralactivities
will

and rates of infection

the seasonal

behaviourand degree of aggregationof adult

between andfocus on the use a video camera and direct

on mature and immature foliage
and plant

oilof D. dtri and the spread

spray

under a range ofon mortality,of huanglongbing.

feedingandIn the controlledenvironment glasshouses, the impact of horticulturaland agricultural mineral oil (HMO andAMO) deposits on the feeding and oviposition
behaviour of adult psyllids,
nymph mortality ofD. dtri and the transmissionof L. asiaticus

of HMOs and AMOs on the ingress
aggregationand population

are being observed.
of psyllid

growth in relation

adultsof timing

In field experiments, the impact

into orchards

and their

subsequentof sprays and plant phenology

will bestudied.
Progressto date•EHuanglongbing
is widely
citrus centres throughout

spread with different
severities
the Indonesianarchipelago.

•EThe distributionof D. citri follows that of Citrus
koenigii.
The psyllid
has been found in lowland
orchards.

at different

locationsin thespp., Murrqya spp. and Bergera
and upland (1200m asl) citrus•ECitrus
indica,allies found in Indonesia include Aegle marmelos, Bergera koenigii,
Clausena
Feroniellalucida, Limonia acidisima,
Glycosmis
pentaphylla,
G citrifolia,

Melicope spp., Murraya paniculata,
M. exotica, Swinglea glutinosaand Triphasia
trifolia.
•EThe natural enemies ofD. citri found include two parasitoids(Tamarixia radiata andDiaphorencyrtus aligarhensis),several generalist
insect predators and three species ofentomopathogens(Hirsutella
citriformis,
Paecilomyces
fumosoroseus
andMetarhizium sp.).
•EA study on the transmissionof L. asiaticus to susceptible
Siem mandarin found thatbacterium spread unevenly in the leaf midribs, stem bark and root bark of host plants.
In one infected plant, not every site assayed by PCR was found to be positive for L.
asiaticus.
•ELiberibactersuggesting

asiaticus
was found in the head, thorax and abdomenthat the bacterium is circulatedaround the psyllid' s body.

of D. citri•EAMOdeposits affected the feeding behaviour of D. citri. During a week of observations,
psyllids
prefer to feed on untreated plants compared to on plants treated with 0.250.50% oil.
No psyllids
were seen to feed on plants treated with higher oilconcentrations.
Acknowledgements
This research is being funded by the Australian
Research (ACIAR)

and Horticulture

Australia

Ltd. (HAL).

Centre for International

Agricultural

A preliminary
on citrus
Siti

study of a toxin associatedSubandiyah1.

Rina Ediati1,

with Huanglongbing

diseaseAchmad Himawan1, Andi Trisyono1, ArmanWijonarko1,

Zamir

Hossain2, Paul Holford2 and Toru Iwanami3
1: Dept.

of Plant

Yogyakarta,

Protection,

55281,

Western Sydney,
Agricultural

Faculty

Indonesia.

of Agriculture,

GadjahMada University,

Bulaksumur,

and Plant Sciences,

University

South DC, NSW 1797, Australia.

3: National

2: Centre for Horticulture

Locked Bag 1797,

PenrithResearch Centre for Kyushu Okinawa Region, Suya 242 1, Nishigoshi,

ofKikuchi-gun

Kumamoto-ken86 1-1 192, Japan
E-mail : ssubandiyah@yahoo.com

Introduction
Huanglongbing(HLB),

Indonesia,

is a very destructive

Candidatus

Liberibacter

diseaseasiaticus.

pathogennor on its interactions

interveinal

chlorosis

trees or be confined
bitterdegeneration

caused by a phloem-limited,uncultivable

There have been few studies
withof thisinfectedand may only yield

effectively

within the host (Himawan unpublished,suggests

bacterium,or a general mottle on leaf laminas that may occur throughout
abscise.

Symptoms of thisinincludeto certain branches.

its host plants.

(CVPD)

on the characteristics

Fruit produced by infected

trees are small, malformed,

If they remain attached to the tree, the fruit

Under poor management, most trees die from huanglongbing

planting
diseasevein phloem

diseaseand may prematurely

properly.

known as citrus

for 1 year.

that a toxin may be involved in disease

was conducted as a preliminary

study to determine

withinThe uneven distribution

Gamier et al 1983) together

do not color
5 years ofof the bacterium

the symptomatologyof thedevelopment.

Therefore, this research

if toxic substance(s)occur in //LB-infectedmaterial.
Materials

and Methods
Leaf midribs were removed from//LB-infected

Siem mandarins {Citrus

reticulata).

temperature

with either

phosphateevaporated

to dryness

mL. Aliquots

Freshbufferedand then re-suspendedof both extracts

were diluted

heat-treatedat 40, 60 and 100°C for 15 min.

infiltrated

into leaves of citrus

inhibitionof bacterial

midribssalineand healthy(bothconfirmed by PCR)

were ground and extracted(PBS)

or ethanol.

These

in PBS to give the equivalent2-, 5- and 10-fold
Control,

dilutedin PBS.

extracts

wereof 5 g midribs

perFurtheraliquots

wereand heat-treatedextracts

weretabacum).

Anyof Siem mandarin and tobacco (Nicotiana

cell growth was tested by placingat room

5 mm diameter filter paper discs soakedcitrifor 30 min in various extracts on lawn growths of Escherichia
Xanthomonas axonopodis pv.
lOg/L

peptone,phosphate,

Results

coli, Ralstonia

on nutrient agar? CPG ( 10 g/L mannitol,pH 7), and SPA (20 g/L sucrose, 5.0 g/L peptone

0.25 g/L magnesium sulphate,solanacearum and1 g/L casamino acid,

0.5 g/L dibasic

potassium

midribs caused chlorosis

in citrus

15 g/L agar, pH 7.2) media.

and Discussion
The PBS and ethanolicleaves and hypersensitive,caused by extracts

extractsof #LB-infectedlesions

in tobacco:necrotic

from healthy

PBS and ethanolic

extracts

also caused zones of growth inhibition

when tested

E. coli, R solanacearum, and X.

axonopodis (Table

zone was greatest

1).

The inhibition

axonopodis:

no inhibition

bacteria tested.

Extracts

cause a hypersensitive
treatment

and to inhibit

bacterial
the activity

their

Table

1. Mean diameter

undiluted,

HLB-infected

B a cte ria l sp e c ie s
E . co li
R . s o la n a c ea ru m
X . a x o n o p o d is p v . c itri

Means followedby different

Duncan's Multiple

Range Test.

lettersallto induce chlorosis,

toof extracts

againstdilution.

Heat

R. solanacearum^by treatment at 60°C. The

was lost by a heat treatment at 100°C (Table 2).

(n = 4) of inhibition
extracts

ability

midribs against

growth up to a 10-foldeffect against X axonopodis was abolishedagainst both species

midribsfor R. solanacearum and least for X.

made in PBS and ethanol retained

at 40° and 60°C did not affectactivity

againstof /7LB-infected

zones were produced by extracts from healthy

reaction

however, the inhibitory
inhibitory

leaves.

no symptoms on citrus or tobacco werezones inducedby filter

discs

soakedinat the 5% level accordingtoof midribs.
M ea n o f in h ib itio n z o n e s (m m )P B S ex tra ctio n
E th a n o lic e x tra c tio n
9 .7 5 a
9 .5 0 a
1 7 .5 0 ‑14 .5 0 ‑7 .0 0 c
6 .5 0 c

are significantly

differentHLB-infected

Table

2. Mean diameter

ethanolicextracts

(n=4)

of

B a c te ri a l s p e c ie sof inhibitionzone induced

and healthy

midribs.

E x tr a c t io n

T e m p e ra t u r e (‑ C )R . s o la n a c e a r u m
E th a n o l

P B S

PBS and

D ia m e t e r o f in h ib itio n z o n e ( m m )H L B

H e a lt h y

4 0

7 .0 00 .0 0

6 0

7 .0 00 .0 010 0

0 .0 00 .0 0

4 0

6 .5 00 .0 0m eth o dP B S

by heat-treated

6 0

7 .0 00 .0 010 0

0 .0 00 .0 0

4 0

6 .3 00 .0 0

6 0

0 .0 00 .0 0X . a x o n o p o d is

10 0

0 .0 00 .0 0p v . c i tr i

4 0

6 .0 00 .0 0

6 0

0 .0 00 .0 010 0

0 .0 00 .0 0E th a n o lConclusion
chlororsis

Extracts of HLB-infected tissue caused a necrotic, hypersensitive
reaction in tobacco,of citrus leaf tissues and inhibitedthe growth of E. coli, R. solanacearum, and X

axonopodis pv. citri. The extracts remained active up to a 10-fold dilution and heat treatment
for 15 min at 60C abolishedthe inhibitory
activity
against X. axonopodis but not R.
solanacearum whilst treatment

at 100°C abolished

Together,these data suggest L. asiaticus

the disease

syndrome.

Reference:
Gamier, M and Bove, J.M. (1983)
diseaseactivity

againstboth organisms.

produces one or more toxic substances

Transmission

from sweet orange to periwinkle

the inhibitoryof the organism

by dodder. Phytopathologythat are part ofassociatedwith greening

73 : 1 358-1363.

Acknowledgement
This researchTechnology.

was funded by a grant from the Indonesian

Ministryof ResearchandPhylogeny
symbionts
Hiroyukiandtaxonomy

Sawada1. Toshiki

ofbacterial

Uchiumi2, Mikiko

nitrogen-fixing

legumeAbe2, Masahito Hayatsu3,

Yukari Ichiman4, Katsumi Akutsu4, Takanobu Yoshida1 and Kenichi Tsuchiya1
1 National

Institutefor Agro-Environmental

Sciences (NIAES),

3- 1-3 Kannondai, Tsukuba,

Ibaraki 305-8604, JAPAN2 Kagoshima University,
1 -2 1 -35 Korimoto, Kagoshima, Kagoshima 890-0065,
3 Shizuoka
4 Ibaraki

University,
University,

836 Ohya, Shizuoka,
3-21-1

Shizuoka

Chuo, Ami, Ibaraki

422-8529,

300-0393,

JAPAN

JAPAN

JAPAN

Email : sawada@affrc.go.jp
Keywords : evolution,
Phylogenetic

rhizobia

Analysis
As of September

2004, bacteriathat form nitrogen-fixing

legumes have been confirmed

in more than 49 species

containing

based(Figure)legume symbionts

Alphaproteobacteria

andanalysisin one lineage

Betaproteobacteria,

associationsof 13 genera. Phylogenies

on the comparative

show that they are not clusteredsymbiotic

and

with

of these taxa

of 16S rDNA sequences

but are distributed

dispersed

overin the classes

thefollowingtenmonophyletic groups, being intermingledwith other taxa that do not contain legume symbionts(shown in parentheses below): Group 1, which comprises Rhizobium and Allorhizobium
species
containing
which

legume symbionts

are nonsymbionts);

nonsymbionts);
Brucella,Group(intermingled

Group 2, Sinorhizobium
3, symbiotic

species

Agromonas, Nitrobacter,speciesand Blastobacterand their

denitrificans

relatives);(withnodulans" (with nonsymbiotic

Methylobacterium

species);nonsymbiotic

Xanthobacterand Aquabacter

neptuniae(withsymbiotic

Burkholderia

Ralstonia

taiwanensis

speciesnonsymbiotic
species

Devosia species(withnonsymbiotic(with nonsymbiotic

Ralstonia

unclassified
Ochrobactrum,
nonsymbiotic

Group5, symbiotic

Bradyrhizobium,

Group 6, "MethylobacteriumGroup 7, Azorhizobium
species);and unclassified(with

Burkholderia
species).

species,(withnonsymbiotic

Afipia, and Rhodopseudomonas species);

Ancylobacter,speciesstrains (with nonsymbiotic

Group 4, Mesorhizobium

and Pseudaminobacter

Bradyrhizobium

and Blastobacterand Ensifer

Ochrobactrum

and Mycoplana species);

Aminobacterwith Agrobacterium

speciesGroup

nonsymbionts);
species);(with

8, Devosia
Group 9,and Group 10,

Taxonomy
For Groups 6, 9, and 10, thepresentclassification,

monophyletic

in which"eachgroup comprises one genus

whereinlegumenonsymbionts

symbionts

are intermingled

other," is consideredandwith eachto be retained

because they are clearly

as is,

separatedfromother genera at high bootstrap

values andhave

sufficiently

already

been

characterized

based

on

polyphasic

taxonomy. As for the remaining
monophyletic
there

seven

groups, on the other hand,are currently

three

options

current

foremending

their(definitions

and circumscriptions)generic level:A) the current classification

shallbe retained

withineach monophyleticclassification

as is; B) all the genera
group shall beamalgamatedinto one single

conformity

withphylogenetic

analysis;

subordinate

at thelineagetheandgenus inresults

of

C)

each

in each monophyletic

group shall be proposed

as a genus. It isconsideredthat researchand discussions

will

continuously

conducted

beemendingmonophyletictheclassificationof thesegroups based

chiefly

Options B and C as preferable

Figure.

forPhylogeneticoncandidates.

relationships

among legume symbionts

and their relatives

divergence.
The names of the genera contained

inferredin each monophyletic

phylogenetic

positionsof legume symbionts

symbionts

are indicatedby the symbol #.

based on the 16S rDNA sequencegroup are listedon the right. The

and the names of the genera which contain legume

Gene-expression controlled by a conidiophore
Acrl In Magnaporthe grisea

patterning

regulator

Marie Nishimura
National

Instituteof Agrobiological

Scieneces(NIAS),

Kannon-dai 2- 1 -2, Tsukuba, 305-8602,

Japan
Email : raarie@affrc.gGjp.
Keywords:

conidiogenesis

Rice blast
thepatterning, conserved domain, medA,

disease,most devastating

diseasesmechanism of conidiationa functional
Results

caused by the filamentous
to global

fungus Magnaporthe oryzae, is one ofrice production.

in M oryzae, a morphologicalTo understandmutant, acrl, has been studied.

homolog of MedA, a conidiophore-developmentalof a protein-protein

homology search (BlastX)

the regulation

regulator

indicatedin Aspergillus

that the central

Acrl isnidulans.

domain in Acrlis conserved among manyfungus.
BlastX Mts against

The lossK>f-function

Acrl protein sequence

Acrl mutants produce chains of elongatedtail array, while the wild type conidia

The sequence of conidia

conidia

are produced in a sympodialarray.

formationand tie acrl mutant

In tie wild-typestrainIn a head-to*In acrl, appressoria
plant

penetration.

The reductions

mutant are not complemented
To investigate
subtraction

libraries

were subjected
subsequent

production

is greatly

in the appressoriumby the addition

formationfrom conidial

by Acrl9

glycogenghosphorylase

Expression

inrates in thethata nylon membrane arrayedanalysis.

The results

theexpressionof a Gl-cyclin(MGPH1 ) genes is positively

regulatedby Acr l.

patternindicateand penetration

mRNA of acrl and its isogenic

to a reverse Northern dot blot hybridization
RT-PCR analyses

are defectiveof exogenous CAMP.

the genes upregulated

constructed

reduced and the appressoriaof genes iip-regulated

by ACR1

wild-typewithstrainfrom this and(MGCl)

andStudygrisea)on durabilityof resistancein the Mekong delta

genes to blast

diseasePham Van Du and Le Cam Loan
Plant pathology Department, Cuu Long Delta Rice Research Institute,{Pyricularia

Co Do (O Mon) Can Tho

Email : Phamvandu@hcm.vnn. vn
Keywords : durability,

Pyricularia

grisea, resistancegenes,

Introduction
Rice blast caused by Pyricularia
rice diseases

grisea Sacc. (Rossman et al. 1990),

in Mekong delta of Vietnam.

dry season 02-03, 189,000of new releasing varieties
races using 129 blast

Infectedarea increased

is one of majorfrom 1999 to 2003. During

ha was infected in a total of 1.6 M ha of direct seeded rice crop. Most
were found to be susceptible
to the disease. Identificationof blast

isolatescollected

in many places of Vietnam (Noda et al. 1999)

12 pathogenic groups based on their virulencealso shown that none of isolates were virulent
Pikt Piz, Pita-2, Piz-t and Pik-p, therefore,

to 12 Japanese
to differentials
this study

indicateddifferential
rice varieties. It waswhich carries genes Pik-sf Pish,intended

to determine

which resistancegenes is more durable against blast over time and space. Study conducted using the same
Kiyosawa varieties and 3 1 monogenic lines under natural condition of blast nursery (Ou 1965).
Materials

and Methods
Blast nursery was laid out in 10 different

placesof Mekong delta which includes

Tho (CT), An Giang (AG), Tra Vinh (TV), Soc Trang (ST), Tien Giang (TG),Can

Kien Giang (KG),

Ca Mau (CM), Ben Tre (BT), Long An (LA), Dong Thap (DT) on two different seasons: wet
and dry season. Twelve Japanese Kiyosawa differential
varieties
and 31 monogenic lines
providedby Dr Fukuta

subceptibilityof varieties

under IRRI-Japanand lines

based on 9 scale of IRRI (SES
susceptible
Results
previously

which carry different1988):

releasedvarieties

nurseries

blast

project.

resistanceResistanceorgenes was scored(R) , 4-6 was moderate(S).

conducted

were susceptible

and cultivatedresearch

Score from 0-3 was resistance(MS) and 7-9 was susceptible
and Discussion
Results from blast

collaborativein largesince

to blast,

1980-2004

indicatedsome of them are still

durableresistancevarieties

are overcome by blast pathogen with average of 1-2 years after releasedthatmost ofconsideredasscale for more than 15 years. Nowaday, resistant(Table1).

We found that in one locationfrom resistantblast

to susceptible

for almostof timestested,reactionand vice versa. Some resistancegenes could express resistancesuch as Pi-km (Kanto-51),

Pish(PiNo.4),

Piz-t (Toride1), Pik-p (K60)(Tablelocationsthen reaction

was also different,

resistancesuch as Piz, Pish (Fukunishiki),susceptible)(Table 3).
Some interchanges

Piz-t

Pia (Aichi

susceptible),P-ik, Pi-sh (Kusabue,

Pish (PiNo.4,

susceptible).

Asahi,

Kiyosawa's

reaction

inoculation

test with 540 isolates

gene pyramiding

was conductedin 10

genes found in almost 10 locations

testedWhen observation

1), Pik-p

20 % suceptible),of 3 1 monogenic

(K60)

Pii,

and Pik-m (Tsuyuake,10 %such as Pi-s, Pish (Shin2, 40 %

Pik-s (Ishikari

30 % ofPi-ta (Yashiro-mochi,

Pi-b, Pi-sh (BL1,such as P ik-m (Tsuyuake),

Recently,

Pish

2).

(Fukunishiki)

Shiroke,

40 % susceptible),

20 % susceptible),

genes have been identified
Piz, Pish (Fukunishiki),differential,

toPita-2f

70 % of susceptible),

20 % susceptible),

However, using

Piz;

and susceptibility

In this study some blast resistanceto blast pathogens(K60).

(Torideof resistancesusceptible),
Pita-2,can be changed over time (8 seasons)Pi-t (K59,

to be more durability

Piz-t (Toride

1) and Pik-p

some varieties

carry two resistanceinfection(multilocationlines by natural

have been conducted(data

is going to be set up for blast resistancebreeding

Table 1 : Reaction of promising lines and varieties to blast (Pyricularia
at OMon,Can tho from 1980-2004

grisea)of blast

not shown here).

genes
test)

and

In near future

program at CLRRI.
scored50 %Table 2 : Reaction of Kiyosawa differential

varieties

with different

resistancegenes at Can Tho, fromdry season 2000 to wet season 2003

Table 3 : Reaction of Kiyosawa differential
genes at 10 different

varieties with resistanceplaces in Mekong delta, wet season 2003

References
Kiyosawa,

S. (1978)Identificationof blast

resistancegenes in some rice varieties.

Japan. J.

Breed. 28: 287-296.
Ou. S.H. (1965)

A proposal

for an international

program of research

The Rice Blast Disease.

The Johns Hopkins

Press, Baltimore,on the rice blast disease.

Maryland,pp. 441-446.

In

Rossman, A.Y., Howard, R.Y. and Valent,
the rice blast disease
Sasaki,R. (1922)

fungus, Mycology

Existence

B. (1990)

Pyricularia

grisea, the correctname forfungus (in Japanese).

J. Plant Disease

and Pests

82: 509-5 12.

of races in rice blast

9:631-644.

Standard Evaluation
System For Rice (1988)
International
International
Rice Research Institute. 3rd June 1988.
Takahito

Noda, Nagao Hayashi

Distribution
65:526-530.

of pathogenic

Rice

Testing

Program.

The,Pham Van Du, Hoang Dinh Dinh and Lai van E (1999)

races of rice blast fungus in Vietnam. Ann. Phythopathol.

Soc.Jpn.

Current status of asparagus
Baharuddin

Saileddiseasesin Southeast

Siti Nurdijati* , Fachri

Djas^,

Sciences,

Universiti

Sains Malaysia

Asia (SEA)

Pangeran InsanuP, Kasmal^

and Lahmuddin^
1 Schoolof Biological^Department

of Plant Pests and Diseases,

Medan, Indonesia.
department of Agriculture,

Faculty(USM),of Agriculture,

1 1 800 Penang, Malaysia.
North Sumatera University,

Bandar Seri Begawan, Brunei Darussalam

E-mail sallehb@usm.my
Keywords: asparagus, diseases,

Southeast

Asia

Introduction
Asparagus (Asparagus
and quickly

became preferred

Malang (Indonesia)

officinalis) was introduced
vegetable.

Shootsand Malacca (Malaysia)

canning factories have ceased operation

SEA are facing serious problems,Materials

and Methods

that produced
especiallythem for canning in 1980s.
supplyby diseasesof shoots.
(Salleh

A continuous survey was conducted on 24 farms in Malaysia,
Brunei Darussalam for the last 15 years (Table
symptoms were collected1984).

Pathogenicity

fungal isolates

representative

rust species i.e. Puccinia

asparagi, obtained

from naturally

on healthy

asparagus seedlings

varieties

10 month-old

asparagus

varieties

planteddiseased

in SEA(Sallehof every species,

Thailand,andor disease& Sulaiman
except one

asparagus were carried out

Mary Washington

the greenhouse at the SBS, USM.
Table 1: Nineteen

et al. 1996).

Indonesia,

and identified

The

All plantations

1). Plant parts showing abnormalities

and the causal organisms isolated

tests of selected(Anon. 1985)

are consumed fresh, except a few farms indue to insufficientthroughout

into SEA in 1950s(MW) and UC 157 in

Results

and Conclusion
Our survey showed that fungal

caused by bacteria
regions

and viruses.

were recorded{Fusarium proliferatum)1),

anthracnose

Phomopsis

blight(Phytophthora

diseases

Nineteen

were more frequent and serious than those

introduced

varieties

from the farms. The most destructive(Salleh1990),

followedby wilts

from subtropicaldiseasecapsici),brown rot (Curvularia{Phomopsis

asparagi),

stem canker(Alternaria

tenuissima)vesicarium),and Cercospora

rust (Puccinia

gray mold (shootblight(Fusarium

spp.)

crown spot

(Botrytis

cinerea),(Cercospora

asparagi).

diseases

were mosaic, crinkles

bacteria

was slimy rot {Pseudomonas solanacearum).

reported

fromthis region.

(Sallehspp.),

asparagi),

blight)and witches

was crown and root rot(F. oxysporum f. sp. asparagi){Colletotrichum
megasperma\

and temperate

The three

et al.

1996),

Phytophthorarotand shootpurple(Fig.

die-back

spot (Stemphylium

most importantviraldiseasebybroom. The most prevalent

Most of these diseases

causedhave not been

References
Anon. (1985)

Tanaman Asparagus Tanah Rendah. Risalah

Semenanjung Malaysia,

PertanianBil. 55. Jabatan

Pertanian

Kuala Lumpur. 27pp.

Salleh, B. (1990)
Crown rot caused by Fusarium proliferatum, a new disease of asparagus in
Malaysia. In: Proceedingsof the 3r(* International
Conference on Plant Protectionin theTropics,
Salleh,Genting Highlands.

B., Safinat,A., Julia,

newdisease of asparagus.
Salleh,343.
L. and Teo, C.H. (1996)
Biotropia

B. and Sulaiman, B. (1984)

Journal of Plant Protection

Brown spot caused by Curvularia

spp., a9: 26-37.
Fusariain the Tropics

associated1 : 47-53.

with naturally

diseasedplantsin Penang.

Table 2: Asparagus

sampling

plots in SEA

Table 3: Diseasesof asparagus

Taxonomic and phylogenic
study on Bipolaris
their productivityof ophiobolins

fungi from Japan andTakao Tsukiboshi1.

Wen Hsin Chung2 and Shigenobu

Yoshida2

National

Instituteof Floricultural

Fujimoto

2National

Institutefor Agro-Environmental

Ibaraki

305-8602,

Science(NIFS),

Sciences

2-1, Tsukuba, Ibaraki,(NIAES),

KannondaiJapan

3-1-3, Tsukuba,

Japan

Email : tuki@affrc.gojp
Keywords:

Cochliobolus,

grasses,host non-specific toxin, leaf spot

Introduction
The genus Bipolaris
Helminthosporium)(teleomorph:

is an important

Cochliobolus

plant

pathogenic

fungus causing

many kinds of gramineous plants and crops. Recently,that Bipolaris

is close

Exserohilum

and Drechslera,study, the Japanesethe productivity
Materials

to Curvularia

isolates

molecularof Bipolaris

were classified

phylogeneticon

analyses

showeddifferent

fromet al. 1999).

In thisand phylogenically

with

clade

genera (Berbeetaxonomicallysynonym:leaf spot and blightand both formed a phylogenetic

the other 'Helminthosporium7

of ophiobolins,

host non-specific toxin of sesterterpene.

and Methods
Collection

and phylogenic

analysis:

We collected

fromsouthern area and examined them taxonomically
DNA was extracted from each isolate
S rDNA were amplified
sequenced. Phylogenic
Productivity
extracted

and Pseudocochliobolus,of the genus mainly

under a light microscope.

and used as a templatewith the PCR conditions
analysis

153 isolates

Whole genomic

for PCR. The ITS regions

using a primer pair of ITS1

and 5.8

and ITS4 andwas made by NJ method.

of ophiobolins:

CM (completewith ethyl acetate. The extracted

media)

dried materials

liquidcultureswere

were extended on TLC with standardof ophiobolins

by a 1 : 1 solventof ethyl acetate and benzene. The Rfvalueof ophiobolins

were determinedby spraying

vanillineof the isolates

in sulfic

acid followed

and color of the spots
by heating.

Results

and Discussion(Petch)We described a new species, Co. heveicola Tsukib. & W. H. Chung [=B. heveaeArx], causing brown stripe on bermudagrass and Zoysia grass (Fig.-®, Tsukiboshi
et al.

2004).
Four groups of Bipolaris
spp. were supposed to be new species based on theirmorphology and phylogeny ((D(3)®(D).
Cochliobolus
peregianensis
was newly recorded inJapan causing eye spot, a new disease,of bermudagrass(®).

The other four species

were alsothought

to be new to Japan. rDNA-

ITS regions of Co. nodulosus and Co
setariae at first sequenced (®,(D).
Cochliobolus

sativus was found tocause a new leaf spot of orchardgrass( ® ). In resultsof phylogenic
analysis,bethe Bipolarisclassified

producing
reported

into

isolates

could

the

groupsshort or long conidia
before (Berbeeas

et ah 1999).

The groups were geneticallydistant

to each other with 84% bootstrap
value in a MP tree reflecting theirdifferencein theteleomorph,conidia)(shortmorphology

Cochliobolus
andconidia(long

Pseudocochliobolus

conidia).
All the species

ophiobolins

ofbelonged
Bipolaris

producingto the long-group

and wereclassified

in closecladesin thephylogenic

tree.

They

producecurved and spindle-shaped

conidia

and were supposed to be geneticallydistant

from Bipolahs

producingstraight

and clavate conidiathat never produced ophiobolins.

The species of the short conidia group never produced ophiobolins.
References
Berbee, M. L., Pirseyedi
of known, highly
dehydrogenase
Tsukiboshi,{Bipolarispress).

M.and Hubbard, S. (1999)

virulent

pathogens,gene sequences.

inferred

Mycologia

T., Chung, W. H. and Yoshida,
heveae)Cochliobolus

phylogenetics

from ITS and glyceraldehydes-3

and the origin-phosphate

9 1 : 964-977.
S. (2004)

Cochliobolus

causes brown stripe of bermudagrassheveicola

sp. nov.

and Zoysia grass. Mycoscience

45 (inStudies on phytopathological,
morphologicalof Plectosporium tabacinum in Japan
Toyozo Sato \ Jun Takeuchi

2, Hideyuki

and molecularNagao * and Keisuke Tomioka *1 Genebank, National

Institute305-8602, Japan
2 Tokyo Metropolitan

Agricultural

Experiment

Station,Keywords : fungi, host specificity,

identification,

plant pathogen,

190-0013,

Japan

Email:

s l 043@afifrc.go.jp

Introduction
which was validly

of Agrobiological

Sciences,

Kannondai 2-1 -2, Tsukuba, Ibaraki
Fujimi-cho

3-8-1 , Tachikawa,Tokyo

taxonomy,

Plectosporium tabacinum (van Beyma) M.E. Palm, W.Gams & Nirenberg,
described
in 1995 (Palm et al. 1995), had been known as a commonsoil

fungus at rhizospheres,and Ito 1975).

variations

and was isolated

We obtained

found that it contained

from lake sediment for the first time in Japan (Tubaki

several isolates

variousstrainsof the fungus pathogenic

phytopathologically,using the isolates.

We present the details

Phytopathologicalvariation

to plants

morphologicallyin Japan, andand molecularly,

here.

Pumpkin (7 cultivars),garden ranunculus (1 cultivar)

andlotus ginger (1 cultivar)
were inoculated with 10 isolates (Table 1). Conidia of each isolatethat had formed in culture on potato dextrose agar (PDA) at 25°C in the dark for 2 weeks weresuspended in sterilized
distilledwater at 5 x 105 conidia/ml to be sprayed onto 2-4 healthy plants
per cultivar.
Healthy plants sprayed with sterilized
distilledwater served as controls.
treated plants were covered with polyethylenebags and kept in a greenhouse at 22-25°C.
bags were removed after 2-3 days.
fromthe treated plants

to the originalhost

plants,but

theotherscausednodiseasein the plants

tested (Table

1).

was thoughtthat theIt

isolatesofP. tabaci-

num

had

specificity.

were performed.
Table 1.

host

Isolates

Observationof symptoms and re-isolation

As a result, the respective

used and their pathogerucity

isolates

to pumpkin, garden ranunculus and lotus ginger.

All
The

of the fungus

were virulent

onlyMorphologicaland molecular
as follows (Plate 2). Colonies

variations
In culture

Morphologicalcharacters of P. tabacinum areon PDA at 20°C in the dark are flat with little aerial

mycelium, smooth and cream to salmon or pale brown in color.

•Eoroccasionally
cells

often

vegetative

branched,arisingmonophialides

anglesConidlogenous

from

cells

formed at the apices,

short (adelophialides)

are
or as

or long blanches

hyphae,cylindricalto obclavate,

sometimes crooked or

sinuous

at

tips,

often

apertures,andoccasionallyConldlaare produced

conidiogenous

hyaline,fromvegetative

thewith second apertures.
In colorlessphialides,
usuallyslime

myceliawithsingleof thesmooth, oblong-ellipsoidal,

asymmetrical to slightly

are 1-septatesmooth,masses at the tips

hyaline,are unbranchedwith conidiogenous

at right

hyphae,Conidiophores

curved, multiguttlate

and most

and witha few aseptate.

Growth speed

and appearance

rates of aseptate

conidiaofon PDA at

20°C in the dark varied from 2.7-4.7 mm/day and 0-28.6%,
respectively,
conidia

in 6-8 isolates.

Sizes

of septate and aseptatethat had formed on synthetic

low nutrient agar (SNA)um

at 20°C in the dark ranged from 4.0-12.0x1.0-5.5

um5

2.5-8.5x 1.0-3.5
Ten isolates

respectively,

were also classified

depending(>94%)

the DDBJ (Fig.

1).

on the isolates.

into 2 groups based on theirsequence data of rDNA ITS regions,were highest

andthoughtheir homologies

with those of P tabacinum registeredThus3 P tabacinum

contain various strains morphologicallyin

was also found toand molecularly.

References
Palm, M.E., Gams, W. and Niraiberg, H.I.
(1995)
Plectosporium,a new genus for
Fusarium tabacinum, the anamorph of
Plectosphaerella
cucumerina. Mycologia
87: 397-406.
TiibaM, K. and Ito, T. (1975)
Descriptive
catalogueof IFO fimgus collectionIV.
IFO Res. Comm.7: 113-142.

Fig
1. Groupingof P. tdhamnum isolates
basedonneighbor-joining
analysisfor sequence of iDNA ITS regions
ITSIF(5«-CTTGGTCATTTAGAGGAAGTAA»3')
and ITS4(S'-TCCTCCGCTTATTGATAT
GC~3?) were used as pnmers for
PCR. Numbers above branches are bootstrap values (%)

Morphology(Teleomorph
Jouji

and molecular taxonomy of Colletotrichum: Glomerella glycines) and related species

Moriwaki1. Toyozo Sato2, Takao Tsukiboshi3,

1 National

Agricultural

2 National

InstituteofAgrobiological

3 National

Institutefor Agro-Environmental

Masako Noguchi1

Research Center, Joetsu, Niigata
Sciences,

destructivum

and Kazuyuki Hirayae1

943-0 1 93, Japan

Tsukuba, Ibaraki

305-8602,

Japan

Sciences,

Tsukuba, Ibaraki

305-8603,

Japan

C. linicola,

C juscum, rDNA sequences

Email : moriwaki@aflfrc.go.jp
Keywords:

Colletotrichum

higginsianum,

Introduction
Colletotrichum

destructivum

Wolf),

Sacc, C. linicola

C. higginsianum

morphologicalcharacters,(Sutton1992).

1980,

morphological

O'gara (Teleomorph:e.g. conidia

Pethybr.

and molecularMorphology
They produced

glycines

Lehman && Lafferty and C. fuscum Laub. have similar

and appressoria,

We think those species

Glomerella

however their pathogenicity

as C. destructivum

are different

sensu lato on the basis of thecharacterization.

conidiastraight

or slightly

curved,fusiformto cylindrical,

taperedtoeach end, 12.4-22.2 x 2.3-4.8 jam and formed appressoriaclavate to obovate, dark brown,
6.5-12.9 x 4.1-7.9 ^im on PCA slide culture at 25°C under black light.
Colonies on PDA,
orange, pale orange, salmon pink, brown to sepia brown or rosy buff, with white felted
mycelia.

Conidia

formed on acervuli

or on stromata covered with hyphaeaerialand with sparsesetae.
Molecular

taxonomy
The intraspecificof C. destructivum

DNA homologies

of rDNA ITS2 and 28S rDNA domain2 sequences

sensu lato were 98.9 to 100%, but interspecifically

94.8 to 96.8% with C.

acutatum Simmonds ex Simmonds, C. coccodes (Wallr.)

S. Hughes and G cingulata

Spauld.

using& von Schrenk.

In phylogenetic

analysisneighbor-joining

examined strains of C. destructivum

sensu lato made a clade with 92% bootstrap

Thus C. destructivum

speciesand the related

are the same species.

(Stonem.)method,thevalue (Fig.

1).

Pathogenicity
Resultsofinocu lation
showedexperiments
hostspecificity

some of the isolates
species.

Althoughofin thethe isolates

from cruciferand from flax

were virulent

only

to their

host plants,

they didn't

cause

any diseasesof other plants.

Colletotrichum

destructivum

from legumes also
host plants
spots

attacked

and made small

on theseedlings

ofcruciferand flax.

Someisolatesof the species

werefound to be host specific,

and

the

othersappeared

to beomnivorous pathogens.

Fig. 1. Tree illustrating
relatednessof Colletotrichum species, based onneighbor-joining
analysis of the ITS2 and 28S rDNA D2 regions.
Percentages of neighbor-joining
analysis of 1 000 bootstrapped
datasets that support specific branches are indicated at the respective
nodes. Bootstrap values greater than 90% are shown. Bar-distancecorresponding to one base changes per 1 00 nucleotide positions.

References
Sutton, B.C. (1980)

The Coelomycetes.

Commonwealth Mycological
Sutton, B.C. (1992)
Jeger, M.J. (eds)
Wallingford,

Fungi imperfecti

Institute,with pycnidia,

acervuli

Kew, pp 523-537.

The genus Glomerella

and its anamorph Colletotrichum.

Colletotrichum:

pathology

pp 1-26.

biology,and stromata.

In: Bailey,and control. CAB International,

J.A.,

Pathogenicity
1of some Colletotrichum

speciesto petalsof Antirrhinum

majus
Keisuke Tomioka \ Jouji
1 Genebank,

National

Moriwaki 2 and Toyozo Sato

Instituteof Agrobiological305-8602,
Japan
2 Hokeriku Department of Lowland
1-2-1, Joetsu, Niigata

943-0193,

Sciences,

Farming,National

Kannondai 2- 1=2, Tsukuba, IbarakI
Agricultural

ResearchCenter,Inada

majus, scrophulariaceous(Nanbu, 1916; Kobayashi

plants)et al.,

Japan

Email : tomloka@affic.gojp

Keywords:

anihracnose,

flower disease,fungi, host range, plant pathogen,Introduction
Anthracnose of snapdragon (Antirrhinum
was reported to be caused by Colletotrichum
gloeosporioides
1992).

However, no details

on the Identification

as Its symptoms ha¥e been described,

and no Isolates

Moriwakl et al. 2002,

2003).

of the causal fungus as wellof the pathogenha¥e been preserved.

We

only to petalsof the plant (Tomloka

et al.

In this study, the pathogenicityof C. gloeosporfoides,

C.

reported previously that C. destructivum
2000;

and pathogenicity

was virulent

acutatum, C. dematium and C. circinans to the plant was Investigated.
Materials
Table 1.

and Methods
Conldlal suspension

plantssprayedwithsterilized

distilledwater

were served as controls.
All plants

were kept andobserved

in

a

green

house at 24^28°C

Re^

isolationof theftmgi

from the treatedplantswas also

performed.

The experiments

werereplicatedtwiceplantsat both the seed»ling

and the flowering

stages.

for

Table

Snapdragon was Inoculated with each fimgal Isolate(ix lQ4^5 conidia/ml)
was sprayed onto healthy plants.
1

Colletotnchum

isolatesused.

listed In
HealthyTsukibosM,

Results

Not only C. destructivum but also C gloeosporioides,

and C circinans

were found to be pathogenic

floweringstage were inoculatedwith the isolates,

only (Fig.

1).

irregularly

diameter

Water-soaked,

to snapdragon.

When healthy

plantsthey developed

brown blotcheson their petals

shaped blotches

appeared on petals 3-4 days after inoculationand coalesced,resultingin early blight

Control

plantsdemonstrating

had no symptoms.
that the isolates

Fie 1

Petal blieht

Discussion

petals,Neitherstage.

of snapdragon

Neithercaused bv inoculation

and/ornecrotic2).

The lesions

enlargedby 10 days after inoculation.

Eachfrom the control

to snapdragon.

No isolate produced

fungus was re-isolated

fromthe plants.

on leaves and/or stems were noted to result in

early

death

diseased(Nanbuhere differ from those described1916).

Symptoms

In the report.

But, thisstudy supports the presence of the disease by C gloeosporioides,
indicatesthat

not only

C. gloeosporioides

and

C.

destructivum

but also C acutatum, C. dematium and C

circinanscan.

Colletotrichum
following
the

other

correspondtoclassified

into

the

two groups:

one produces

curved conidia,

andgloeosporioides,

straight

conidia.

Colletotrichum

are the former,

and C

C. destructivum and C. acutatum are

the latter.

Colletotrichum

snapdragon

and other scrophulariaceous

may be generally

are important as ornamental plants
speciespathogen.

are broadly

dematium and C circinansinterestingits

speciesforms

whether the virulenceoccur only in petals

or not.

symptoms

anthracnose,that appeared

Identified

plants,with the Colletotrichum isolates

lesions

plantsspots 0.5-1 mminbut not from stems and leaves of the plants.

In the first report for snapdragonof theat thegraduallyfungus was re-isolated

were pathogenic

on the plants at the seedling(Fig.

of whole petals

fungus was re-isolated from the diseased

C acutatum, C. demattium

virulenttoplants,which

in agriculture.

It isof other Colletotrichum

andFig. 2 Lesions on a petal of
snapdragon inoculated
withC. gloeosporioides.

References
Kobayashi,T., Katumoto, K., Abiko, K., Abe, Y.
and KakisMma, M. (1992).
Illustrated
Generaof Plant Pathogenic
Fungi in Japan. Zenkoku
Noson Kyoiku Kyokai, Tokyo, 685 p.
Moriwaki, J.,
T. and Sato, T. (2002)
Grouping of Colletotrichum
species In Japan
based on rDNA sequences.
J. Gen. Plant
Pathol. 68: 307-320.
Morlwaki,J., Sato, I., TsuMboshi,
T. andNakajima, T. (2003)
A taxonomic revision ofColletotrichum
destructivum
and Its closely
related species and their pathogenicities.
Jpn. J.
Phytopathol.
69: 259.
Nanbu, N. (1916).
Anthracnoseof snapdragon.
Byotyegal zasshi 3: 419.
Tomloka, K., Nakamura, H. and Sato, T. (2000)
Pathogenicltyof Colletotrichum
coccodes andC. fiiscum to snapdragon (Antirrhinum
majus).
Proceedings of 7th International
symposium of
the mycologlcal
society of Japan p. 63.

Virulenceof Fusicoccum aesculi3 Phomopsis phomoides,and Stemphylium lycopersici to sweet pepper fruits

lateritium

Fusarium

Keisuke Tomioka and Toyozo Sato
Genebank,

National

305-8602,

Japan

Instituteof Agrobiological

Sciences,

Kannondal

2-1-2, Tsukuba,

Email : tomloka@affrc.go.jp

Keywords : fungi,

Identification,

new disease, plant pathogen,Introduction

Four postharvest

diseasesofsweet pepper (Capcicum annuumvar. grossum)
were frequently
Japan,

found In Kagawa Prefecture,in 1999-2001.

We repeatedly

anamorphic

fungifrom theIdentified

them

as

Isolateddiseased

fruits,

Fusicoccum

aesculi,

Phomopsis phomoides,

Fusarium lateritium

andStemphylium

lycopersici

and demonstrated

theirpathogenicity

to sweet pepper.

details

We present thehere.

Symptoms

Diseasescaused by E aesculi,

E phomoides and S. lycopersici
ripe fruits(Plateslateritium

was found on non-ripe fruits (Plate

The diseases

1, 3),

were found on

and disease

by E aesculi

were recognizedof the two fungi

diseases,

water-soaked

lesionsappearedenlargedand softened, resulting

aesculiand

Initially.

Elateritium

conidlophores

andnecrotlc
gradually

In entire

rot ofIn the fruits diseasedby itphomoides,
and

or
They

appeared to exude conldialFby mixed

In all four of thelesions

the affected fruits.

2).

and E phomoides

on fruits diseased

Infection

by E

their

masses.

pycnldiaIn those byS.

lycopersici,

theirconldlaappeared.

The

postharvest

diseaseIbarakiisolates of each fungus produced
dextrose agar (PDA) or synthetic

conidla, which were as same as those on the lesions, on potato
low nutrient agar (SNA) at 25°C under a black light (BLB)9

and were Identified

as the fungal

speciesA

suspension of
was dripped

basedconidiogenesis on
conidial

Pathogenlclty
each Isolate(Ix lG4"6 conidia/ml)

onto the surface of healthy

fruits, or InjectedIntohealthy

fruitswith

fruits.

Healthyinsterilized distilled
fruits were kept

conidialTheF

seedlings.

with sterilizedF

kurata,Studiesgreen
Inst

was also sprayed

Healthy

distilledseedlings

onto

sprayedwater served as controls.

All seedlings
were kept in a green house at
24-30cC.
As a result3 the natural symptoms
were reproduced
Isolate.

Paescult,
Flateritium

water served as controls.
Allmoist condition at 24-28°C.

suspension

healthytreatedsuccessfulbyinoculation

with

each

The reproduction

by the Isolatesofphomoidesand S. lycopersici

wason ripe fruits but not on non-ripe

fruits.

That ofwas successfulonboth ripe and non-ripe fruits.

Control fruits had

no symptom.

Each fungus

was re-isolated

fromthe diseased

fruits, but not fromthe control

fruits,demonstrating

that

the

isolateto sweet pepper

fruits.

No isolate

produced any symptom on seedlings,

and neither

pathogenic

fungus was re-IsolatedDisease

name

every

specieslateritium.

wasfromthem.
Sweet pepperofThe diseasealso reported previously,

aesculi,

is a new host forP. phomoides

by S. lycopersici
thoughandhad not beenleaf spot of the plantcaused by the fungus has been noted (Saito et al. 1968,1970).

We coined the term "fruit rot" to refer to all

four of the diseases.

Reference s
Saito, M., Kurata, M. and Yamamoto,I.
(1968)A new disease of sweet
pepper caused by Stemphylium sp.,white leaf spot. Ann. Phytopath.
Soc. Japan 34: 182-183.
Saito, M,
M and Yamamoto,I.
(1970)
on Stemphylium
leaf spot of
pepper. Bull
Kochi
Agr. & Sci. 3: 1-8.

Saccharomyces cerevisiae genome-wide mutant screen for antifungal
activities
of yeasts, Williopsis
mrakii, Kluyveromyces lactis and S.
cerevisiae
Hiroko Kuze Kitamoto
National

Instituteof Agrobiological

Sciences

2-1-2 Kannondai,

Tsukuba,

Ibaraki

305-8602Japan
E-mail : kitamoto@aflfrc.go.jp
Keywords:

chitin,glucan, killer

Introduction
Antagonistic
quite highinteractionsin natural habitat.

screened a collection
alteredsensitivity

The sequenced

againstsensitivity
2003),

To know the molecularpurifiedand analyzed

proteins

S. cerevisiaedeleted(Winzelera collectionofet al. 1999).

Suchroles of genes, but also the mechanismof each killer

to WmKT and K1KP with the results

weyeast genes, forto constructgene disruptionof not only cellular

To know the characteristic

for 4901

proteins,of W.mrakii (WmKT) and K. lactis (K1KP).

genome has enabledin each gene by targeted

toxins appear to bemechanisms of yeast killer

mutants, individually

killerpromotes the discovery

of toxin action.

RNA polymerase

between yeasts by secreted proteinaceousof S. cerevisiae

mutant strains deficienta collection

protein,action,we compared the resultsof S. cerevisiae

Kl killer

proteinkillerwas concentrated

(Page

of

et al.

respectively.

Materials

and Methods
Purificationof killer

proteins:

from 1.3 L of YPD culture broth
chromatography
purified

as described

K. lactisof K. lactisin (Sugisaki

IFO1267

et al

proteinand purified

to 13ml by hydroxyapatite

1984).

Killerproteincolumnof W. mrakii wasfrom 50 ml of YNB culture broth to 3.5 ml by Macro Prep High S support

column(BIORAD).
Analysis
inoculatedbottomedof sensitivity

to killer

protein:Approximately

1 x 103 yeast mutant cells werein 60 yl of 1M sorbitol-YPD liquid medium with diluted K1KP and WmKTin round
96 well microplates,
respectively.
The growth of mutants at 30°C was obsreved for3days.
Results

and Discussion
The yeast cell wall is made principallyof four components:

mannoproteins,chitin,antifungal

p-l?3-glucan

and p-l,6-glucan.

-1,6-glucan

and chitin,-1,3-glucan

synthase

gene disruptants
cells resistantCellrespectively.

ofS. cerevisiae

ofN-glycosylation

receptors(Kimura el al 1997).
of olygosaccharide

to Kl and WmKT,respectively.
of chitin.

of Kl and K1KP are reported

And WmKT was reported

As glucan mutants accumulate chitin,to the increase

wall

Killerdeletion

could easily

actions, which is obtained

References
Kimura el al (1997)
139-147.
Page et al. (2003)

linear DNA plasmidWinzeler et al (1999)
and parallel

analysis.

to K1KP.

the specificIIcell wallmechanisms involved

during the course of its evolution.

toxin that inhibits

A Saccharomyces

sensitivity
to Kl killer
Sugisaki, Y. et al (1984)

Therefore,

be one of the potential

A novel yeast gene, RHK1, is involved

receptor for the HM-1 killerpmake the yeastof the gene for RNA polymerase

structureinwallof the mutants may be attributed

to HMK, K1KP and Kl.

machinery

to protein

However, these mutants are sensitivemachinery makes yeast cells resistantand transcription

cell

spectrum ofWmKT on theand its transfer

the increase in sensitivity

On the other side,

to inhibit

as ptoxin. Genetics
Charecterization

p-l,3-glucan

cerevisiaein the synthesis
synthesis.

of the cell wall

Mol. Gen. Genet. 254:

genome-wide mutant screen for altered

163: 875-894.
of a novel killer

toxin encoded by a double-stranded

ofKluyveromyces lactis. Eur. J. Biochem. 141, 241-245.
Functional characterizationof the S. cerevisiae genome by gene deletion
Science285:

901-906.

Isolationand characterizationin northern part of Vietnam
Yasuhiro Inatsu and Shinichi
Food Hygiene
Ibarakiin fermented

vegetables

sold

Kawamoto

Team, National

305-8602,

of bacteria

Food Research Institute(NFRI),

Kannondai 2-1-12,

Tsukuba,Japan

Email : inatu@affrc.go.jp
Keywords: bacteriocin,

coliform bacteria, lactic acid bacteria

Introduction
Fermented vegetables

are an integral

part of the diet of people in many countries. The

organic acids or other metabolites

produced by lactic acid bacteria during fermentation

give the raw vegetables

desirable

taste, flavor or texture. In addition,

substanceswith vitamins,

biologicallyorganic acids or bacteriocins
pathogenicor spoilage

proteins,The

may suppress the growth ofor Japanese fermented vegetables

In high salt concentration,

low water activity

and high ionicactivity

are influencedby

the growth of bacteria tends to be repressedbyin general.

of undesirable

bacteria without

However, not many reports related

to the fermentation

fermented (salted)

vegetables

microfloraof lightly

fermented vegetables

Materials

and Methods
The lightly

acids.

bacteria.

salt concentration.

the growthenriches foodamino acids and fatty

produced by long period fermentation

The micro floras of Sauerkrauts

suppressessential

fermentation

processare published.

fermented

In the suitableaffectingconcentration,

salt willthat of lactic

acid bacteria.

bacteria in the South-East

Asian lightly

So we investigatedabout the chemical

propertyin the northern part of Vietnam.

vegetables

sold in Hanoi city were collectedin March 2003.

Each ten grams of samples were stomached with 90 grams of phosphatebuffered

In case of the measurement of pH or salt concentration,water for stomaching

insteadof using PBS. One milliliterorof solution

we used distilled

was decimallyserially

dilutedsaline (PBS).
and used forenumeration according to the standard method.
The coliform

bacteria grown in the desoxicolate

agar plate were purified and identified

by api20

kit followedby biochemical

The lactic

acid bacteria

identified

by using

production

by LAB was assayed by agar welldiffusionassay using Lactobacillus

api50

tests.

kit. Bacteriocin

The nisA genes in the bacteriocin

plantrum or Listeria
producing(LAB)

were similarly

monocytogenes as an indicatorstrains were confirmed by PCR method.

strain.

Bacteriocinkuipers,

Results

and Discussion
Salt concentrations,salt concentrations

pH and micro flora of 5 kinds of 37 samples

tend to be lower than that of Japanese lightly

samples did not ferment enough to increase the acidity.
5 to 8 log CFU/g.

Most of them were lactic

were analyzed. The

fermented

vegetables

and some

The aerobic plate count varied between

acid bacteria(LAB),

including

Lactobacillus,

Leuconostoc, Lactococcus and Pediococcus.

Two to five log CFU/g of coliform

detected;

to the known natural flora on fresh vegetables,

all of Identified

however.
isolatedstrains

were similar

producingstrains

effective for L. monocytogenes were screened from the

200 LABs. Three Lactococcus lactis subsp. lactis strains producing

The DNA sequence of nisA gene (structural
same as that of previously

bacteria also

nisin were obtained.

gene of nisin A) of each strain was completely

reported one. One of the Isolatedacid was found to produce antimicrobial

theBacillus

subtilis

strain producing

compound(s)againstGram-positive L.

monocytogenes.

( i)

Nlsk

A producing strain,(C)

Control,(2) Nlsin(H) Heat treatment,

Z producing(P) Proteasestrain,(3) NFR17426,(4) NFRI7427

of lactic

bacteria

treatment

References
Merita,H. ei al. (1990)

Jap.J.Dairy

Isolationand Identification

acidfrom pickles.

Food Sci 39, 183493.

Nakagawa, H. et al. (2001)
Food Microbiol.

Lacticacid bacteria

flora Isolated

from salted

vegetables.

Jpn. J.

of the nisin

gene

nisABTCTPR

18, 61-66.

O.P. et al.

(1993)

Characterization

Lactococcus

lactis. Requirementof Immunity.

Eur. J. Blochem.

of expression
216,

281-291.

clusterof the nisA and nisi genes for developmentofA newmethod to obtain plasmid

variants

from Lactococcus lactis

Miho KobayashL Masaru Nomuraand Hiromi Kimoto-Nira
National

Instituteof Livestockand Grassland

Science, Norin-danch,

Tsukuba, Ibaraki

305-090

1,

Japan
Email : mihokoba@affrc.

gojp

Keywords : incompatibility,

plasmid

curingIntroduction
Lactococcal

strains

generally

carry a number of theta (O)-replicatingplasmids

which

are essential for fermentation or remain cryptic. Plasmid curing technique was performed toinvestigate
the diverse propertiesof plasmids, and the treatment was currently performed byculturingwithmutagenic

exposureof cells

chemicalto elevatedas acridine

orange,growth temperatures,

culturing

regenerationin unbufferedof bacterial

medium,protoplastorcomposite those methods. However, plasmids can not be chosen for excluding by these current
methods. Moreover, there is a great risk of mutation that spoils property good for fermentationby the aforesaid
selectively

treatmentsfor plasmid

exclude a residentG-plasmid

damages and to obtain the plasmid
Materials

and Methods
Bacterial strains,

vector pBluescript

curing.

In this poster

we present

a new method to

fromLactococcus lactis without producing
variants

plasmids

any genomic

as starters for products.

and culture:

II were used for plasmid

E. coli XL1-Blue

construction.

N7, 527 and 712 were used for preparation(Stratagene)

Lactococcus lactis

of nativeG-plasmids

and a plasmid
ssp. lactis

and for bacterial

DRC 1,
hosts oftransformation. E. coli was grown on LB agar or LB medium at 37°C, supplemented with 50 (igampicillin(Ap) ml'1 when required. Lactococcus lactis were grown in TYG agar or TYG
medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride,
1% glucose and 1% sodium
succinate; pH 6.8) at 30°C, supplemented with 5 fig erythromycin (Em) ml"1 when required.
An outline
major in lactococci,of strategy

for the selective

was as follows:

exclusion(1) Constructionof universal

containing

variable

artificial

PCV(X)sbeing incompatible

to a resident

plasmid

pDBl.

(3)

of L. lactis

strainwith

FV(X)serythromycin
cultivation

into

resistantof native

Transformation(Emr)-gene.

(4)

in TYG-Em medium = Selective

9-plasmid

9-plasmids,

which arereceptor vector PDB l for PCR

fragment FV(X)replicons

regionof resident
replicon.

(2)

Construction

in lactococciwith

PCV(X)by cloning
carrying

Isolationof Emr-transformant.

(5)

exclusionof the resident(6) Isolation

plasmid.

ofContinuousofderivative

excluded

the resident

Em to exclude the PCV(X).

(8)

plasmid.

(7) Continuous

cultivation

Isolationof Em-sensitive

in TYG medium without

variant containing

no exogenousDNA.

The construction
detailin Results

electroporation.
Resultsof PCV(X)sand Discussion.

and plasmidTransformation

Emr-transformants were selected

and Discussion
Three sets of oligonucleotide

amplify

three parts(FC13

ofL. lactis

primers P1-P2,

FC2 and FV(X))

pDRl-1,are describedwith a PCV(X)was performed by

P3-P4 and P5-P6 were designedof major lactococcal

0-plasmid

were amplified

The middle part (FV(X))

that contained

purifiedby electrophoresis

inserted

into Nru I-Xho I site of pDBl,of PCV(X)scould

in 4 lactococci.
pCVm6)

Templatesfrom total plasmids

a variable region of each repliconartificial

pCVl

The Emr-isolates

pDBl,

which

of FV(X)swere

The FV(X)shybrid replicons
strains

Eightfrom resident

with those PCV(X)s,and pCV5, N7 carrying
were subcultured

could bePCV(X)s.

which were amplified

ofL lactis wild-type( DRC1 carrying

were isolated.

for PCR amplification

with FV(X)s,After transformationII, generatingin the above lactococci.

generating

be constructedfour kinds of transformants
carrying

The

with P5-P6. The FC1, FC2 and an Emr-gene were cloned one after another into

was used as a receptor vector for FV(X)s.

plasmids

replicons.

toby PCR from a lactococcal

Ban lll-Eco RI site, Pst l-Xba I site and Sac I site of pBluescript

kinds

in

which were fromL lactis DRC 1. The FC 1 and FC2 contained the conserved

region of 0-replicon.
was amplified

with PCV(X)son TYG-Emagar.

upstream part (FC 1) and the downstream part (FC2)0-plasmid

exclusionpCVc8 and 712

in TYG-Em media for

selective exclusion of the resident plasmid, which were incompatible to co-existing PCV(X).
Presence of the resident plasmid was examined by PCR analysis. After 100 generations, thevariants losing

the resident

plasmid

exclusions(pCVl,pCV5,

PCV(X),

the plasmid

generations,plasmidof wild-typewere appeared

lactococci

pCVc8 and pCVm6)

variants with PCV(X)each PCV(X)without

selective

starters

for food-productsin thispressure.

at 20 to 50% of a rate. The selectively

were ultimately

succeededwith above 4 PCV(x)smanipulation.

Finally,in order to exclude

were subcultured

was completely

excluded

The plasmid

variants produced

thein TYG media. After 100

because the artificial

replicon

was instableby this method are able to use forbecause they contain no exogenous DNAs.

Effect of plasmids of Lactococcus
murine macrophage cells
Hiromi Kimoto-Nira1.

Miho Kobayashi1,

strains

on cytokine

Koko Mizumachi1,

production

Jun-ichi

Kurisaki2,

fromand Takashi

Okamoto1
Departmentof Animal

Products,

National

Instituteof Livestockand Grassland

Science,

Tsukuba Norin-danchi, P.O. Box 5, Ibaraki 305-090 1, Japan
2 National Institute of Agrobiological
Sciences, 2-1-2, Kannondai,

Tsukuba, Ibaraki

305-8602,

that exhibita beneficial

Japan
Email : anne@affrc.go.jp
Keywords : probiotics,

plasmid-curing,

Introduction
Probiotics

are commonly defined

effecton the healthof the host when they are ingested(Lee and Salminen

effective

probiotic

properties

host immunity. However, only limited

information

is available

is theirimmunomodulatory activity

ability(Kitazawa

harbor various plasmids

utilized

microorganisms

to stimulate

on the probiotics

of such microorganisms

as viable

factors involved

et al. 1992;

focused on. In the present study, the effects

et al. 1996).

the immunologicalof a plasmid

One of thein the immunomodulatory

De Ambrosini

for food industry,1995).

Since

functionslactococciroles on the host are to bein lactococcion the cytokine

responseof a murine immunocompetent cell line are examined.
Materials

and Methods
The lactococcal

supplemented
dried.

strains

used in thisstudywith 0.5% glucose.

The cultures

were heated at 100°C

line

was obtained

A murine macrophage cell

Culture Collection).

(J774.1)

The cells were routinely

were grown in M17

broth(Difco)for 50 min and freeze

from ATCC (AmericanTypegrown at 37°C in a 5% CO2-95% air atmospherein

RPMI-1640 supplemented
with 10% inactivated(30 min, 56°C) fetal calf serum, 100 U/ml
penicillin,
100 ^ig/ml streptomycin and 5 x lO"5 M 2-mercaptoethanol. For the assay, J774. 1 cells
were seeded at 5 x 105/mlcells

into 24-welltissueculture48 hours. Then the medium was refreshed and the cells
lOOjxlof additional

stimulisuch as lactococcifor 24 hours. E coli LPS (Sigma,
J774. 1 cells.

After the culture,l|xg/ml)platesin 1 ml/wellwere further

incubatedor their cell wall fractions
was used as a positive

the supernatants

were harvested

and incubatedcontrolforwith or without( 10|xg/RPMI

medium)for the stimulationofand stored at -80°C for cytokine

assay by ELISA. Each experiment was conducted over two passages of the cells.

Results

and Discussion
The plasmid-cured

DRC1 (strain

DRC1021)variant
stimulated

times more than the wild-typebiovar diacetylactis
The

strain.

lactissubsp.

IL-12p40

and IL-6 production

In contrast,

plasmid-cured

lactisbiovardiacetylactis

by J774. 1 cells

variant ofL. lactis

nearlytwo

subsp. lactis

N7 did not show such kind of effects.

introduction

immunostimulatory
the cytokine

of Lactoocccus

of pDRSE5

activity

production

pDRSE5 or inherent

plasmids

cytokineproduction

were obtained

production

by the stimulation

rearranged the plasmid

composition

activityof lactococci

includingtheirsuggestcells.

results

prepared

It was reported

and changed the properties

that the plasmids

it is not clear at present

how the plasmid

and the insertion

suggests

to create immunologicallyfunctional

probiotics.

Acknowledgement
The authors thank to the late Dr. Yasuhito Fujita

for providing

plasmidS. (1995)

that some stresscellularstudy strongly

References
Lee, Y.K., and Salminen,

from strains
cells

would affect the immunostimulatory

cell wall. Although

plasmid-curing

in IL-6of the bacterialof the hosts'of cell wall, the present

such asMoreover, similarchanges in the properties

composition

theof host strains

via possible

and/or structural

reducedmodulate the ability

with the cell wall fractions

in bacteria

N7or curing of some plasmids

not the case for IL-12 production.

These results

andof those by the wild types in terms of

could apparently

by macrophage

while

DRC1021Thus, the insertionin lactococci

DRC1 and DRC1021,(Kim et al. 2001).

strains

to 1/8 and 1/4, respectively,

by J774 cells.

to stimulate

into

The coming of age ofprobiotics.

componentschanges chemical
the potentials

ofvariants.

Trends Food Sci. Technol.

6:241-245.
Kitazawa, H., Yamaguchi, T, and Itoh, T. (1992)
producedfrom slime-forming,

encapsulated

B-cellmitogenic

Lactococcus

lacitsactivityof slime products

ssp. cremoris. J Dairy

Sci.

75 :2946-2950.
De Ambrosini,
ChemicalV. M., Gonzalez, S., Perdigon,compositionof the cell

G, De Ruiz Holgado, A. P., and Olover, G (1996)

wall of lactic

acid bacteria

and related

species.

Chem.

Pharm. Bull. 44:2263-2267.
Kim, W. S., Park, J. H., Ren, J., Su, P., and Dunn, N. W. (2001)
rearrangementof plasmid

Environ. Microbiol.

DNA of Lactococcus

67:4594-4602.

lactisduringSurvival

long-term

response
starvation.

and
Appl.

ƒÀ-lactoglobulin,

ƒÀ-lactoglobulin

Effects of a probiotic
antigens in micestrainon the cellular

Koko Mizumachi1. Hiromi Kimoto1 and Jun-ichi
department
Scienceof Animal(NILGS),
305-8602,

Kurisaki2

Research, National

2 Ikenodai,

305-0901,

Instituteof Agrobiological

Department, National
Ibarakiproducts

immune response to foodInstitute

Tsukuba,of Livestock

Ibaraki,

Sciences

Japan,(NIAS),and Grasslandand 2Genetic

Diversity

2- 1-2 Kannondai, Tsukuba,

Japan

Email : koko@affrc.go.jp
Keywords : allergy,

IgE,

Introduction
Probiotic

bacteria,

ovalbumin,such as lactic

immune responses.

However, theimmunomodulatory

effects

administrationof Lactococcus

lactisantigen-specificIgE antibody

response

examined the effectsof G50 feedingacid bacteria,

mechanisms

are not fully

probiotics

have been known to modulate the hostbyelucidated.
subsp.

lactistheprobiotics

exert

their

Our previous

studies

showed that oral

G50

could

suppressin mice (Kimotoon the cellular

which
(G50)

et al. 2004).

In the present

immune response

the

food

study, wein mice to the potent

allergens,ovalbumin (OA) and(LG).

Materials

and Methods
Mice: Female BALB/c mice were purchased from Charles River Japan. The mice wereused at 6 - 8 weeks of age.
Preparationof lactic acid bacteria and its feeding:
G50 was grown in M17 brothsupplemented with 0.5% glucose for 18 hr at 30°C. The bacteria (0.2 mg/ml) in saline or saline
alone were fed to mice once a day for 7 days by gastricintubation

with an animal feedingneedle.
Cell cultures: The spleen cells were harvested from the mice fed G50 or saline alone.
The cells were seeded into 24-well plates (4 x 106cells/well)and stimulated with 100 |ig/ml of
LG or OA. The culture medium was RPMI-1640

containing

50 \\M 2-mercaptoethanol,

10 mM

HEPES, 10 U/ml of penicillin,
100 ^ig/ml of streptomycin and 10% fetal calf serum. After 72 hr
of culture at 37°C in a 5% CO2 atmosphere, the culture supernatants were collectedandcytokine productions
effects of G50-feeding

were measured by commercial ELISA kits. To examine the suppressiveon the antibody response, the spleen cells (2 x 106 cells /well) from mice

immunized with LG or OA and the spleen cells(2 x 106cells/well)

from mice fed lactic

acidbacteria or saline alone were co-cultured
culture insertswith 0.45 |mn filters.

72 hr and additional
supernatant
Resultseitherdirectly

in a 24-well

The cells were cultured

72 hr in the absence of the antigens.

plate

or separately

using

in the presence of the antigens

The antigen-specificantibodies

forin thewere detected by ELISA.

and Discussion
The spleen cells from mice fed G50 or saline were stimulated

and cytokines

with OA or LG in vitro(IL-4, IL-6, IL-10, TNF-a and IFN-y) were measured in the culture supernatants.

The IFN-y production by the OA- or LG-stimulated
significantly
higher than those of control mice (Fig.

spleen cells from mice fed G50 was1). The cells from mice fed G50 also

showed a slight increase in the production of IL-6 and TNF-a. The IL-4 and IL-10 secretions
were not detected. The antigen-specificantibody production in vitro by the spleen cells from
OA-or LG-immunized mice was suppressed
mice fed G50 (Tablein the case of the co-culturing1). Moreover, the suppressive

system with culture inserts.

with the cells from

effect was reduced in the separate co-culture

On the other hand, the levelsof IgG2a antibodies(Thl-dependent)

were increased in the co-culture with the cells from mice fed G50 (Table 1). Then, the IFN-ysecretion in the supernatant was increased, while the IL-13 secretion was slightly decreased(Table1). These findings

immunosuppressive
The probiotic
suppression

suggestthat the cells

stimulatedroles in the response to food antigens

strain such as G50 can be used as innovativeof Th2-dominatingwith G50 throughvia Th1-type

the gut playcytokineproduction.

tools to treat or prevent allergy

byimmune responses.

Table. 1 Effects
OA-specificof G50-feeding
antibodyon the in vitro

responseand cytokine

production

Fig.

1. IFN-ysupernatants

productionin thecultureof the spleen

cellsfrom themice fed G50.

References
Kimoto H, Mizumachi
immunomodulatory
Immunol. 48(2):75-82.

K, Okamoto T? Kurisaki
activity:enhancement

J. (2004)

New Lactococcus

strainwithof Thl-type

immune response.

Microbiol

4. TRAINING

COURSE

Training Course"Identificationand preservation
16 Octoberof plant

pathogens"2004 in MAS Genebank

Schedule
9 : 00-9 : 20

Orientation

9 : 20-9 : 55Introductionof MAFF Genebank(The whole system and the plants section)
9 : 55-10: 05

Taking of a group photo

10 : 05-10: 25

Introductionof MAFF Genebank(The microorganisms
10 : 25-10: 40

Break andQ&A

10 : 40-12: 00

Practice(continued)

section)DNA sequencing for molecular identificationofplant pathogens
12 : 00-13

: 00

Lunch

13 : 00-16: 30

Practice

16:30-17:00

13 : 00- 14 : 00

Preservationof plant pathogenic

fungi

14 : 00- 15 : 00

Preservationof plant pathogenic

bacteria

15 : 00-15: 20
15 : 20- 16 : 30

Break
DNA sequencing

for molecularplant pathogens(continued)

Q&A

identificationofPa rticipants
Anchalee Chiengkul

Agricultural

Plant & Microbial

Department of Agriculture,
Apirusht

SomrithPlant Protection

Research & Development Office,Department ofThailand

Jingang Gu

Agriculture

Culture Collection

Ying Mei Liang

Collegeof China, China

of Forestry, Northwest Sci-Tech University

and Forestry,

Research & Development Office,Department ofThailandPham van Du

Cuu Long Delta Rice Research Institute,Pornpimon Athipunyakom

Plant Protection
Agriculture,

Tariq Mahmood

ThailandInstituteof Biotechnology

Agricultural

University,and Genetic Engineering,
Pakistan

Yezin Agricultural

University,

Yuling Li

Qinghai

China

Jun-ichicommittees
Kurisaki(National

VietnamResearch & Development Office,Department of

Thein Lwin

Executive

University,

InstituteofAgrobiological

MyanmarSciences)

Toyozo Sato

Nagao Hayashi

Keisuke Tanaka

Toshirou Nagai

Kasumi Takeuchi

Hideyuki

Makoto Kawase

K

F

Motoko Iida
eisuke Tomioka

of Agriculture

China

Plant Protection
Agriculture,

Office,ThailandAgriculture,

Nuttima Kositcharoenkul

Genebank, Biotechnology

Nagaoukuhiro Yamasaki

Hisako Nakamura

NWFP

印刷発行

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