United States 

Department of 

Agriculture 

Forest 

Service 

Northern 

Region 

200 East Broadway 

P.O. Box 7669 

Missoula, MT 59807 

Numbered Report 07-04 July 2007

PATHOGENICITY OF FOUR FUSARIUM SPECIES 

ON ACACIA KOA SEEDLINGS 

N.S. Dudley1, R.L. James2, R.A. Sniezko3, and A. Yeh1

1

Hawaii Agriculture Research Center, Aiea, HI; 2USDA Forest Service, 

Coeur d’ Alene, ID; 3USDA Forest Service, Cottage Grove, OR 

ABSTRACT 

Fusarium isolates obtained from diseased koa plants, rhizosphere soil and seeds/seedcoats may or may 

not be pathogenic on young seedlings under greenhouse conditions. This includes isolates of F. 

oxysporum, the putative cause of koa wilt/dieback disease in Hawaii. We tested ten Fusarium isolates, 

comprising four different species, for their pathogenic potential on Acacia koa seedlings under 

greenhouse conditions. Tested isolates were obtained in Hawaii from either diseased Acacia koa

seedlings, soil adjacent to seedling roots, or seeds/seedpods. All tested Fusarium isolates completely 

colonized seedling root systems and became systemic, spreading to above-ground plant tissues (stems, 

branches, and leaves). Virulence was quantified on the basis of production of disease symptoms 

(mortality, wilting, foliar chlorosis or necrosis) and effects on seedling height, diameter and root volume. 

Of the five tested F. oxysporum isolates, one exhibited high virulence, another was non-pathogenic, and 

the other three were moderately-virulent. One tested F. solani isolate was quite virulent, whereas the other 

was only slightly virulent. One isolate of F. subglutinans was non-pathogenic and the other tested isolate 

was moderately virulent on inoculated seedlings. The one tested isolate of F. semitectum displayed 

moderate virulence. Pathogenic screening of many more isolates, particularly those classified within the 

F. oxysporum species complex, will be necessary to identify pathogens that can be effectively used to 

screen families of Acacia koa for potential resistance to the wilt/dieback disease that is seriously 

impacting this important Hawaiian tree species. 
2 

INTRODUCTION 

Native Acacia koa A. Gray (Fabaceae) is a 

dominant canopy tree in many Hawaiian forest 

ecosystems. Koa is present on the main 

Hawaiian Islands, growing in moist habits at 

elevations from 90 to 2100 m. Koa has always 

been an important part of Hawaiian culture; koa 

wood was used for building many original 

Hawaiian structures, as well as being prized for 

sea-faring canoes. This important tree species is 

currently of primary importance in an expanding 

Hawaiian wood industry, being used for 

producing furniture, musical instruments, bowls, 

surf boards, and handi-crafts. 

The major factor limiting establishment and 

maintenance of koa is a wilt/decline disease that 

adversely affects tree survival and growth. High 

tree mortality following koa plantings due to this 

disease has restricted koa establishment, 

particularly in certain areas where disease 

severity is high. Koa wilt/dieback is putatively 

caused by the fungus Fusarium oxysporum f.sp. 

koae, which was first described in Hawaii by 

Gardner (1980). Information on disease impacts 

and biology was subsequently investigated 

(Anderson and others 2002). Limited genetic 

analysis of several Hawaiian isolates of F. 

oxysporum indicated potentially low genetic 

diversity, possibly due to recent introductions of 

pathogenic strains of this fungus into the state 

(Anderson and others 2004). If pathogenic 

strains were recently introduced and non-native 

to Hawaii, the severe disease impacts currently 

occurring may be a reflection of an invasive, 

well-adapted, pathogen. Improved techniques 

for detection and management of this disease are 

urgently needed to limit pathogen spread and 

reduce disease impacts. Several other Fusarium

species, particularly F. solani, are routinely 

isolated from diseased koa seedlings and trees 

(Daehler and Dudley 2002; James 2004). In 

addition, 13 different Fusarium spp. were 

recently isolated from koa seeds and seedpods 

(James and others 2006). Some of these 

organisms may be involved in disease etiology 

and epidemiology and require testing for their 

ability to induce disease. 

Family variation in Acacia koa field trials 

strongly suggests presence of resistance to F. 

oxysporum f.sp. koae (Dudley 2002). Although 

wilt/dieback disease has significantly impacted 

koa plantings on certain sites, some seed sources 

appear mostly unaffected. Therefore, genetic 

disease resistance likely occurs and may be 

widespread within some families. Frequency of 

resistance in natural Acacia koa populations is 

unknown, but is expected to be low. Rapid 

screening of many koa families would provide 

valuable information on resistance potential that 

could be validated with outplanting performance 

tests. Inoculation methods for screening 

potential disease resistance in seedlings are 

available (James and others 1989; James 1996; 

Anderson and others 2002). Following 

laboratory and greenhouse experiments, field 

validation of resistance screening results will be 

needed (Sniezko 2004). 

The present work was designed to evaluate 

pathogenic potential of selected Fusarium

isolates associated with diseased koa plants and 

nearby soil in order to help clarify their potential 

roles in disease etiology. Tests were conducted 

under controlled conditions on koa seedlings 

within a greenhouse. 

MATERIALS AND METHODS 

Many isolates of Fusarium were obtained during 

routine isolations from koa seedlings, large trees 

exhibiting wilt/dieback disease symptoms, and 

seeds and seedpods. Isolates comprised several 

different Fusarium species (James 2004; James 

and others 2006), determined on the basis of 

morphological characterization (Nelson and 

others 1983). Some of these species have not 

previously been described on Acacia koa and 

their potential role in disease etiology is 

unknown. Therefore, seedling inoculation tests 

were initiated to help elucidate disease potential 

of selected representative isolates. Ten isolates 

encompassing four different Fusarium species 

were evaluated for their pathogenic potential 

(Table 1). 
3 

Fungal inoculum was prepared using the 

procedures of Miles and Wilcoxin (1984). 

Perlite, an inert, inorganic, siliceous rock of 

volcanic origin commonly used in potting 

mixtures, was the matrix for fungal growth. 150 

g of yellow cornmeal was moistened with 300 ml 

warm 1% potato dextrose agar (PDA), to which 

75 g of perlite were added. The perlite-cornmealPDA matrix was autoclaved at 121o

C for 60 min, 

cooled, inoculated with spore suspensions of test 

fungi, and incubated at about 24o

C in the dark 

for at least 15 days. After incubation, inoculum 

was dried in open petri plates within a cabinet. 

Inoculum dried within 5-7 days and did not 

become contaminated with other microorganisms 

because the food base was completely colonized 

by inoculated fungal isolates. Once dry, 

inoculum was refrigerated until needed. 

Inoculum was ground to a fine powder and 

thoroughly mixed with commercial peat 

moss/perlite growing media (Sunshine Mix 4, 

Aggregate Plus, Sungro Horticulture, Belleview, 

WA) at a concentration of 1:50 (w/w). Inoculum-growing media mixtures were placed into 

plastic containers (“dibble tubes” – 115 mm3

) 

which had previously been sterilized by 

immersion in hot water (71oC for 5 min). 

Seeds of Acacia koa from one family were 

nicked at their distal end with nail clippers to 

break dormancy, soaked in water for about 12 

hrs and sown into flats containing a 50:50 (v/v) 

mixture of vermiculite (Sta-Green Horticultural 

Vermiculite, St. Louis, MO) and perlite (Redco 

II, North Hollywood, CA), periodically watered, 

and monitored for germination. Following 

germination, when radicals were approximately 

the same length as cotyledons, they were 

carefully extracted from the flat and transplanted 

into the plastic containers containing inoculumgrowing media mixtures. Following 

transplanting, seedlings were watered to activate 

inoculum. For each tested isolate, four 

replications of six seedlings each were evaluated. 

A fully replicated set of 24 seedlings was 

included as a control, which were transplanted 

into peat/perlite growing media without fungal 

inoculum. 

Transplanted seedlings were monitored for 

development of wilt and/or foliar chlorosis and 

necrosis. When seedlings were considered dead, 

they were carefully extracted from plastic containers, their roots thoroughly washed to remove 

adhering particles of growing media, and 

analyzed in the laboratory for root colonization 

by inoculated isolates. For this analysis, ten root 

pieces, each approximately 5 mm in length, from 

each seedling, were surface sterilized in a 10% 

bleach solution (0.525% aqueous sodium 

hypochlorite; 1 part standard household bleach in 

10 parts water), rinsed in sterile, distilled water, 

and placed on a selective agar medium for 

Fusarium spp. (Komada 1975). Plates were 

incubated under diurnal cycles of cool, 

fluorescent light at about 24o

C for 7-10 days. 

Emerging fungi were compared with inoculated 

isolates to determine whether they were the same 

morphological species. 

At the end of the experiment (120 days), all 

surviving seedlings were examined for aboveground disease symptoms. Each seedling was 

rated for vigor based on severity of disease 

symptoms using the following numerical system: 

0 = no disease symptoms; seedling appears 

healthy; 1 = seedling with less than 50% of its 

crown with wilt or foliar chlorosis/necrosis 

symptoms; 2 = seedling with greater than 50% of 

its crown with wilt or foliar chlorosis/necrosis 

symptoms, but still alive; 4 = seedling dead 

[entire crown with disease symptoms]. Seedling 

diameter just above the ground line and height 

from the ground line to the highest point of the 

main stem were measured on each surviving 

seedling. Seedling roots were washed 

thoroughly, surface sterilized as described 

previously, and analyzed for colonization by 

inoculated isolates as described above. 

Remaining roots were dried for at least 24 hrs at 

100o

C and weighed (oven-dry weight = root 

volume). Means of seedling height, diameter, 

root volume and average vigor rating were 

compared and significant differences (P=0.05) 

found among isolates using the Waller DuncanK Ratio t test (SAS Institute 1982). 

A numerical system was developed to 

quantitatively compare virulence among tested 

isolates using all measured parameters: seedling 
4 

mortality, height, diameter, root volume, and 

vigor rating. This system assigned numbers for 

each parameter based on mean comparisons 

associated with each isolate, with lower numbers 

representing less fungal effects, i.e., less 

mortality, greater seedling height, diameter, root 

volume and higher vigor. Using this numerical 

system, Fusarium isolates were ranked on the 

basis of their overall virulence on Acacia koa

seedlings. 

RESULTS 

Low levels of Acacia koa seedling mortality 

resulted from inoculating with most tested 

Fusarium isolates (Table 2). However, 

significant differences (P=0. 05) were found 

among different isolates for the other measured 

parameters, including seedling height, diameter, 

root volume and average vigor rating. When all 

parameters were collated, isolates were ranked as 

to their relative virulence on inoculated seedlings 

(Table 3). This ranking indicated that the five 

tested isolates of F. oxysporum varied widely in 

their relative virulence, including the most 

(0431B) and least (0429K) virulent of all 

evaluated isolates. The two isolates of F. solani

we tested likewise exhibited relatively low 

(0424E) and high (0424I) virulence. One tested 

F. subglutinans isolate (0421A) was weakly 

virulent, whereas the other isolate of this species 

(0421J) was moderately virulent. The only tested 

F. semitectum isolate (0431J) was moderately 

virulent, although it was associated with the 

highest amount of seedling mortality (Table 2). 

DISCUSSION 

Fusarium isolates obtained from either diseased 

Acacia koa plants, soil adjacent to diseased 

plants, or seeds/seedpods varied widely in their 

virulence on inoculated koa seedlings under 

greenhouse conditions. This included several 

isolates of F. oxysporum, the putative cause of 

koa wilt/dieback disease. Apparently, not all 

isolates of this Fusarium species are equally 

virulent on koa seedlings, even though they may 

be isolated from diseased plant material. This is 

not surprising because isolates morphologically 

classified as F. oxysporum likely comprise 

several different biological species based on their 

genetic differences (Gordon and Martyn 1997; 

Kistler 1997; Di Peitro and others 2003). Large 

numbers of F. oxysporum associated with either 

healthy or diseased plants are likely saprophytic 

(Gordon and Martyn 1997; Bao and others 2002; 

Lori and others 2004); a much smaller number 

are capable of eliciting disease (Bao and 

Lazarovits 2001; Roncero and others 2003). Our 

results indicated that at least one tested isolate of 

F. oxysporum (0431B) was quite virulent on 

inoculated seedlings, whereas another isolate 

(0429K) was definitely saprophytic (Table 3). 

The former isolate was obtained from a disease 

koa seedling, whereas the latter was isolated 

from rhizosphere soil around the roots of a 

diseased plant. We plan to re-test both isolates 

in subsequent greenhouse inoculation trials to 

confirm their pathogenic or saprophytic 

behavior. The other tested F. oxysporum isolates 

were moderately virulent, indicating that these 

isolates were probably not aggressive pathogens, 

at least under our experimental conditions. They 

may be facultative pathogens that require high 

levels of plant stress to cause disease (Nelson 

and others 1981; Bao and others 2002). 

We have routinely isolated high levels of F. 

solani from diseased Acacia koa plants (James 

2004). This species is most often isolated from 

the interior of stems, branches, and large roots of 

plants displaying crown wilt/dieback symptoms, 

particularly from tissues adjacent to typical 

interior greenish-charcoal staining that is often 

associated with diseased plants. Fusarium solani

has also commonly been associated with 

infestation by black twig borers (Xylo-sandrus 

compactus Eichhoff) [Coleo-ptera: Scolytidae], 

insects that routinely attack diseased trees 

(Daehler and Dudley 2002). The two isolates 

evaluated in this test displayed either relatively 

high or relatively low virulence (Table 3). Roles 

of F. solani in koa disease etiology are currently 

unknown, but may be important based on the 

relative frequency it is isolated from diseased 

plants. Further evaluation of its potential 

importance is warranted. 

Fusarium subglutinans was commonly isolated 

from insect-predated seeds and seedpods 

collected from koa trees displaying wilt/dieback 

symptoms (James 2004). This fungal species has 
5 

previously been reported in Hawaii on plants in 

the genus Aglaonema (Uchida and Aragaki 

1994). Isolates morphologically classified as 

this species may actually comprise several 

genetically-distinct species (Nirenberg and 

O’Donnell 1998;O’Donnell and others 1998). 

For example, two isolates obtained from 

diseased koa plants morphologically classified as 

F. subglutinans were genetically identified as F. 

sterilihyphosum Britz, Marasas & Wingfield 

sp.nov. (Hyphomycetes) (O’Donnell 2005), a 

species previously described only on malformed 

mangos in South Africa (Britz and others 2002). 

The two F. subglutinans isolates we tested were 

either non-pathogenic or exhibited either 

moderate virulence on koa seedlings. We 

suspect that this species (or species complex) 

may be associated with or vectored by seedinfesting insects and may not necessarily be 

important in disease etiology. 

Fusarium semitectum is relatively common 

fungal species within subtropical or tropical 

environments (Nelson and others 1981, 1983). 

This species can be pathogenic on several 

different plant species (Onyike and Nelson 1992; 

Santou and others 2001; Dhingra and others 

2002; Bokshi and others 2003) and often 

produces mycotoxins that may adversely affect 

plants and animals (Abbas and others 1995; 

Jimenez and others 1997; Logrieco and others 

2002). The one isolate we tested was rated as 

moderately virulent on Acacia koa seedlings, 

although a quarter of the seedlings inoculated 

with this isolate died during our test. This 

relatively high mortality compared with other 

tested Fusarium isolates may have been because 

seedlings inoculated with this isolate were the 

last to be collected from germination trays. 

Therefore, they may have been the least vigorous 

of all tested seedlings. Fusarium semitectum is 

frequently isolated from disease koa plants and 

its potential role in disease etiology warrants 

further investigation. 

We were able to identify at least one candidate 

Fusarium isolate (0431B) that may have some 

potential for screening Acacia koa families for 

resistance to the wilt/dieback disease. Our 

inoculation study indicated that Fusarium

isolates obtained from diseased koa seedlings, 

adjacent soil, and seeds/seedpods are not 

necessarily pathogens. Five tested isolates of F. 

oxysporum showed a wide range of virulence on 

seedlings in our test. This would indicate that 

both pathogenic and nonpathogenic strains of 

this species, the putative cause of koa 

decline/wilt disease, commonly colonize koa 

plants. Therefore, all isolates of F. oxysporum

obtained from diseased koa plants are not 

necessarily the pathogen capable of eliciting koa 

disease. Isolates will have to be genetically 

analyzed to locate potential markers associated 

with pathogenicity. Such markers may be 

effective to quickly and easily locate pathogenic 

strains from within fungal populations (Bao and 

others 2002; Lori and others 2004; Stewart and 

others 2004). Further pathogenicity tests, 

particularly with isolates of F. oxysporum, are 

planned to obtain additional isolates that can be 

effectively used for resistance screening. When 

mixtures of pathogenic strains are available, we 

should be successful in screening for disease 

resistance. 

ACKNOWLEDGEMENTS 

We acknowledge the USDA Forest Service, 

Forest Health Protection, Special Technology 

Development grant R5-2005-02 for financial 

assistance for this research.
6 

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N.S. Dudley is Forester, Hawaii Agriculture Research Center, 99-193 Aiea Heights Drive, Aiea, HI 96701 

[email: NDudley@harc-haspa.com]; R.L. James is Plant Pathologist, USDA Forest Service, Forest Health 

Protection, Northern Region, 3815 Schreiber Way, Coeur d’Alene, ID 83815 [email: rjames@fs.fed.us]; 

R.A. Sniezko is Geneticist, USDA Forest Service, Dorena Genetic Resource Center, 34963 Shoreview 

Road, Cottage Grove, OR 97424 [email: rsniezko@fs.fed.us]; A. Yeh is Plant Propagation Specialist, 

Hawaii Agriculture Research Center, 942 Kawailani Street, Hilo, HI 96778. 

 Table 1. Fusarium isolates evaluated for pathogenicity on Acacia koa seedlings 

Isolate 

Number 

Fusarium Species Isolation Location 

0421A F. subglutinans (Wollenw. & Reinking) 

Nelson, Toussoun & Marasas 

Insect-predated koa seeds and seedpods 

0421J F. subglutinans Insect-predated koa seeds and seedpods 

0424E F. solani (Mart.) Appel & Wollenw. Stem of diseased koa seedling 

0424I F. solani Stem of diseased koa seedling 

0425K F. oxysporum Schlechten.:Fr. Roots of diseased koa seedling 

0429K F. oxysporum Rhizosphere around roots of diseased 

seedling 

0430B F. oxysporum Soil near roots of diseased koa seedling 

0431B F. oxysporum Stem of diseased koa seedling 

0433D F. oxysporum Roots of diseased koa seedling 

0431J F. semitectum Berk. & Rav. Stem of diseased koa seedling 
9

Table 2. Effects of selected Fusarium isolates on mortality, height, diameter, root volume, and vigor of 

inoculated Acacia koa seedlings. 

Isolate 

Numbe

r 

Fusarium

Species 

Percent 

Mortality

1

Average 

Height2

Average 

Diameter2

Root 

Volume3

Vigor 

Rating4

0421A F. subglutinans 0 30.6 BC5 38 B 0.63 AB 0.91 DC 

0421J F. subglutinans 0 22.9 E 30 E 0.53 BC 0.96 BCD 

0424E F. solani 0 29.2 BC 33 DE 0.52 BC 0.54 E 

0424I F. solani 0 23.8 E 30 E 0.48 CD 1.23 AB 

0425K F. oxysporum 4.2 24.3 DE 35 CD 0.55 BC 0.79 DE 

0429K F. oxysporum 4.2 35.6 A 42 A 0.73 A 0.59 E 

0430B F. oxysporum 0 27.2 CD 32 DE 0.52 BC 1.22 ABC 

0431B F. oxysporum 8.3 22.6 E 29 E 0.39 D 1.36 A 

0433D F. oxysporum 8.3 31.2 B 38 BC 0.61 AB 1.05 BCD 

0431J F. semitectum 25.0 28.3 BC 36 BC 0.52 BC 1.18 ABC 

- CONTROL 0 38.8 A 42 A 0.60 BC 0 F 

1

Percent of inoculated seedlings (24 per isolate) that died during the test (120 days) 

2

Average height (cm) and diameter (mm) at the end of the test. 

3

Average oven-dry weight (g) of root volumes at the end of the test. 

4

Average vigor rating at the end of the test; see text for descriptions. 

5

Within each column, means followed by the same capital letter are not significantly different (P=0.05) 

using the Waller-Duncan K-ratio t test 

Table 3. Comparative virulence of selected Fusarium isolates on inoculated Acacia koa seedlings1. 

Isolate 

Number 

Fusarium

Species2

Mortality Height Diameter Root 

Volume

Vigor Virulence Rating 

Control - 1 1 1 2 1 6 - 

Nonpathogenic 

0429K F. oxysporum 2 1 1 1 2 7 - 

Nonpathogenic 

0421A F. 

subglutinans 

1 2 2 1 3 9 - Low 

0424E F. solani 1 2 3 2 2 10 - Low 

0433D F. oxysporum 3 2 2 1 3 11 - Moderate 

0425K F. oxysporum 2 4 3 2 2 13 - Moderate 

0430B F. oxysporum 1 3 3 2 4 13 - Moderate 

0431J F. semitectum 4 2 2 2 4 14 - Moderate 

0421J F. 

subglutinans 

1 5 5 2 3 16 - Moderate 

0424I F. solani 1 5 5 3 5 19 - High 

0431B F. oxysporum 3 5 5 4 6 23 - High 

Maximum - 4 5 5 4 6 24 - High 

1

Numbers in table compare averages for each isolate; higher numbers reflect greater virulence.