United States 

Department of 

Agriculture 

Forest 

Service 

Northern 

Region 

200 East Broadway 

P.O. Box 7669 

Missoula, MT 59807 

Numbered Report 07-08 November 2007

COMPARATIVE VIRULENCE OF HAWAIIAN 

FUSARIUM OXYSPORUM ISOLATES ON 

ACACIA KOA SEEDLINGS 

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

1

Hawaii Agriculture Research Center, Aiea, HI; 

2

USDA Forest Service, Coeur d’Alene, ID 

ABSTRACT 

Sixty-one fungal isolates within the Fusarium oxysporum species complex isolated from Acacia koa

plants or rhizosphere soil in Hawaii were tested for pathogenicity on young koa seedlings. Only about 

15% of these were considered highly- or moderately-virulent in greenhouse inoculation tests. On the other 

hand, more than 50% of the tested isolates either exhibited low virulence or were considered nonpathogenic. Highly-virulent isolates were easily identified because they killed nearly all inoculated 

seedlings during the 90-day tests. Non-pathogenic isolates often did not cause disease symptoms on any 

of the 24 seedlings exposed per isolate. We have confidence that our testing procedure can be used to 

easily and consistently identify highly-virulent and non-pathogenic isolates within Hawaiian populations 

of F. oxysporum and may be used to screen koa families for potential disease reistance. 

INTRODUCTION 

Fusarium oxysporum Schlecht. is a taxon 

containing a complex of morphologically-similar 

Fusarium species that cause important plant 

diseases worldwide. Strains may vary widely 

genetically, although they usually cannot be 

differentiated morphologically (Gordon and 

Martyn 1997; Kistler 1997). Pathogenic strains 

of F. oxysporum are often quite host-specific and 

those capable of eliciting disease on particular 

host species are designated by a sub-specific 

taxon called a formae specialis (Armstrong and 

Armstrong 1975; Nelson and others 1981). 

However, saprophytic strains incapable of 

causing diseases are widespread and often 

occupy the same environmental niches as 

pathogenic strains (Gordon and Martyn 1997; 

Nelson and others 1981). Saprophytic strains 

cannot be differentiated from pathogenic strains 

unless either tested on susceptible hosts or 
2 

separated on the basis of specific genetic markers 

associated with virulence genes (Alves-Santos 

and others 2002; Bao and others 2002; 

Chiocchetti and others 1999; Gordon and 

Okamoto 1992). Pathogenicity testing is 

expensive and time-consuming, whereas genetic 

differentiation can usually be done more rapidly 

and can be less costly if genetic markers for 

specific pathosystems have been identified. 

We have recently been investigating an 

important wilt/dieback disease of Acacia koa A. 

Gray in Hawaii. Koa is the most economicallyimportant tree in many Hawaiian forest 

ecosystems, where it grows in moist habitats at 

elevations from 90 to 2100 m. High tree 

mortality following koa plantings due to this 

disease has restricted koa establishment, 

particularly in certain areas (Daehler and Dudley 

2002). Koa wilt/dieback was first described in 

Hawaii by Gardner (1980). He completed Koch’s 

postulates and determined that the primary cause 

of the disease was pathogenic strains of F. 

oxysporum which he designated f.sp. koae. 

Information on several aspects of disease impact 

and biology was subsequently investigated 

(Anderson and others 2002). An initial genetic 

analysis of several pathogenic Hawaiian isolates 

of F. oxysporum from diseased koa plants 

indicated potentially low genetic diversity, which 

may indicate recent introduction(s) of the 

pathogen into Hawaii (Anderson and others 

2004). Subsequent genetic characterization of a 

much larger population of F. oxysporum from 

different sized hosts and islands indicated that 

the overall Hawaiian population within this 

species complex is likely quite diverse; at least 

six genetically-distinct clades were identified 

within a population representing more than 100 

isolates from throughout Hawaii (Yang and 

others 2007). 

We have routinely isolated F. oxysporum from 

roots and occasionally from stems and branches 

of large diseased koa trees (James and others 

2007a), either diseased or healthy-appearing 

seedlings (James 2004; James and others 2007b), 

rhizosphere soil and occasionally from seeds and 

seedpods (James 2004; James and others 2006). 

Most of these isolates appear very similar 

morphologically, based on presence and 

structure of particular spores (microconidia, 

macroconidia, chlamydospores) and conidiophores (short, unbranched monophialides) 

(Nelson and others 1983). Most isolates also 

appear similar when grown in culture (aerial 

hyphae, sporodochia, pigment formation). 

Although preliminary characterization of F. 

oxysporum populations associated with Hawaiian 

koa has been done (Anderson and others 2002; 

Yang and others 2007), specific genetic markers 

associated with pathogenic strains have not yet 

been identified. We initiated greenhouse tests to 

determine pathogenic potential of selected F. 

oxysporum isolates obtained from Hawaiian koa 

in an effort to identify specific isolates that might 

be used to screen koa families for potential 

disease resistance and to quantify the prevalence 

of potential virulence within fungal populations. 

MATERIALS AND METHODS 

Sixty-one isolates identified as F. oxysporum

based on morphological characteristics were 

selected for testing in greenhouse seedling 

inoculation experiments (Table 2 - Appendix). 

Three tests were conducted sequentially; two in 

2006 and one in 2007. 

For each selected isolate, 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-cornmeal-PDA mixture 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. 
3 

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). Inoculumgrowing media mixtures were placed into plastic 

containers (“dibble tubes” – 115 mm3

) which 

were either new or had previously been sterilized 

by immersion in hot water (71o

C 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 

they same length as cotyledons, they were 

carefully extracted from the flat and transplanted 

into the plastic containers with inoculumgrowing media mixtures. Following 

transplanting, seedlings were watered to activate 

inoculum. For each tested isolate, four 

replications of six seedlings each were evaluated. 

Two fully replicated sets of 24 seedlings each 

were included as controls. These contained 

seedlings 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 

randomly-selected 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. 

Tests were run for a maximum of 90 days. 

Heights of seedlings surviving to the end of the 

test were measured and their biomass (oven-dry 

weight - 100o

C for a minimum of 24 hrs - of 

roots and tops) determined. A few root pieces 

from surviving seedlings were incubated on the 

selective agar medium to confirm reisolation of 

the inoculated isolates. 

RESULTS 

Virulence ratings for individual isolates were 

assigned primarily on the basis of disease 

production, but also included average survival 

(number of days seedlings lived during the 90-

day test) and average height of non-diseased 

seedlings. Categories of virulence ratings and the 

proportion of isolates falling within each 

category are summarized in table 1. A listing of 

individual isolates in the approximate order of 

the virulence based on the above criteria is 

compiled in table 3 (Appendix). 

Only about 15% of the tested isolates exhibited 

high- or moderately-high virulence on young koa 

seedlings under our greenhouse inoculation 

conditions. More than half of the isolates 

exhibited low virulence or were considered nonpathogenic. The other tested isolates were 

somewhere in between. As expected, isolates 

classified as highly-virulent caused extensive 

disease and seedlings survived for shorter time 

periods. Differences in average seedling height 

among the six virulence categories were 

inconsistent. Our results indicated that the 

majority of the tested F. oxysporum isolates were 

not highly-virulent strains and therefore probably 

not responsible for the wilt/dieback disease of 

koa (table 1). 
4 

Table 1. Virulence of Hawaiian Fusarium oxysporum isolates on Acacia koa.

Virulence 

Rating 

Number of 

Isolates 

Percent of 

Isolates 

Average 

Disease1

Average 

Survival2

Average 

Height3

High 5 8.2 89.2 53.2 10.8 

ModeratelyHigh 4 6.6 65.6 62.0 12.3 

Moderate 15 24.6 50.5 70.5 13.0 

ModeratelyLow 4 6.6 34.4 81.4 12.9 

Low 6 9.8 20.9 85.0 11.3 

Nonpathogenic 27 44.2 4.2 88.8 12.8 

Controls 2 Sets - 4.2 87.7 12.6 1

Percent of inoculated seedlings that became diseased 

2

Average number of days seedlings survived; maximum = 90 days 

3

Average height (cm) of surviving seedlings

DISCUSSION 

The protocols we used to test virulence of F. 

oxysporum isolates on young Acacia koa

seedlings were developed initially for use on 

conifer seedlings (James 1996; James and others 

1989). Results on conifers were consistently 

reliable in locating virulent Fusarium isolates 

and separating them from non-pathogens (James 

and others 1989, 1997, 2000). Therefore, these 

procedures were adapted for use on koa 

seedlings with the ultimate goal of developing a 

reliable inoculation system that could routinely 

be used to screen koa families for resistance to 

highly-virulent F. oxysporum isolates. 

Classification of isolates into virulence ratings of 

“high”, “moderately high”, etc. were fairly 

arbitrary. However, we believe that we were able 

to reliably identify those isolates at the extremes 

of our established virulence categories: highlyvirulent and non-pathogenic. Highly-virulent 

strains identified by our inoculation protocols 

can be used to screen koa families for potential 

disease resistance. 

We found that only a small percent of tested 

isolates could be classified as highly- or 

moderately-virulent. These isolates were readily 

identifiable because they killed the majority of 

inoculated seedlings. Most seedling mortality 

usually began about one month after inoculation 

and extended for the next 3-4 weeks. We suspect 

that it takes about a month for virulent isolates to 

infect roots, grow into vascular tissues, spread 

systemically throughout seedlings, and begin to 

initiate wilt symptoms by occluding water 

transport within vascular cells of inoculated 

seedlings (Beckman and others 1989, 1991; 

Nelson and others 1981). After this period of 

mortality, surviving seedlings often did not 

develop disease symptoms for the remaining test 

period. 

We found that all tested isolates, even those 

considered non-pathogenic, always infected roots 

of inoculated seedlings. Inoculated roots 

exhibited no noticeable necrosis or discoloration, 

i.e., they were white and appeared completely 

healthy. However, they were extensively 

colonized by inoculated isolates, even to the 

point where no other fungi were detected during 

root assays. We suspect that non-pathogenic 

isolates were unable to successfully colonize 
5 

vascular systems and thus spread systemically 

throughout inoculated seedlings (Beckman and 

others 1989; Nelson and others 1981). Such 

isolates may have been restricted to root cortical 

cells where they existed as endophytes which did 

not adversely affect seedling health (Bloomberg 

1966; Dhingra and others 2000). 

Our results indicated that most F. oxysporum

isolates obtained from koa trees displaying wilt 

or dieback symptoms were not highly specialized 

pathogens of koa. Isolates within the F. 

oxysporum species complex that are specifically 

capable of causing koa diseases are classified as 

F. oxysporum f.sp. koae (Gardner 1980). Highlyspecialized pathogenic strains have usually 

evolved from non-pathogenic F. oxysporum

populations (Gordon and Martyn 1997; Kistler 

1997). Such isolates are so specialized that they 

are usually incapable of causing disease on other 

plant hosts, even those closely-related to their 

primary hosts (Buxton 1958; Ficcadenti and 

others 2002; Roncero and others 2003). In some 

cases, specific races of F. oxysporum within 

particular formae speciales evolve to the point 

that they can only elicit disease on distinct host 

cultivars (Awuah and others 1986; Baayen and 

others 1988; Ben-Yephet and others 1997). 

Pathogenic and non-pathogenic F. oxysporum

isolates can often be differentiated using 

particular molecular genetic markers (Bao and 

Lazarovits 2001; Bao and others 2002; Di Pietro 

and others 2003; Lori and others 2004). These 

can be used to quickly locate highly-virulent 

isolates within particular fungal populations 

associated with host plants (Bao and others 

2002; Di Pietro and others 2003) and within soil 

(Edel and others 2001). Genetic analyses using 

amplified fragment-length polymorphisms 

(AFLPs) were completed on a population of F. 

oxysporum isolated from diseased koa plants, 

healthy seedlings, and rhizosphere soil (Yang 

and others 2007). Four highly-virulent isolates 

were included in this analysis; they clustered 

together in groups of two within two distinct 

clades. Pathogenicity testing of other geneticallycharacterized isolates is current underway. We 

hope to relate virulence on seedlings to genetic 

differentiation with the goal of identifying 

specific molecular markers that can be used to 

easily identify highly-virulent isolates within 

natural koa-associated F. oxysporum populations. 

The high percentage on non-pathogenic F. 

oxysporum isolates identified in our tests may 

indicate high potential for biological control of 

highly-virulent strains. Non-pathogenic strains of 

this fungus have successfully been used as 

biocontrol agents in several cropping systems 

(Alabouvette and others 1993; Benhamou and 

Garand 2001; Blok and others 1997; Damicone 

and Manning 1982; Duijff and others 1999; 

Fravel and Larkin 2002; Fravel and others 2003). 

These strains successfully compete with and 

occupy niches normally colonized by pathogenic 

strains (Alabouvette and others 1993; Bao and 

Lazarovits 2001; Fravel and others 2003). They 

may often be more specifically adapted to 

control pathogenic strains of F. oxysporum than 

other, commercially-developed microorganisms 

(De Cal and Melgarejo 2001; Dumroese and 

others 1998; El-Hassan and Gowen 2006; 

Mousseaux and others 1998). Biological control 

may particularly be attractive in nurseries where 

non-pathogenic strains can be easily introduced 

into seedling production systems (James and 

Dumorese 2006; Stewart and others 2004). Such 

an approach holds promise in Hawaiian nurseries 

producing koa seedlings because we have found 

very high levels of infection by F. oxysporum on 

seedling stock (James 2004; James and others 

2007b). 

This work confirmed our expectation of the wide 

variability of fungal strains within the F. 

oxysporum species complex in Hawaii. Although 

most of these organisms appear morphologically 

similar, there are extensive genetic differences 

(Yang and others 2007) that affect pathogenicity 

on koa plants. We plan to expand our screenings 

to include many more F. oxysporum isolates to 

confirm proportions of populations capable of 

eliciting wilt or dieback disease of Hawaiian koa. 

We also want to have as many highly-virulent 

isolates as possible for screening koa families for 

potential disease resistance. This will improve 
6 

our confidence that selected resistant families 

will perform well after planting. 

ACKNOWLEDGEMENTS 

We acknowledge the USDA Forest Service, 

Forest Health Protection, Special Technology 

Development grant R5-2005-02 for financial 

assistance for this research. 

REFERENCES 

Alabouvette C, Lemanceau P, Steinberg C. 1993. 

Recent advances in biological control of 

Fusarium wilts. Pesticide Science 37:365-373. 

Alves-Santos FM, Ramos B, Garcia-Sanchez 

MA, Eslava AP, Diaz-Minguez JM. 2002. A 

DNA-based procedure for in planta detection 

of Fusarium oxysporum f.sp. phaseoli. 

Phytopathology 92:237-244. 

Anderson RC, Gardner DE, Daehler CC, 

Meinzer FC. 2002. Dieback of Acacia koa in 

Hawaii: ecological and pathological 

characteristics of affected stands. Forest 

Ecololgy and Management 162:273-286. 

Anderson RC, Bentley TB, Correll JC. 2004. 

Population diversity of Fusarium oxysporum

f.sp. koae, the pathogen of Koa dieback in 

Hawaii. Phytopathology 94:S4. 

Armstrong GM, Armstrong JK. 1975. 

Reflections on the wilt fusaria. Annual Review 

of Phytopathology 13:95-103. 

Awuah RT, Lorbeer JW, Ellerbock LA. 1986. 

Occurrence of Fusarium yellows of celery 

caused by Fusarium oxysporum f.sp. apii race 

2 in New York and its control. Plant Disease 

70:1154-1158. 

Bao JR, Fravel DR, O’Neill NR, Lazarovits G, 

van Berkum P. 2002. Genetic analysis of 

pathogenic and nonpathogenic Fusarium 

oxysporum from tomato plants. Canadian 

Journal of Botany.80:271-279. 

Bao JR, Lazarovits G. 2001. Differential 

colonization of tomato roots by nonpathogenic 

and pathogenic Fusarium oxysporum strains 

may influence Fusarium wilt control. 

Phytopathology 91:449-456. 

Baayen RP, Elgersma DM, Demmink JF, 

Sparnaaij LD. 1988. Differences in 

pathogenesis observed among susceptible 

interactions of carnation with four races of 

Fusarium oxysporum f.sp. diathi. Netherlands 

Journal of Plant Pathology 94:81-94. 

Beckman CH, Morgham AT, Mueller WC. 1991. 

Enlargement and vacuolization of the 

cytoplasm in contact cells of resistant and 

susceptible tomato plants following inoculation 

with Fusarium oxysporum f.sp. lycopersici, 

race 1. Physiological and Molecular Plant 

Pathology 38:433-442. 

Beckman CH, Verdier PA, Mueller WC. 1989. A 

system of defence in depth provided by 

vascular parenchyma cells of tomato in 

response to vascular infection with Fusarium 

oxysporum f.sp. lycopersici, race 1. 

Physiological and Molecular Plant Pathology 

34:227-239. 

Benhamou N, Garand C. 2001. Cytological 

analysis of defense-related mechanisms 

induced in pea root tissues in response to 

colonization by nonpathogenic Fusarium 

oxysporum Fo47. Phytopathology 91:730-740. 

Ben-Yephet Y, Reuven M, Shtienberg D. 1997. 

Complete resistance by carnation cultivars to 

Fusarium wilt induced by Fusarium oxysporum

f.sp. dianthi race 2. Plant Disease 81:777-780. 

Blok WJ, Zwankhuizen MJ, Bollen GJ. 1997. 

Biological control of Fusarium oxysporum

f.sp. asparagi by applying non-pathogenic 

isolates of F. oxysporum. Biocontrol Science 

and Technology 7(4):527-542. 

Bloomberg WJ. 1966. The occurrence of 

endophytic fungi in Douglas-fir seedlings and 

seeds. Canadian Journal of Botany 44:413-420. 

Buxton EW. 1958. A change of pathogenic race 

in Fusarium oxysporum f.sp. pisi induced by 

root exudate from a resistant host. Nature 

181:1222-1224. 

Chiocchetti A, Bernardo I, Daboussi MJ, 

Garibaldi A, Gullino ML, Langin Migheli Q. 

1999. Detection of Fusarium oxysporum f.sp. 

dianthi in carnation tissue by PCR 

amplification of transposon insertions. 

Phytopathology 89:1169-1175. 
7 

Daehler CC, Dudley N. 2002. Impact of the 

black twig borer, an introduced insect pest, on 

Acacia koa in the Hawaiian Islands. 

Micronesica Supplement 6: 35-53. 

Damicone JP, Manning WJ. 1982. Avirulent 

strains of Fusarium oxysporum protect 

asparagus seedlings from crown rot. Canadian 

Journal of Plant Pathology 4:143-146. 

De Cal A, Melgarejo P. 2001. Repeated 

applications of Penicillium oxalicum prolongs 

biocontrol of fusarium wilt of tomato plants. 

European Journal of Plant Pathology 107:805-

811. 

Dhingra OD, Netto RAC, Rodrigues FA, Silva 

GJ, Maia CB. 2006. Selection of endemic nonpathogenic endophytic Fusarium oxysporum

from bean roots and rhizosphere competent 

fluorescent Pseudomonas species to suppress 

Fusarium-yellows of bean. Biological Control 

39:75-86. 

Di Pietro A, Madrid MP, Carcuel Z, DelgadoJarana J, Roncero MIG. 2003. Fusarium 

oxysporum: exploring the molecular arsenal of 

a vascular wilt fungus. Molecular Plant 

Pathology 4:315-325. 

Duijff BJ, Recorbet G, Bakker PAHM, Loper JE, 

Lemanceau P. 1999. Microbial antagonism at 

the root level is involved in the suppression of 

Fusarium wilt by the combination of 

nonpathogenic Fusarium oxysporum Fo47 and 

Pseudomonas putida VCS358. 

Phytopathology 89:1073-1079. 

Dumroese RK, James RL, Wenny DL. 1998. 

Interactions among Streptomyces griseoviridis, 

Fusarium root disease, and Douglas-fir 

seedlings. New Forests 15:181-191. 

Edel V, Steinberg C, Gauthero N, Recorbet G, 

Alabouvette C. 2001. Genetic diversity of 

Fusarium oxysporum populations isolated from 

different soils in France. FEMS Microbiology 

Ecology 36:61-71. 

El-Hassan SA, Gowen SR. 2006. Formulation 

and delivery of the bacterial antagonist 

Bacillus subtilis for management of lentil 

vascular wilt caused by Fusarium oxysporum

f.sp. lentis. Journal of Phytopathology 

154:148-155. 

Ficcadenti N, Sestili S, Annibali S, Campanelli 

G, Belisario A, Maccaroni, Corazza L. 2002. 

Resistance to Fusarium oxysporum f.sp. 

melonis race 1,2 in muskmelon lines Nad-1 

and Nad-2. Plant Disease 86:897-900. 

Fravel DR, Larkin RP. 2002. Reduction of 

fusarium wilt of hydroponically grown basil by 

Fusarium oxysporum strain CS-20. Crop 

Protection 21:539-543. 

Fravel DR, Olivain C, Alabouvette C. 2003. 

Fusarium oxysporum and its biocontrol. New 

Phytologist 157:493-502. 

Gardner DE. 1980. Acacia koa seedling wilt 

caused by Fusarium oxysporum. 

Phytopathology 70:594-597. 

Gordon TR, Martyn RD. 1997. The evolutionary 

biology of Fusarium oxysporum. Annual 

Review of Phytopathology 35:111-128. 

Gordon TR, Okamoto D. 1992. Population 

structure and the relationship between 

pathogenic and nonpathogenic strains of 

Fusarium oxysporum. Phytopathology 82:73-

77. 

James RL. 1996. Technique for quantifying 

virulence of Fusarium and Cylindrocarpon

spp. on conifer germinants. Missoula (MT): 

USDA Forest Service, Northern Region, Forest 

Pest Management. Nursery Disease Notes No. 

132. 8p. 

James RL. 2004. Fusarium colonization of 

seeds, seedpods, and diseased seedlings of 

Acacia koa from Hawaii. Missoula (MT): 

USDA Forest Service, Northern Region, Forest 

Health Protection. Nursery Disease Notes No. 

159. 22p. 

James RL, Dumroese RK. 2006. Investigations 

of Fusarium diseases within inland Pacific 

Northwest Forest Nurseries. In: Guyon JC 

[compiler]. Proceedings of the 53rd Western 

International Forest Disease Work Conference: 

2005 August 26-29; Jackson, WY; USDA 

Forest Service Intermountain Region. pp. 3-11. 

James RL, Dumroese RK, Gilligan CJ, Wenny 

DL. 1989. Pathogenicity of Fusarium isolates 

from Douglas-fir seed and container-grown 

seedlings. Moscow (ID): Idaho Forest, Wildlife 

and Range Experiment Station Bulletin No. 52. 

10p. 
8 

James RL, R. Dumroese RK, Wenny DL. 1997. 

Pathogenicity of Fusarium proliferatum in 

container-grown Douglas-fir seedlings. In:

James RL (editor). Proceedings of the third 

meeting of IUFRO Working Party S7.03-04 

(Diseases and Insects in Forest Nurseries). 

USDA Forest Service, Northern Region, Forest 

Health Protection. Report 97-4. pp. 26-33. 

James RL, Dudley NL, Yeh A. 2006. 

Investigating koa wilt in Hawaii: Examining 

Acacia koa seeds and seedpods for Fusarium 

species. Native Plants 7(3):307-310. 

James RL, Dudley NL, Yeh A. 2007. 

Colonization of diseased Acacia koa trees with 

Fusarium species. USDA Forest Service, 

Northern Region, Forest Health Protection. 

Report 07- . 

James RL, R. Perez R, Dumroese RK, Wenny 

DL. 2000. Virulence of Fusarium oxysporum

on Douglas-fir germinants: comparison of 

isolates from nursery soil and roots of healthy 

and diseased seedlings. In: Lilja A, Sutherland 

JR (eds.). Proceedings of the 4th Meeing of 

IUFRO Working Party 7.03.04 – Diseases and 

Insects in Forest Nurseries. Finnish Forest 

Research Institute, Research Papers 781. pp. 

49-64. 

James RL, Yeh A, Dudley NL. 2007. 

Colonization of Hawaiian Acacia koa seedlings 

with Fusarium species. USDA Forest Service, 

Northern Region, Forest Health Protection. 

Report 07- . 

Kistler HC. 1997. Genetic diversity in the plantpathogenic fungus Fusarium oxysporum. 

Phytopathology 87:474-479. 

Komada H. 1975. Development of a selective 

medium for quantitative isolation of Fusarium 

oxysporum from natural soil. Review of Plant 

Protection Research (Japan) 8:114-125. 

Lori G, Edel-Hermann V, Gautheron N, 

Alabouvette C. 2004. Genetic diversity of 

pathogenic and nonpathogenic populations of 

Fusarium oxysporum isolated from carnation 

fields in Argentina. Phytopathology 94:661-

668. 

Miles MR, Wilcoxin RD. 1984. Production of 

fungal inoculum using a substrate of perlite, 

cornmeal, and potato dextrose agar. Plant 

Disease 68:310. 

Mousseaux MR., Dumroese RK, James RL, 

Wenny DL, Knudsen GR. 1998. Efficacy of 

Trichoderma harzianum as a biological control 

of Fusarium oxysporum in container-grown 

Douglas-fir seedlings. New Forests 15:11-21. 

Nelson PE, Toussoun TA, Cook RJ. 1981. 

Fusarium Diseases, Biology, and Taxonomy. 

University Park (PA): The Pennsylvania State 

University Press. 457p. 

Nelson PE, Toussoun TA, Marasas WFO. 1983. 

Fusarium species: an illustrated manual for 

identification. University Park (PA): The 

Pennsylvania State University Press. 193p. 

Roncero MIG, Hera C, Ruiz-Rubio M, GarciaMaceira FI, Madrid MP, Caracuel Z, Calero F, 

Delgado-Jarana J, Roldan-Rodriguez R, 

Martinez-Rocha AL, Velasco C, Roa J, MartinUrdiroz M, Cordoba D, Di Pietro A. 2003. 

Fusarium as a model for studying virulence in 

soilborne plant pathogens. Physiological and 

Molecular Plant Pathology 62:87-98. 

Stewart JE, Kim MS, James RL, Dumroese RK, 

Klopfenstein NB. 2004. Molecular 

characterization of Fusarium oxysporum from 

tree nurseries: Tools for early detection of 

pathogens. Phytopathology 94:S99. 

Yang B, James RL, Uchida J, Zhong S. 2007. 

Molecular characterization of Fusarium 

oxysporum isolates from koa (Acacia koa) in 

Hawaii. Phytopathology 97:S127. 

APPENDIX 
9 

Table 2. Number and source information for Hawaiian Fusarium oxysporum isolates tested for virulence on Acacia koa

seedlings. 

Isolate Number Isolation Location 

0424F Maui; diseased seedling; fine roots 

0429D Maui; diseased seedling; rhizosphere soil 

0429K Maui; diseased seedling; rhizosphere soil 

0430B Maui; diseased seedling; rhizosphere soil 

0431B Maui; diseased seedling; stem 

0433A Maui; diseased seedling; fine roots 

0433E Maui; diseased seedling; fine roots 

0433F Maui; diseased sapling; secondary roots 

0433K Maui; diseased seedling; fine roots 

0433M Maui: diseased seedling; fine roots 

0433N Maui; diseased seedling; fine roots 

0503I Big Island; diseased tree; fine roots 

0503J Big Island; diseased tree; fine roots 

0503L Big Island; diseased tree; fine roots 

0503M Big Island: diseased tree; fine roots 

0504B Big Island; diseased tree; secondary roots 

0505A Big Island: diseased tree; fine roots 

0528A Oahu; diseased tree; fine roots 

0528D Oahu; diseased tree; secondary roots 

0528F Oahu; diseased seedling; fine roots 

0531A Big Island; diseased tree; fine roots 

0531B Big Island; diseased tree; secondary roots 

0531F Big Island; diseased tree; secondary roots 

0531M Big Island; diseased tree; fine roots 

0531O Big Island; diseased tree; tertiary roots 

0531W Big Island; diseased tree; secondary roots 

0532A Big Island; diseased tree; fine roots 

0532P Big Island; diseased tree; fine roots 

0532V Big Island; diseased tree; secondary roots 

0533C Big Island; diseased tree; fine roots 

0533F Big Island; diseased tree; secondary roots 

0540A Big Island; diseased tree; secondary roots 

0540H Big Island; diseased tree; secondary roots 

0540K Big Island; diseased tree; secondary roots 

0540R Big Island; diseased tree; secondary roots 

0542F Big Island; diseased tree; fine roots 

0542H Big Island; diseased tree; secondary roots 

0542J Big Island; diseased tree; secondary roots 

0542O Big Island; diseased tree; secondary roots 

0543H Big Island; diseased tree; secondary roots 

0544G Big Island; diseased tree; secondary roots 

0544J Big Island; diseased tree; fine roots 

0544M Big Island; diseased tree; secondary roots 

0545A Big Island; diseased tree; fine roots 

0545G Big Island; diseased tree; secondary roots 
10 

0545K Big Island; diseased tree; fine roots 

0554A Big Island; diseased tree; secondary roots 

0554H Big Island; diseased tree; rhizosphere soil 

0554L Big Island; diseased tree; fine roots 

0554N Big Island, diseased tree; secondary]roots 

0608A Big Island; diseased tree; fine roots 

0608G Big Island; diseased tree; fine roots 

0618A Big Island; slightly diseased seedling; fine roots [nursery] 

0619A Big Island; slightly diseased seedling; fine roots [nursery] 

0620A Big Island; slightly diseased seedling; fine roots [nursery] 

0621A Big Island; slightly diseased seedling; fine roots [nursery] 

0622A Maui; diseased tree; fine roots 

0623J Big Island; slightly diseased seedling; fine roots [nursery] 

0624A Big Island; healthy seedling; fine roots [nursery] 

0625J Big Island; healthy seedling; fine roots [nursery] 

0629A Big Island; slightly diseased seedling; fine roots [nursery]] 

Table 3. Virulence of Hawaiian isolates of Fusarium oxysporum on Acacia koa seedlings. 

Isolate 

Number 

Virulence Rating Percent 

Disease1

Average 

Survival2

Average 

Height3

Average 

ODW4

Average 

Vigor5

0505A High 95.8 49.7 8.0 0.170 1.00 

0544J High 91.7 46.8 10.0 0.130 2.00 

0503J High 91.7 56.5 12.0 0.130 1.00 

0503I High 87.5 56.4 11.3 0.143 0.67 

0433M High 79.2 56.4 12.6 0.252 0.60 

0540K Moderately High 70.8 51.7 12.9 0.289 1.00 

0533C Moderately High 66.7 70.3 14.7 0.373 0.71 

0532A Moderately High 62.5 62.3 11.2 0.283 0.89 

0433A Moderately High 62.5 63.8 10.2 0.161 1.00 

0503M Moderate 58.3 65.9 12.2 0.264 0.50 

0431B Moderate 58.3 66.2 12.4 0.219 0.50 

0532P Moderate 58.3 76.7 16.1 0.388 1.00 

0528A Moderate 54.2 61.5 12.0 0.221 0.82 

0528D Moderate 54.2 63.6 12.6 0.234 0.73 

0429K Moderate 50.0 68.6 10.5 0.172 1.00 

0433K Moderate 50.0 68.8 12.5 0.232 0.83 

0433E Moderate 50.0 69.5 14.8 0.323 0.50 

0533F Moderate 50.0 72.6 13.9 0.261 0.75 

0540H Moderate 50.0 77.9 12.6 0.277 1.00 

0503L Moderate 45.8 69.5 12.1 0.241 0.54 

0532V Moderate 45.8 72.8 12.6 0.217 0.85 

0540R Moderate 45.8 73.7 14.8 0.294 0.67 

0540A Moderate 45.8 80.2 15.5 0.352 0.71 

0504B Moderate 41.7 69.6 10.5 0.195 1.07 

0531B Moderately Low 37.5 81.8 13.9 0.253 1.07 

0424F Moderately Low 33.3 76.2 11.5 0.199 0.94 

0531A Moderately Low 33.3 83.3 11.8 0.260 1.06 

0531F Moderately Low 33.3 84.0 14.5 0.275 0.88 
11 

0531M Low 25.0 85.9 14.5 0.274 0.74 

0531W Low 25.0 86.0 13.1 0.255 0.53 

0531O Low 20.8 83.0 14.9 0.285 0.75 

0554A Low 20.8 84.6 9.6 0.105 1.58 

0542O Low 17.4 84.2 8.2 0.093 2.00 

0544M Low 16.7 86.2 7.8 0.095 2.00 

0545K Nonpathogenic 8.3 84.3 7.7 0.095 2.09 

0625J Nonpathogenic 8.3 86.4 17.8 0.631 1.23 

0433F Nonpathogenic 8.3 87.0 7.5 0.098 1.95 

0433N Nonpathogenic 8.3 88.5 8.3 0.100 3.50 

0430B Nonpathogenic 8.3 89.3 8.7 0.098 2.09 

0554H Nonpathogenic 8.3 89.5 7.6 0.095 2.00 

0623J Nonpathogenic 4.2 87.3 20.2 0.824 1.13 

0629A Nonpathogenic 4.2 87.7 14.5 0.452 1.17 

0608G Nonpathogenic 4.2 87.7 18.1 0.540 1.26 

0624A Nonpathogenic 4.2 87.7 19.1 0.782 1.13 

0608A Nonpathogenic 4.2 88.0 17.8 0.604 1.13 

0542H Nonpathogenic 4.2 88.4 7.6 0.098 2.08 

0545G Nonpathogenic 4.2 88.6 9.1 0.100 1.96 

0545A Nonpathogenic 4.2 88.8 7.6 0.091 2.09 

0542J Nonpathogenic 4.2 89.3 8.5 0.096 2.00 

0528F Nonpathogenic 4.2 89.3 8.8 0.098 2.00 

0543H Nonpathogenic 4.2 89.4 9.1 0.093 2.09 

0544G Nonpathogenic 4.2 89.6 8.9 0.100 1.91 

0429D Nonpathogenic 4.2 89.8 8.3 0.093 1.65 

0542F Nonpathogenic 4.2 89.8 9.3 0.100 2.00 

0554N Nonpathogenic 4.2 90.0 6.5 0.098 2.09 

0554L Nonpathogenic 0 90.0 8.2 0.098 1.96 

0618A Nonpathogenic 0 90.0 19.4 0.646 1.00 

0619A Nonpathogenic 0 90.0 20.5 0.824 1.00 

0620A Nonpathogenic 0 90.0 21.0 0.855 1.04 

0621A Nonpathogenic 0 90.0 23.4 1.010 1.13 

0622A Nonpathogenic 0 90.0 23.5 1.060 1.04 

Control 1 - 4.2 87.8 10.9 0.099 0.61 

Control 2 - 4.2 87.6 14.3 0.385 0.91 1

Percent of 24 inoculated seedlings per isolate displaying foliar wilt, chlorosis or necrotic symptoms. 2

Average number of days [maximum = 90 days] inoculated seedlings survived without displaying disease symptoms. 

3

cm 

4

oven-dry weight (g) of both tops and roots 

5

average vigor rating based on a 5-point numerical system [0-4] 

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

96701; NDudley@harc-haspa.com.; R.L. James is Plant Pathologist, USDA Forest Service, Forest Health 

Protection, 3815 Schreiber Way, Coeur d’Alene, ID 83815; rjames@fs.fed.us. A. Yeh is Plant 

Propagation Specialist, Hawaii Agriculture Research Center, 942 W. Kawailani Street, Hilo, HI 96778; 

akfwyeh@hawaii.rr.com.