Interactions Between Fusarium Oxysporum F. Sp. Lycopersici

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1994

Interactions Between Fusarium Oxysporum F. Sp. Lycopersici and Interactions Between Fusarium Oxysporum F. Sp. Lycopersici and

Nonpathogenic Strains of Fusarium Oxysporum. Nonpathogenic Strains of Fusarium Oxysporum.

Monica Michelle Lear Louisiana State University and Agricultural & Mechanical College

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O rder N um ber 9502122

Interactions between Fusarium oxysporum f. sp. Lycopersici and nonpathogenic strains of Fusarium oxysporum

Lear, Monica Michelle, Ph.D.

The Louisiana State University and Agricultural and Mechanical Col., 1994

U M I300 N. ZeebRd.Ann Arbor, MI 48106



INTERACTIONS BETWEEN FUSARIUM OXYSPORUM F. SP. LYCOPERSICI AND

NONPATHOGENIC STRAINS OF FUSARIUM OXYSPORUM

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree of Doctor of Philosophy

in

The Department of Plant Pathology and Crop Physiology

byMonica Michelle Lear

B. S., Louisiana State University, 1982 . M. S., Louisiana State University, 1986

May 1994


ACKNOWLEDGEMENTS

I would like to thank the following people:

My committee, including my major professor, Dr. R. W. Schneider, Dr. C.A. Clark, Dr. J. Hoy, Dr. M. C. Rush, Dr. E. Dunigan, and Dr. T. Bricker, for serving and helping me complete this dissertation.

The office staff of the Department of Plant Pathology and Crop Physiology, including Mildred Charles and Jill Atwood but especially Mrs. Patricia Hives, for staying after me.

My family for their encouragement and support.

All my friends, whom I will not name for fear of forgetting someone very important, who helped me keep working at it especially when I didn't want to.

My co-workers at the Louisiana Department of Agriculture and Forestry, Horticulture and Quarantine Division, for their patience, support, and help.

Commissioner Bob Odom for pressing me until I finally finished this.

ii


TABLE OF CONTENTS

page

ACKNOWLEDGEMENTS ........................................................................................................... ii

LIST OF TABLES .................................................................................................................... jv

LIST OF FIG URES.................................................................................................................... vi

A B S T R A C T ..................................................................................................................................vii

CHAPTER

1 REVIEW OF LITERATURE.................................................................................. 1Literature Cited in Chapter 1 ..................................................................... 10

2 SELECTION OF AN ORANGE MUTANT OF FUSARIUMOXYSPORUM F. SP. LYCOPERSICI.......................................................... 14Introduction...................................................................................................... 15Materials and Methods ................................................................................ 16Results .............................................................................................................. 20Discussion.........................................................................................................28Literature Cited in Chapter 2 .....................................................................31

3 COMPETITIVE INFECTION STUDIES BETWEEN PATHOGENICAND NONPATHOGENIC ISOLATES OF FUSARIUM OXYSPORUM 33Introduction ......................................................................................................3 4Materials and Methods ................................................................................36Results ..............................................................................................................42Discussion.........................................................................................................50Literature Cited in Chapter 3 ................................................................ 54

4 ISOZYME ANALYSIS OF SELECTED VEGETATIVE COMPATIBILITYGROUPS OF NONPATHOGENIC FUSARIUM O X Y S P O R U M 58Introduction ......................................................................................................59Materials and Methods ................................................................................60R e s u lts ..............................................................................................................65Discussion........................................................................................................ 66Literature Cited in Chapter 4 .....................................................................71

APPENDICES

A MEDIA RECIPES...............................................................................................74B BUFFER SYSTEMS AND ENZYME STAINING SYSTEMS ..................81

VITA ............................................................................................................................................88


I

LIST OF TABLES

2.1. Mycelial growth and production of microconidia of orange mutants of Fusarium oxysporum f . sp. lycopersici andwild-type c o n tro ls ............................................................................. 22

2.2. Pathogenicity of orange mutants of Fusarium oxysporum f. sp. lycopersici and wild-type controls .................................................. 25

2.3. Evaluation of orange mutants of Fusarium oxysporum for sectoring following serial transfers on potatodextrose a g a r ..................................................................................... 26

2.4. Evaluation of orange mutants of Fusarium oxysporum for sectoring following serial transfers on 10% potatodextrose a g a r ..................................................................................... 27

3.1. Strains of Fusarium oxysporum used in the present studyand their designation by vegetative compatibility group ............. 40

3.2. Rate of infection by the orange mutant (ORA-22) in the presence of nonpathogenic isolates of £. oxysporum formembers of VCG 2028 ................................................................... 44

3.3. Rate of infection by the orange mutant (ORA-22) in the presence of nonpathogenic isolates of £. oxysporum formembers of VCG 2035 .................................................................... 45

3.4. Relative competitive infection of nonpathogenic Fusarium oxysporum isolated from tomato ro o ts ........................................... 46

3.5. Rate of infection by the orange mutant (ORA-22) in the presence of nonpathogenic isolates of E. oxysporum formembers of VCG 2050 ................................................................... 47

3.6. Rate of infection by the orange mutant (ORA-22) in the presence of nonpathogenic isolates of E. oxysporum formembers of VCG 2061 ................................................................... 48

4.1. Strains of Fusarium oxysporum used in the present study andtheir designation by vegetative compatibility group .................... 62

4.2. Enzymes, abbreviations, Enzyme Commission (E.C.) numbersand buffer systems used in this study ........................................... 64

iv


4.3. Putative allele distribution for 15 putative enzyme loci in 36 isolates of Eusarium oxysporum for symptomless tomato ro o ts .....................................................................

v


LIST OF FIGURES

2.1. Orange mutant (ORA-21) of Fusarium oxvsporum f. sp. lycppersici compared to wild-type Fusarium oxysporum

f. sp. lycopersici (LSU 3) (d) on (a) potato dextrose,(b) water, and (c) cornmeal agars .................................................. 23

2.2. Orange mutant (ORA-22) of Fusarium oxysporum f . sp. lycopersici compared to wild-type Fusarium oxysporum f. sp. lycopersici (LSU 3) (d) on (a) potato dextrose,

(b) water, and (c) cornmeal agars .................................................. 24

2.3. Orange mutants of Fusarium oxysporum f. sp. lycopersici and nonpathogenic £. oxysporum emerging from tomato roots on Komada's m edium ............................................................................. 30

3.1. Location of sites samples in Louisiana for nonpathogenic strainsof Fusarium oxysporum ................................................................... 38

3.2. Rate of infection by an orange mutant (ORA-22) of Fusarium oxysporum f. sp. lycopersici in the presence of nonpathogenic isolates of £. oxysporum in all VCGs ............................................. 49

4.1. Principle components analysis of isozyme polymorphisms innonpathogenic populations of Fusarium oxysporum .................... 68

vi


ABSTRACT

Race 1 isolates collected from tomato roots were used to develop an

orange mutant of Fusarium oxysporum f. sp. lycopersici (FPL) for use in

competitive infection tests against nonpathogenic £. oxvsporum isolates. Eighty

one color mutants were induced by exposure to ultraviolet light. The mutant ORA-

22 retained pathogenicity and growth habit similar to the wild-type.

Preliminary competitive infection tests indicated differences within the

nonpathogenic population in the ability to suppress or enhance infection by FPL

orange mutant ORA-22. Four vegetative compatibility groups (VCGs) were tested

to determine if competitive infection ability was associated with VCG designation.

Results from these tests revealed a wide range of effectiveness, ranging from no

effect to suppression or enhancement of root infection by the pathogen, FPL. All

members of a VCG showed the same reaction. The results suggest that

competitive infection ability and VCG are closely related traits. Isozyme analysis

by starch gel electrophoresis was used to estimate genetic diversity within and

among the four VCGs. Principal components analysis of the results showed that

each population was unique and clearly separated from the pathogenic controls.

These results provide strong evidence that the genetic traits associated with

competitive infection ability are confined to genetically isolated populations of

nonpathogenic E. oxvsporum as delimited by VCG.

vii


CHAPTER 1

REVIEW OF LITERATURE

1


I

2

Fusarium oxysporum (Schlect.) Snyd. & Hans, is a soilborne fungus

which includes strains that cause vascular wilts of numerous cultivated

plant species including vegetables, flowers, herbaceous perennial

ornamentals, and plantation crops (1.1). Infection also has been reported

on noncultivated species (1.4, 1.7,1.10, 1.16, 1.20). The pathogen is

worldwide in distribution, and it is the most frequently isolated of all

Fusarium species in agricultural and nonagricultural soils (1.12). It may

exist as a saprophyte, parasite, or pathogen (1.10, 1.12, 1.32). The

fungus produces chlamydospores which allow it to persist in soils for many

years without a susceptible host (1.10). Agricultural soils may contain

between 103 and 105 propagules of £. oxysporum per gram (1.12).

Tomato (Lycopersicon esculentum L. Mill.) is one of the most widely

grown vegetable crops worldwide (1.19, 1.35). Fusarium w ilt, caused by

Fusarium oxysporum (Schlect.) f. sp. lycopersici (Sacc.) Snyder & Hansen

(EQL), is a recurring and devastating disease of tomato that has been

reported in at least 32 countries (1.19). It is particularly serious because of

the development of new races of the pathogen (1.14, 1.19, 1.21, 1.34).

There are three known races of FPL that are distinguished by their

pathogenicity to cultivars with specific dominant genes for resistance

(1.24). Race 1 was first described in 1886 (1.9), and race 2 was initially

reported in 1945 but did not cause serious crop damage until 1961 in

Florida (1.31). Race 3 was reported in Australia in 1978 (1.17) and was


found in Florida in 1982 (1.34) and California in 1987 (1.14). Race 3 must

now be considered a major threat to the tomato industry because of limited

sources of resistance to this race (1.34) and the time required to

incorporate resistance into horticulturally acceptable cultivars.

Control of Fusarium wilts is limited to the use of resistant varieties,

cultural practices, and soil fumigation (1.1, 1.30). Soil fumigation has been

used with success, but it is expensive and the effect does not last long.

As costs of petrochemicals rise and the environmental effects of these

pesticides are elucidated, use of soil fumigation will probably have to be

abandoned. Cultural practices effective in reducing pathogen populations

include deep plowing, crop rotation, leaving the soil fallow, and the flooding

of fields (1.1). For greenhouse crops, soil sterilization and sanitation are

effective control measures. When available, the use of resistant varieties is

the most desirable and effective means of control.

Drawing momentum from the growing public concern regarding the

widespread use of hazardous chemicals in disease control, biological control

of plant pathogens has recently received considerable attention throughout

the world (1.2, 1.3, 1.6, 1.7, 1.11, 1.12, 1.23, 1.25, 1.26 1.27, 1.29,

1.36). Biological control is defined as the total or partial destruction of

pathogen populations by other organisms; or the use by man of an

organism for pathogen control (1.1, 1.12). Cook and Baker (1.12) listed

three general approaches to biological control of plant pathogens: (i)


control of pathogen inoculum, (ii) protection against infection, and (iii)

cross-protection or induced resistance. Mechanisms of biological control by

antagonists are antibiosis, competition, and parasitism (1.11, 1.12). The

form of biological control which has been the most successful against

formae species of £. oxysporum is competition. Competition occurs when

two or more organisms require the same resource and the use of that

resource by one reduces the amount available to the other (1.11).

Disease suppressive soils are defined as those soils in which disease

development is suppressed even though the pathogen and a susceptible

host are present (1.30). Examples of soils suppressive to Fusarium wilts

have been found in California, Florida, and the Chateaurenard region in

France (1.3, 1.23, 1.29, 1.33). Toussoun (1.33) showed that some soils

are suppressive to pathogenic formae speciales, but not to nonpathogenic

populations of E. oxysporum. Alabouvette et al. (1.3) considered soil to be

a living entity which manifests a certain resistance to infestation and to the

expression of the pathogenic capabilities of an organism. Using E.

oxysporum f. sp. melonis (FOM). the causal agent of Fusarium w ilt of

muskmelon, they showed that soil suppressiveness to FOM could be

transferred to other soils and that chlamydospore germination was lowered

in these suppressive soils. They also concluded that nonpathogenic strains

of E. oxysporum and E. solani were the organisms responsible for soil

suppressiveness in soils in the Chateaurenard region in France. They


5

proposed that the mechanism of suppressiveness to Fusarium w ilt of

muskmelon involved competition for the same ecological niche between the

pathogenic forma and saprophytic strains of £. oxysporum. Schneider,

working with celery, (1.29) was the first to demonstrate that some

nonpathogenic, parasitic strains of £. oxysporum predisposed plant roots to

infection by the pathogenic forma, £. a. f. sp. apii. while others

competitively suppressed infection. Furthermore, he provided evidence for

and concluded that naturally-occurring disease suppression was caused by

relatively high populations of effective, nonpathogenic root-infecting strains

of £. oxysporum. Because the pathogen is morphologically

indistinguishable from the competitors, Schneider used a pigmented mutant

of the pathogen in his investigations (1.29). Colonies growing from the

roots were easily differentiated on the basis of pigmentation. Larkin et al.

(1.22) reported the development of a soil suppressive to Fusarium w ilt of

watermelon, Fusarium oxysporum f. sp. niveum. which was induced by

monoculture to the moderately resistant Crimson Sweet cultivar. A

population of nonpathogenic £. oxysporum as well as bacteria,

actinomycetes, and fluorescent pseudomonads were present in this

suppressive soil which were not present in the conducive soils. Paulitz,

Park and Baker (1.27) isolated nonpathogenic strains of £. oxysporum from

symptomless surface-sterilized cucumber roots. They showed that some of

these isolates were effective while others were ineffective in reducing the


rate of infection by the pathogen. They found one isolate which

significantly reduced the rate of infection by the pathogen, £. £. f. sp.

cucumerinum (FOCI, in raw soil. Park et al. (1.26) later showed that

nonpathogenic isolates of £. oxysporum and fluorescent pseudomonads,

which are ineffective alone, induced suppressiveness against FOC when

used in combination. Based on the work of Schneider (1.29) and Baker

(1.5), Alabouvette (1.2) reassessed his suppressive isolates and compared

them for saprophytic and parasitic competitive effectiveness. He found

that some isolates were saprophytically effective, while others were

effective parasitically, there being no correlation between the two.

Cross-protection is a form of biological control in which a biocontrol

agent induces resistance in a host rather than being directly antagonistic to

the pathogen. Resistance to a pathogen may be induced by the previous

inoculation of a plant with an incompatible race of the pathogen or

nonpathogenic form of the fungus. However, it should be noted that cross

protection is generally achieved following direct inoculation of the

susceptible plant organ with extremely high inoculum levels and is short

lived (1.8, 1.36). Ogawa and Komada (1.25) used £. oxysporum to pre

inoculate sweet-potato cuttings. They found that these inoculated plants

were protected from soilborne inoculum of £. £. f. sp. batatas as well as

that transmitted from infected roots. Biles and Martyn (1.8) pre-inoculated

watermelon seedlings with either FPL or avirulent races of £. q. niveum 24


or 72 hr prior to challenge with a virulent race of £. £>. f. sp. niveum. All

treatments significantly reduced w ilt symptoms, but pre-inoculation with

avirulent races of £. q . niveum induced a higher level of resistance than the

other treatments.

Nonpathogenic strains of £. oxysporum. which survive in soil as

saprophytes and parasites, constitute the largest proportion of the soilborne

population (1.10, 1.33). These highly competitive saprophytes are able to

utilize a wide variety of organic substrates, including cellulose, pectin,

lignin, and other complex materials (1.11). Currently, nonpathogenic

saprophytes and parasites are included in a single taxonomic group because

they are morphologically indistinguishable from each other and from

pathogenic strains of £. oxysporum. Both pathogenic and nonpathogenic

strains of £. oxysporum are known to infect roots of weeds and other

nonsusceptible hosts, including cultivated and noncultivated species (1.4,

1.7, 1.10, 1.16, 1.18, 1.20).

Until recently, the role of nonpathogenic strains has been largely

ignored, probably because of the difficulties involved in identification. In

1983, Puhalla (1.28) developed a technique to assign isolates of £.

oxysporum to vegetative compatibility (heterokaryon) groups (VCG) using

nitrate metabolism (nil) mutants. When two different n il genotypes are

paired on minimal medium, complementation may occur. Complementation

is demonstrated by the formation of a dense aerial mycelium at the


interface of the two colonies. Heterokaryon formation occurs when the

tw o o il mutants have identical alleles at all vegetative compatibility loci.

Heterokaryons or vegetatively compatible isolates are known to be closely

related if not clonal derivatives (1.15, 1.28). Correll, Puhalla and Schneider

(1.13) showed that VCG could be used to separate populations of

nonpathogenic root-infecting strains of £. oxysporum from celery roots. In

a more detailed study, Elias, Schneider and Lear (1.15) conducted a large

survey and VCG analysis of nonpathogenic isolates of £. oxysporum from

tomato roots collected from eight sites in Louisiana and found a more

diverse population than that of celery. Isolates within a VCG were not

vegetatively compatible with isolates from other VCGs or pathogenic

isolates of FPL.

A primary conclusion to be drawn from the results discussed above

is that the population of nonpathogenic strains of £. oxysporum associated

with tomato is quite diverse. It was hypothesized that if certain segments

of the nonpathogenic population of £. oxysporum can effectively compete

with the pathogenic forma for the same ecological niche, then it could lead

to the reduction or suppression of infection and disease severity, a form of

biological control by antagonism. The objectives of this study were to (i)

determine the influence of nonpathogenic strains of £. oxysporum on

tomato root infection by the pathogen, £. q. f. sp. lycopersici: (ii) to use

vegetative compatibility groups (VCGs) as a qualitative measure of genetic


diversity within the nonpathogenic population; and (iii) to obtain

quantitative measures of genetic diversity within and among VCGs (clonal

populations) of nonpathogenic strains of £. oxysporum.


10

Literature Cited in Chapter 1

1.1. Agrios, G. N. 1988. Plant Pathology. 3rd ed. Academic Press, San Diego. 803 pp.

1.2. Alabouvette, C. 1990. Biological control of Fusarium w ilt pathogens in suppressive soils. Pages 27-43 in: Biological Control of Soil-borne Pathogens. D. Hornsby, ed. CAB International, United Kingdom.

1.3. Alabouvette, C., F. Rouxel, and J. Louvet. 1979. Characteristics of Fusarium w ilt suppressive soils and prospects for their utilization in biological control. Pages 165-182 in: Soil-borne Plant Pathogens.B. Schippers and W. Gams, eds. Academic Press, London. 686 pp.

1.4. Armstrong, G. M., and J. K. Armstrong. 1948. Nonsusceptible hosts as carriers of w ilt fusaria. Phytopathology 38:808-826.

1.5. Baker, R. 1980. Measures to control Fusarium and Phialophora w ilt pathogens of carnation. Plant Dis. 64:743-749.

1.6. Baker, R., P. Hanchey, and S. D. Dottarar. 1978. Protection of carnation against Fusarium stem rot by fungi. Phytopathology 68:1495-1501.

1.7. Beckman, C. H. 1987. The Nature of Wilt Diseases in Plants. American Phytopathology Press, St. Paul. 175 pp.

1.8. Biles, C. L., and R. D. Martyn. 1989. Local and systemic resistance induced in watermelons by formae speciales of Fusarium oxysporum. Phytopathology 79:856-860.

1.9. Booth, C. 1971. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England. 237 pp.

1.10. Burgess, L. W. 1981. General ecology of the Fusaria. Pages 225- 235 in Fusarium: Diseases, Biology and Taxonomy. P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. The Pennsylvania State University Press, University Park.

1.11. Campbell, R. 1989. Biological control of microbial plant pathogens. Cambridge University Press, Cambridge. 218 pp.


11

1.12. Cook, R. J., and K. F. Baker. 1983. The Nature and Practice of Biological Control of Plant Pathogens. The American Phytopathological Society, St. Paul. 539 pp.

1.13. Correll, J. C., J. E. Puhalla and R. W. Schneider. 1986.Identification of Fusarium oxysporum f . sp. a p ii on the basis of colony size, virulence, and vegetative compatibility. Phytopathology 76:396-400.

1.14. Davis, R. M., K. A. Kimble and J. J. Farrar. 1988. A third race of Fusarium oxysporum f. sp. lycopersici identified in California. Plant Dis. 72:453.

1.15. Elias, K. S., R. W. Schneider, and M. M. Lear. 1991. Analysis of vegetative compatibility groups in nonpathogenic populations of Fusarium oxysporum isolated from symptomless tomato roots. Can. J. Bot. 69:2089-2094.

1.16. Gordon, T. R., D. Okamoto, and D. J. Jacobsen. 1989.Colonization of muskmelon and nonsusceptible crops by Fusarium oxysporum f. sp. melonis and other species of Fusarium. Phytopathology 79:1095-1100.

1.17 Grattidge, R., and R. G. O'Brien. 1982. Occurrence of a third race of Fusarium w ilt of tomatoes in Queensland. Plant Dis. 66:165- 166.

1.18. Hendrix, D. L. and L. W. Nielsen. 1958. Invasion and infection of crops other than the forma suscept by Fusarium oxysporum f. sp. batatas and other formae. Phytopathology 48:224-228.

1.19. Jones, J. P., and S. S. Woltz. 1981. Fusarium-incited diseases of tomato and potato and their control. Pages 157-168 in: Fusarium: Diseases, Biology and Taxonomy. P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. The Pennsylvania State University Press, University Park.

1.20. Katan, J. 1971. Symptomless carriers of the tomato Fusarium w ilt pathogen. Phytopathology 61:1213-1217.

1.21. Katan, J., and R. Ausher. 1974. Distribution of race 2 Fusarium oxysporum f. sp. lycopersici in tomato fields in Israel.Phytoparasitica 2:83-90.


12

1.22. Larkin, R. P., D. L. Hopkins, and F. N. Martin. 1987. Cultivar- specific induction of soil suppressiveness to Fusarium w ilt of watermelon. Phytopathology 77:607-611.

1.23. Louvet, J., F. Rouxel, and C. Alabouvette. 1976. Recherches sur la resistance des sols aux maladies. I. Mise en evidence de la nature microbiologique de la resistance d'un sol au developpement de la fusariose vasculaire du melon. Ann. Phytopathol. 8:425-436.

1.24. McGrath, D. J., D. Gillespie, and L. Vawdrey. 1987. Inheritance of resistance to Fusarium oxysporum f. sp. lycopersici races 2 & 3 in Ivcopersicon pennelli. Aust. J. Agric. Res. 38:729-733.

1.25. Ogawa, K. and H. Komada. 1985. Biological control of Fusarium w ilt w ith cross-protection by nonpathogenic Fusarium oxysporum. Pages 121-123 in: Ecology and Management of Soilborne Plant Pathogens. C. A. Parker, A. D. Rovira, K. J. Moore and P. T. W. Wong, eds. The American Phytopathological Society, St. Paul.

1.26. Park, C. S., T. C. Paulitz, and R. Baker. 1988. Biocontrol of Fusarium wilt of cucumber resulting from interactions between Pseudomonas putida and nonpathogenic isolates of Fusarium oxysporum. Phytopathology 78:190-194.

1.27. Paulitz, T. C., C. S. Park, and R. Baker. 1987. Biological control of Fusarium w ilt of cucumber with nonpathogenic isolates of Fusarium oxysporum. Can. J. Microbiol. 33:349-353.

1.28. Puhalla, J. E. 1985. Classification of strains of Fusarium oxysporum on the basis of vegetative compatibility. Can. J. Bot. 63:179-183.

1.29. Schneider, R. W. 1984. Effects of nonpathogenic strains of Fusarium oxysporum on celery root infection by Fusarium oxysporum f. sp. apii and a novel use of the Lineweaver-Burk double reciprocal plot technique. Phytopathology 74:646-653

1.30. Schneider, R. W., and D. M. Huber 1982. Suppressive Soils and Plant Disease. The American Phytopathological Society, St. Paul.88 pp.

1.31. Stall, R. 1961. Development of Fusarium w ilt on resistant varieties of tomato caused by a strain different from race 1 isolates of Eusarium oxysporum f. lycopersici. Plant Dis. Reptr. 45:12-15.


13

1.32. Stoner, M. F. 1981. Ecology of Fusarium in noncultivated soils. Pages 276-286 in: Fusarium: Diseases, Biology and Taxonomy. P.E. Nelson, T. A. Toussoun, and R. J. Cook, eds. The Pennsylvania State University Press, University Park.

1.33. Toussoun, T. A. 1975. Fusarium-suppressive soils. Pages 145-151 in: Biology and Control of Soil-Borne Plant Pathogens. G. W.Bruehl, ed. American Phytopathological Society, St. Paul. 216 pp.

1.34. Volin, R. B., and J. P. Jones. 1982. A new race of Fusarium w ilt of tomato in Florida and sources of resistance. Proc. Fla. State Hort. Soc. 95:268-270.

1.35. Walker, J. C. 1971. Fusarium Wilt of Tomato. Monograph No. 6. The American Phytopathological Society, St. Paul. 56 pp.

1.36. Wymore, L. A., and R. Baker. 1982. Factors affecting cross protection in control of Fusarium w ilt of tomato. Plant Dis. 6:908- 910.


CHAPTER 2

SELECTION OF AN ORANGE MUTANT OF FUSARIUM OXYSPORUMF. SP. LYCOPERSICI

14


15

Introduction

Fusarium oxysporum is a soilborne fungus which includes strains that

cause vascular wilts of a wide variety of crop plants worldwide (2.12).

Most isolates from soil are nonpathogenic and exist as saprophytes or

parasites (2.2). Strains of E. oxysporum. including pathogens and

nonpathogens, are generally morphologically indistinguishable.

Nonpathogens have been largely ignored because of the difficulty in

identifying strains even though they are known to interact with pathogenic

strains (2.2). In order to conduct ecological studies, it is necessary to be

able to recognize and quantify populations of introduced strains relative to

other populations of E. oxysporum.

Ultraviolet irradiation of fungi may be used to induce color mutants

(2.6, 2.9, 2.10) as well as mutants which are resistant to fungicides such

as benomyl. Color mutants are advantageous as compared to benomyl

resistant mutants because, following selection and preliminary screenings,

they are well adapted to surviving in the soil and they can be observed

together with the pathogen on the sane plate of selective media. Therefore,

in studies involving introduced strains, these mutants can be used to

provide a fast and accurate means of identification and separation of the

nonpathogenic and pathogenic populations of E. oxysporum. They can then

be used to investigate interactions between the pathogenic and

nonpathogenic populations within the rhizosphere.


16

The objective of this study was to induce pigmented mutants and

select and test selected mutants of race 1 £. oxysporum f. sp. lycopersici

for use in subsequent studies.

Materials and Methods

Mutagenesis and selection of orange mutants. In order to quantitatively

monitor root infection by the pathogen, £. a. f. sp. lycopersici. pigmented

mutants were sought which could be readily distinguished from

nonpathogens on Komada's agar medium (2.4), which is selective for £.

oxysporum. Pathogenic orange mutants of FPL were selected by irradiating

the following race 1 isolates using a modification of Puhalla's method (2.9):

PS-1, BFOL-54, BFOL-56, BFOL-67, LSU-3, LSU-4, LSU-5, and LSU-6.

Isolates were plated on minimal medium (MM) or potato dextrose agar

(PDA) (Appendix A) and incubated for 5-10 days at room temperature. Ten

ml of sterile, distilled water were pipetted onto the agar surface, and the

cultures were scraped with a flame-sterilized bent glass rod. The resulting

conidial suspension was filtered first through sterile cheesecloth into a

sterile beaker to remove pieces of mycelium and then through a 1.0 //m

mesh screen to separate macroconidia and chlamydospores from

microconidia. The microconidia were counted with a hemacytometer,

diluted to 104 per ml with sterile, distilled water, poured into petri dishes

containing a magnetic stir bar, and placed on a magnetic stir plate for

agitation. The plates were irradiated for 70 sec using a short-wave


17

ultraviolet lamp (4 watts) placed 14 cm above the uncovered stir plate in a

darkened room. These parameters were determined previously by

constructing a kill curve and selecting distance and time to give 99% kill

(2.9). After irradiation, all work was performed in darkness. Irradiated

microconidia were plated onto MM at a rate of 30 to 300 colony forming

units per plate. The plates were wrapped in aluminum foil in groups of 10

and incubated for 7-10 days at room temperature. Orange colored mutants

were selected, single-spored, and stored on silica gel at 4 C (2.8).

Inoculum preparation. Orange mutant cultures were grown in PDB on a

rotary shaker for 72 hr at room temperature. One hundred grams of finely

ground corncob grit (Grit O'Cob, Nebraska) in 250-ml Erlenmeyer flasks

were moistened with 10 ml 0.025M L-asparagine, autoclaved for 1 hr on

each of tw o consecutive days, inoculated with a conidial suspension (106 /

ml) obtained from the PDB cultures, and incubated for 2 wk at room

temperature. The ground corncob cultures, hereafter referred to as

inoculum, were dried at room temperature in 100mm by 80mm deep petri

plates covered with cheesecloth, ground with a mortar and pestle, and

refrigerated until used.

Pathogenicity of orange mutants. Greenhouse pathogenicity tests and race

determinations were performed using the orange mutant isolates and

nonmutagenized controls. The tomato cultivars 'Fantastic' (susceptible to

races 1, 2, and 3); 'Supersonic' (resistant to race 1); and 'Walter' (resistant


18

to races 1 and 2) were used as hosts. Fungal strains were grown in

Czapek's solution for 5 days on an orbital shaker at room temperature. The

cultures were filtered through cheesecloth and the resulting spore

suspension was adjusted to about 106 spores per ml. Seedlings were

grown in sterile, commercially prepared potting soil mix for 2 wk until the

first true leaves emerged. The root dip method of inoculation was used

(2.13). Seedlings were gently uprooted, and roots were pruned to about 3

cm in length. Six seedlings per cultivar were dipped into spore suspensions

of each strain for 30 sec and transplanted to plastic cell trays pre-filled with

soil-peat moss mix (50:50, v:v) at the rate of one seedling per cell. Two

controls were used: seedlings with pruned roots were dipped in either

sterile water or inoculum of a race 1 FPL isolate. Disease symptoms were

recorded 10-14 days after inoculation. Pathogenicity tests using the

orange mutant isolates were conducted three times.

Phenotypic tests. Tests were performed using orange mutant isolates to

compare production of microconidia, colony growth and morphology, and

reversion to wild-type. Production of microconidia was determined by

growing orange mutant isolates in potato dextrose broth (PDB) at room

temperature on an orbital shaker for 36 hr. At that time, numbers of

conidia per ml broth were determined with a hemacytometer. Serial transfer

tests were performed using 26 single-spored orange mutant isolates plated

on full strength and 10% PDA and mass transferred weekly to test for


19

reversion of mutants. Appearance of wild-type sectors was taken as

presumptive evidence for reversion. Radial growth of orange mutants and

wild-type FPL was measured at 16 and 24 C on full strength potato

dextrose, cornmeal and water agars for 5 days.

Inoculum density tests. Preliminary experiments were performed to

determine the appropriate amount of inoculum to incorporate into soil for

competitive infection experiments. The objective was to determine the

amount of inoculum to add to soil to give 50% of the maximum attainable

root infection measured as infections per 100 cm root length. In this way,

both increases and decreases in root infection could be determined. One kg

of sterile soil was amended with 0.25, 0.50, 0.75, or 1.0 g of orange

mutant inoculum, blended thoroughly in a large plastic bag, and dispensed

into 10-cm clay pots. Tomato seedlings, cultivar 'Supersonic', were

transplanted into the pots and grown for 4 wk in the greenhouse. Also

included were a noninoculated control and a noninoculated, sterilized

corncob grit control. At harvest, the plants were removed from the pots,

immersed in water, and the soil was carefully washed from the roots. The

roots were surface sterilized for 4 min in 0.21 % household bleach (2.10),

rinsed in three changes of sterile water, plated on Komada’s medium (2.4),

and incubated at room temperature for 1 wk. The number of orange

colonies was determined for each plate. Root lengths on each plate were

estimated using the line intersect grid method (2.7, 2.11). The number of


20

orange colonies and root lengths were used to calculate the number of

colonies per 100 cm of root (2.10). Briefly, plates with roots were placed

on a grid of known line length. The number of root-line intersections was

determined, and the root length was estimated by the following

equation:

7i N AR = ----------

2 H

where R = root length, N = number of intersections, A = area of the grid,

and H = grid line length.

Results

Mutagenesis and selection of orange mutants. Eighty one orange

mutant isolates were obtained. Forty orange mutants were recovered from

isolate LSU-4, 14 from BFOL-67, and 17 from LSU-3. These three wild-type

isolates were all race 1 of FPL. Isolates PS-1, BFOL-54, BFOL-56, LSU-5,

and LSU-6 did not produce any orange mutants. Several yellow, pink, and

purple mutants were generated from these isolates. Orange mutants

selected for final studies had a vigorous growth habit and were medium to

deep orange in color.

Phenotypic tests

Mycelial growth on PDA at 16 and 24 C. A total of 26 mutant isolates and

three controls were grown on PDA at 16 and 24 C. Generally, there was a


21

wide range of colony morphology with most orange mutants having a less

vigorous mycelial growth as compared to the wild type controls, LSU-4,

LSU-3, and BFOL-67, from which they were derived (Table 2.1). Isolates

ORA-6, ORA-21, ORA-22, ORA-23, ORA-24, ORA-25, and ORA-26 had

mycelial growth most comparable to that of the controls (Figures 2.1 and

2.2). Measurements for the orange mutants are significantly different from

the parents.

Pathogenicity of orange mutant isolates. Determination of

pathogenicity in most isolates was straightforward, in that all or none of a

particular cultivar died within 10-14 days after inoculation. The isolates

were variable in pathogenicity. Four of the 27 orange mutant isolates (ORA-

21, ORA-22, ORA-24, ORA-25) were determined to be virulent race 1

pathogens (Table 2.2). All other mutants were nonpathogenic.

Serial transfer studies. Isolates ORA-1 through ORA-20 were subjected to

14 weekly transfers onto PDA and 10% PDA, and isolates ORA-21 through

ORA-26 were subjected to 10 weekly transfers onto PDA and 10% PDA

(Tables 2.3 and 2.4). The rate of sectoring of the isolates was low. In

addition, single-spores were transferred at random from these plates in an

effort to detect changes in color or growth habit. No sectoring or mutation

was observed.


22

Table 2.1. Mycelial growth and production of microconidia of orange mutants of Fusarium oxysporum f. sp. lycopersici and wild-type controls.

Isolate Colony diameter (cm)A 16C 24C

microconidia/mlB

ora-1 3.4 4.1 NDCora-2 2.6 3.8 NDora-3 2.8 4.2 NDora-4 2.0 3.6 NDora-5 3.8 7.3 NDora-6 5.0 8.0 NDora-7 2.8 4.4 NDora-8 2.4 4.2 NDora-9 2.5 4.5 NDora-10 2.6 4.4 NDora-11 2.9 4.2 NDora-12 2.8 4.0 NDora-13 2.9 4.3 NDora-14 2.8 4.4 NDora-15 2.8 3.8 NDora-16 2.6 4.0 NDora-17 2.8 4.0 NDora-18 2.8 4.4 NDora-19 2.8 3.8 NDora-20 2.7 4.5 NDora-21 3.5 7.3 8.49/1 O'ora-22 4.5 7.6 8.83/10'ora-23 4.7 7.5 NDora-24 4.0 7.6 NDora-25 5.0 8.0 1.84/10ora-26 4.7 8.0 NDLSU-4 4.4 7.9 NDLSU-3 4.8 7.5 2.19/10BFOL-67 4.9 8.0 ND

Agrown on potato dextrose agar.BPDB cultures inoculated with 10 ml of inoculum and incubated for 48 hrs. cnot determined.


23

Figure 2.1. Orange mutant (ORA-21) of Fusarium oxysporum f. sp. lycopersici compared to wild-type Fusarium oxysporum f. sp. lycopersici (LSU 3)(d) on (a) potato dextrose, (b) water, and (c) cornmeal agars.


24

Figure 2.2. Orange mutant (ORA-22) of Fusarium oxysporum f. sp. lycopersici compared to wild-type Fusarium oxysporum f. sp. I.ycppersisi (LSU 3)(d) on (a) potato dextrose, (b) water, and (c) cornmeal agars.


25

Table 2.2. Pathogenicity of orange mutants of Fusarium oxysporum f. sp. lycopersici and wild type controls.

Isolate PathogenicityA test results

Origin6

ora-1 0 LSU-4ora-2 0 LSU-4ora-3 0 LSU-4ora-4 0 LSU-4ora-5 0 LSU-4ora-6 0 LSU-4ora-7 0 LSU-4ora-8 0 LSU-4ora-9 0 LSU-4ora-10 0 LSU-4ora-11 0 LSU-4ora-12 0 LSU-4ora-13 0 LSU-4ora-14 0 LSU-4ora-15 0 LSU-4ora-16 0 LSU-4ora-17 0 LSU-4ora-18 0 LSU-4ora-19 0 LSU-4ora-20 0 LSU-4ora-21 6 LSU-3ora-22 6 LSU-3ora-23 0 BFOL-67ora-24 6 BFOL-67ora-25 6 LSU-3ora-26 0 BFOL-67LSU-4 6LSU-3 6BFOL-67 0

A Number of plants wilted or dead after 10 days.B LSU-3 and LSU-4 were isolated from Plaquemines Parish, LA, and BFOL-

67 was isolated from Deltaville, MS.


26

Table 2.3. Evaluation of orange mutants of Fusarium oxysporum for sectoring following serial transfers on potato dextrose agar.

Isolate No. of transfers No. of transfers at which sectors appearedA

ora-1 14 NSora-2 14 NSora-3 14 NSora-4 14 5ora-5 14 NSora-6 14 NSora-7 14 NSora-8 14 2ora-9 14 NSora-10 14 NSora-11 14 NSora-12 14 NSora-13 14 NSora-14 14 NSora-15 14 NSora-16 14 NSora-17 14 3ora-18 14 NSora-19 14 NSora-20 14 NSora-21 10 NSora-22 10 NSora-23 10 NSora-24 10 NSora-25 10 NSora-26 10 NS

ANo sectors were observed during the course of this study.


27

Table 2.4. Evaluation of orange mutants of Fusarium oxysporum for sectoring following serial transfers on 10% potato dextrose agar.

Isolate No. of transfers No. of transfers at which Asectors appeared

ora-1 14 2Bora-2 14 NSora-3 14 NSora-4 14 NSora-5 14 11ora-6 14 NSora-7 14 NSora-8 14 NSora-9 14 NSora-10 14 NSora-11 14 NSora-12 14 NSora-13 14 NSora-14 14 NSora-15 14 NSora-16 14 NSora-17 14. NSora-18 14 NSora-19 14 NSora-20 14 NSora-21 10 NSora-22 10 NSora-23 10 NSora-24 10 NSora-25 10 NSora-26 10 NS

AAII isolates are orange colored except those noted with asterisks which are purple.

BNo sectors were observed during the course of this study.


28

Discussion

The process of secondary metabolism in fungi results in the

production of metabolites with no obvious chemical function in the

organism (2.3). Examples of secondary metabolites produced by fungi

include penicillin, aflatoxins, carotenoids such as P-carotene and

xanthophylls. Carotenoids are lipid compounds and may be colorless or

pigmented yellow, orange, red or purple. In vivo, they are located in

lipophilic, hydrophobic regions in the cell and in association with protein in

membranes(2.1). In fungi, carotenoids function in photoreception and

photoprotection. In addition, p-carotene is a precursor to trisporic acid,

which controls gametogenesis in the fungus Blakeslea trispora (2.5).

Orange mutants have been previously used in ecological studies with

£. oxysporum (2.6, 2.10). Orange mutants, as compared to a drug

(benomyl) resistant mutants, allow for the use of a colored pathogen in

coinoculation studies because the mutant can be separated from

nonpathogenic £. oxysporum and other soil fungi by the use of Komada's

medium (2.4), which is selective for Fusarium spp. Pathogens can be

separated from nonpathogens on the same petri dish by the orange

pigmentation.

Nagao and Hirano (2.6) looked at pathogenicity, morphological

features, and survival in soil of UV-irradiated orange mutants of £. a . f. sp.

cucumerinum (FO_C). They concluded that their mutants retained the


29

features and behaviors of wild-type FOC. The studies described herein and

those conducted previously (2.6, 2.10) show conclusively that

pigmentation is a valid marker for use in ecological studies. A total of

eighty one orange mutant isolates were obtained from UV irradiation of £.

oxysporum f. sp. lycopersici race 1 isolates. In addition, yellow, pink and

purple isolates were obtained. Isolate ORA-22 was selected from the

collection because of its similarity to the wild-type in growth habit and

pathogenicity as well as its deep orange coloration. An example is shown

in Figure 2.3.


Figure 2.3. Orange mutants of Fusarium oxysporum f. sp. lycopersici and nonpathogenic £. oxysporum emerging from tomato roots on Komada's medium.


31

2 .1.

2.2.

2.3.

2.4.

2.5.

2.6.

2.7.

2.8.

2.9.

2 .10.

2 .11.


Britton, G. 1971. The Biochemistry of Natural Pigments.Cambridge University Press, Cambridge. 366 pp.

Burgess, L. W. 1981. General ecology of the Fusaria. Pages 225- 235 in Fusarium: Diseases, Biology and Taxonomy. P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. The Pennsylvania State University Press, University Park.

Garroway, M. 0 ., and R. C. Evans. 1984. Fungal Nutrition and Physiology. John Wiley & Sons, New York. 401 pp.

Komada, H. 1975. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev. Plant Protection Res. 8:114-124.

Krinsky, N. I. 1971. Chapter X. Functions. Pages 669-716 in: Carotenoids. 0. Isler, ed. Birkhauser Verlag, Basel.

Nagao, H. and K. Hirano. 1990. Survival and pathogenicity of the orange mutant of Fusarium oxysporum f. sp. cucumerinum. Ann. Phytopath. Soc. Japan 56:185-193.

Newman, E. I. 1966. A method of estimating the total length of root in a sample. J. Appl. Ecol. 3:139-145.

Perkins, D. D. 1962. Preservation of Neurospora stock cultures with anhydrous silica gel. Can J. Microbiol. 8:591-594.

Puhalla, J. E. 1984. A visual indicator of heterokaryosis in Fusarium oxysporum from celery. Can. J. Bot. 62:540-545.

Schneider, R. W. 1984. Effects of nonpathogenic strains of Fusarium oxysporum on celery root infection by Fusarium oxysporum f. sp. apii and a novel use of the Lineweaver-Burk double reciprocal plot technique. Phytopathology 74:646-653.

Torssell, W. R., J. E. Begg, C. W. Rose, and G. F. Byrne. 1968. Stand morphology of Townsville lucerne (Stylosanthes humilis). Seasonal growth and root development. Aust. J. Exp. Agric. Anim. Hub. 8:532-543.


32

2.12. Toussoun, T. A. 1975. Fusarium-suppressive soils. Pages 145-151 in: Biology and Control of Soil-Borne Plant Pathogens. G. W.Bruehl, ed. American Phytopathological Society, St. Paul.

2.13. Williams, P. H. 1981. Fusarium yellows. Pages 124-129 in: Screening Crucifers for Multiple Disease Resistance. P. H. Williams, ed. University of Wisconsin Press, Madison.


CHAPTER 3

COMPETITIVE INFECTION STUDIES BETWEEN PATHOGENIC AND NONPATHOGENIC ISOLATES OF FUSARIUM OXYSPORUM

33


34

Introduction

Fusarium oxysporum is comprised of more than 120 formae speciales

and races as well as nonpathogenic strains (3.3). The nonpathogenic

strains of £. oxysporum comprise the largest proportion of the soilborne

population (3.7, 3.29). The pathogenic and nonpathogenic strains form a

homogeneous taxonomic group because they are morphologically

indistinguishable from one another. Both are known to infect the roots of

host and nonhost plants (3.2, 3.5, 3.7, 3.13, 3.14, 3.15), and

nonpathogenic strains may colonize the root cortex without causing

disease. Thus, pathogenic and nonpathogenic strains are well adapted to

surviving in the soil, and some nonpathogenic strains are known to occupy

and compete with the pathogen for a specific niche, the cortex of the plant

root (3.12, 3.14, 3.29).

In 1975, Toussoun (3.30) suggested that some soils are suppressive

to pathogenic formae but not to nonpathogenic populations of £.

oxysporum. Schneider (3.27) demonstrated that certain nonpathogenic

strains of £. oxysporum. collected from symptomless celery roots,

effectively predisposed the host to or suppressed infection by the

pathogen, £. oxysporum f. sp. aoii, on celery. He went on to show that a

soil suppressive to Fusarium yellows of celery had a higher proportion of

effective competitive strains as compared to a disease conducive soil.


35

Preliminary competitive infection tests showed that there also are

differences within a collection of nonpathogenic isolates from symptomless

field-grown tomato plants in the ability to suppress infection by E-

oxysporum f. sp. lycopersici on tomato. These isolates may enhance,

suppress, or have a neutral effect on infection. Except for one example

(3.27), the use of nonpathogenic strains of E. oxysporum as biological

control agents against Fusarium w ilt of tomato has been largely unexplored.

The primary question to be addressed in this study is whether or not

competitive effectiveness is confined to discrete subpopulations within the

nonpathogenic, tomato root-infecting population of E. oxysporum. Having

already established the existence of vegetative compatibility groups (VCGs)

in this population (3.10, 3.11), it seemed intuitive that, if competitive

infection ability was to be confined to discrete subpopulations, VCGs

should function in this capacity because they form clonal, genetically

distinct groups.

Vegetative compatibility is assessed in E. oxysporum with

complementary nutritional mutants (3.25). Nitrate metabolism (n il) mutants

are selected from fast growing, chlorate resistant sectors growing on a

chlorate medium. When grown on a minimal medium containing nitrate as

a nitrogen source, the n il mutants grow very sparsely. N il mutants may

then be phenotypically classified by their growth on media which is

amended with one of several different nitrogen sources (3.9). When two


36

complementary mutants are paired on minimal medium, heterokaryosis may

occur and is characterized by a zone of wild-type growth. This occurs as a

result of hyphal fusion, nuclear exchange, and nutritional complementation

(3.25). If two isolates are vegetatively compatible, these isolates must have

identical alleles at each vegetative incompatibility (vie) locus, and they are

considered to be closely related if not clonal derivatives (3.11, 3.25).

Vegetative compatibility groups have been used previously as a genetic

marker to distinguish between £. oxysporum and £. oxysporum f. sp.

vasinfectum (3.16).

The objective of this study was to determine if competitive

effectiveness is associated with VCGs or clonal populations. If effective,

ineffective, or neutral isolates are confined to different specific VCGs, this

would be evidence that the genetic traits for competitive effectiveness are

subject to selection pressures.


Fungal strains. Putative nonpathogenic strains of £. oxysporum were

collected from symptomless tomato roots in locations where tomatoes had

been grown in monoculture for many years. Root samples were taken from

the following sites: Oak Grove, LA (West Carroll Parish), Tombrallo Farm in

Shreveport, LA (Caddo Parish), Calhoun Research Station, Calhoun, LA

(Ouachita Parish), Sweet Potato Research Station, Chase, LA (Franklin

Parish), and the Citrus Research Station, Port Sulphur, LA (Plaquemines


Parish) (Figure 3.1). Root pieces were surface-sterilized with 4% household

bleach (0.21% sodium hypochlorite) for 4 min, rinsed in sterile, distilled

water, plated on Komada's medium, which is selective for Fusarium spp.

(3.17), and incubated at room temperature. Resulting colonies were single-

spored, transferred to carnation leaf agar (CLA) (3.20) for identification,

and isolates of £. oxysporum were stored on silica gel at 4 C (3.23). A

total of 471 isolates was collected and tested for pathogenicity against a

susceptible cultivar by the root-dip method of inoculation (3.31). Seedlings

were grown in sterile, commercially prepared potting soil mix for 2 wk until

the first true leaves emerged. The tomato cultivars 'Fantastic' (susceptible

to races 1, 2, and 3); 'Supersonic' (resistant to race 1); and 'Walter'

(resistant to races 1 and 2) were used as hosts. Fungal strains were grown

in Czapek's solution for 5 days on an orbital shaker at room temperature.

The cultures were filtered through cheesecloth and the resulting spore

suspension was adjusted to about 106 spores per ml. Seedlings were gently

uprooted, and roots were pruned to about 3 cm in length. Six seedlings per

cultivar were dipped into spore suspensions of each strain for 30 sec and

transplanted to plastic cell trays pre-filled with soil-peat moss mix (50:50,

v:v) at the rate of one seedling per cell. Two controls were used: seedlings

with pruned roots were dipped in either sterile water or inoculum of a race

1 FPL isolate. Disease symptoms were recorded 10-14 days after

inoculation.


38

S P H

•.-4 3

'n » v • » /— -1 \ ' *

1 = Oak Grove, West Carroll Parish, LA; 2 = Chase, Franklin Parish, LA; 3 = Shreveport, Caddo Parish, LA; 4 = Calhoun, Ouachita Parish, LA;5 = Citrus Station, Plaquemines Parish, LA.

Figure 3.1. Location of sites sampled in Louisiana for nonpathogenic strains of Fusarium oxysporum.


The susceptibility of tomato cultivars to a particular isolate of E.

oxysporum was recorded after 14 days. Pathogenicity tests were

conducted at least twice. All of the nonpathogenic strains of E. oxysporum

were assessed for VCG (3.10, 3.11). \

Inoculum preparation. Orange mutant cultures were grown in PDB on a

rotary shaker for 72 hr at room temperature. One hundred grams of finely

ground corncob grit (Grit O'Cob, Nebraska) in 250-ml Erlenmeyer flasks

were moistened with 10 ml 0.025M L-asparagine, autoclaved for 1 hr on

each of two consecutive days, inoculated with a conidial suspension {106 /

ml) obtained from the PDB cultures, and incubated for 2 wk at room

temperature. The ground corncob cultures, hereafter referred to as

inoculum, were dried at room temperature in 100mm by 80mm deep petri

plates covered with cheesecloth, ground with a mortar and pestle, and

refrigerated until used.

Competitive root infection studies within vegetative compatibility groups.

Competitive infection experiments were conducted using members of VCGs

2028, 2035, 2050, and 2061 (Table 3.1). These VCGs were selected

because they were multiple member VCGs made up of isolates from more

than one location. One kg of a sterile Mississippi alluvial soil from St.

Gabriel, LA was amended with 0.5 g of ORA-22 inoculum and 0.5 g of

inoculum of each test isolate, mixed in a large plastic bag until well


40

Table 3.1 Strains of Fusarium oxysporum used in the present study and their designation by vegetative compatibility group.

STRAIN ORIGIN

VCG2028

CH-51 * Franklin ParishCH-5 Franklin ParishCH-35 Franklin ParishCH-43 Franklin ParishCH-45 Franklin ParishCAL-11 Ouachita ParishCAL-13 Ouachita ParishCAL-14 Ouachita ParishCAL-17 Ouachita ParishCAL-18 Ouachita Parish

VCG 2035

TOM-32* Caddo ParishTOM-13 Caddo ParishTOM-14 Caddo ParishTOM-23 Caddo ParishTOM-29 Caddo ParishTOM-38 Caddo ParishTOM-44 Caddo ParishTOM-46 Caddo ParishTOM-51 Caddo ParishTOM-59 Caddo ParishCAL-2 Ouachita Parish

‘ Denotes tester strain


41

Table 3.1 (cent.)

STRAIN ORIGIN

VCG 2050

CS3-27* Plaquemines ParishCS3-6 Plaquemines ParishCS3-14 Plaquemines ParishCS3-19 Plaquemines ParishCS3-22 Plaquemines ParishCS3-26 Plaquemines ParishCS3-28 Plaquemines ParishCS3-37 Plaquemines ParishCS3-39 Plaquemines ParishCS3-42 Plaquemines ParishCS3-43 Plaquemines ParishCAL-23 Ouachita Parish

VCG 2061

CAL-64* Ouachita ParishCAL-6 Ouachita ParishCAL-29 Ouachita ParishCAL-32 Ouachita ParishCAL-33 Ouachita ParishCAL-44 Ouachita ParishCAL-47 Ouachita ParishCAL-51 Ouachita ParishCAL-56 Ouachita ParishCAL-69 Ouachita ParishOG-37 West Carroll Parish

* Denotes tester strain


42

blended, and dispensed into 10-cm clay pots. Tomato seedlings, cultivar

'Supersonic', were transplanted into the pots and grown for 4

wk in the greenhouse. At harvest, the plants were removed from the pots,

and the soil was carefully washed from the roots. The roots were surface-

sterilized for 4 min in 0.21 % NaOCI (3.27), rinsed in three changes of

sterile water, plated on Komada's medium (3.17), and incubated at room

temperature for 1 wk. The number of orange and wild-type colonies was

determined on each plate. Root lengths on each plate were estimated using

a line intersect grid method (3.21, 3.29). These data were used to

calculate the number of colonies per 100 cm of root according to the

method of Schneider (3.27). All competitive infection tests were performed

initially within each VCG and repeated using all isolates. Analyses of

variance were performed and means were compared by Duncan's multiple,

range test (E = 0.05) using pc SAS (3.26).

Results

Pathogenicity of fungal strains. None of the putative nonpathogenic strains

of E. oxysporum isolated from symptomless tomato roots caused disease

symptoms on the appropriate differential tomato cultivars used in the

pathogenicity tests. Based on these results, they are assumed to be

nonpathogenic E. oxysporum.


43

Competitive root infection studies within vegetative compatibility groups.

Coinoculation of the pathogen with nonpathogenic members of VCGs 2028

and 2035 resulted in significantly suppressed infection by the pathogenic

orange mutant, ORA-22, at P = 0.05 and P = 0.01, respectively (Tables

3.2 and 3.3). Mean root infection by ORA-22 was reduced by 68.9% and

82.8% across all members of VCGs 2028 and 2035, respectively (Table

3.4). In VCGs 2050 and 2061, individual isolates caused statistically

significant reductions in infection by ORA-22 relative to the inoculated

control, but, as a group, these VCGs did not significantly affect infection by

the pathogen (Tables 3.5 and 3.6). There were no significant correlations

between root infection by the pathogen and nonpathogens in any of the

VCGs (Tables 3.2, 3.3, 3.4, and 3.5). Taken as a whole, members of VCG

2061 had no significant effect on infection by the pathogen; however,

isolate CAL-51 caused a significant increase in infection relative to the

inoculated control. When viewed as subpopulations, VCGs 2061 and 2050

were significantly different from VCGs 2035 and 2028 in their interaction

with the pathogen (Table 3.6). The latter three VCGs caused a significant

reduction in infection by the pathogen with VCG 2035 causing more than

80% reduction in infection by ORA-22. VCGs 2035 and 2028 are not

significantly different; however, they are significantly different from VCG

2050 and VCG 2061.


44

Table 3.2. Rate of infection by the orange mutant (ORA-22) in the presence of nonpathogenic isolates of £. oxysporum for members of VCG 2028.

ISOLATE NUMBER OF COLONIES PER 100 CM ROOT

.0RA:22 NQNRAIHD.GENS

ORA-22 13.68 0.24CH-51 10.45 0.06CH-43 6.76 5.72CH-35 5.51 9.60CH-5 4.12 3.98CAL-11 3.09 6.03CAL-13 2.77 3.64CH-45 2.72 12.49CAL-18 2.70 4.73CAL-17 2.41 3.70CONTROL 2.05 0.49CAL-14 1.96 5.66

LSDa 7.11 5.63

ALeast significant difference ( P = 0.05 ), R2 = -0.185.


45

Table 3.3. Rate of infection by the orange mutant (ORA-22) in the presence of nonpathogenic isolates of F. oxysporum for members of VCG 2035.


QBAi22 NONPATHOGENS

ORA-22 22.49 0.05TOM-46 12.62 11.27CAL-2 5.50 2.30TOM-13 5.20 7.79TOM-29 5.01 8.55TOM-51 3.65 5.83TOM-32 2.67 12.81TOM-44 2.23 6.31TOM-38 1.84 0.37TOM-14 1.62 2.79TOM-23 1.36 4.32TOM-59 0.81 1.31CONTROL 0.58 0.00

LSDA 10.49 6.21

ALeast significant difference ( P = 0.05 )f R2 = -0.001.


46

Table 3.4. Relative competitive infection of nonpathogenic Fusarium oxysporum isolated from tomato roots.

VCG MeanA SEM

2035 0.15 a 0.052028 0.19 ab 0.072050 0.36 b 0.062061 0.96 c 0.18

Source of Variation DF SS MSBetween Treatments 3 4.60 1.53Residual 40 4.81 0.12Total 43 9.41

F P12.73 < 0.001

AMeans with a common letter are not significantly different at P = 0.05 based on the Student-Newman-Keuls test (Sigma Stat).


47

Table 3.5. Rate of infection by the orange mutant (ORA-22) in the presence of nonpathogenic isolates of E. oxysporum for members of VCG 2050.


ORA-22 NONPATHOGENS

ORA-22 6.86 0.00CS3-6 6.62 1.29CS3-37 3.00 1.59CS3-22 2.88 0.50CS3-28 2.87 1.68CS3-39 2.70 4.23CS3-27 2.08 1.78CS3-42 1.99 1.26CS3-14 1.84 2.12CS3-26 1.63 1.47CAL-23 1.54 2.01CS3-19 1.29 1.60CS3-43 0.82 1.35CONTROL 0.00 2.23

LSDa 2.85 2.19

ALeast significant difference ( P = 0.05 ), R2 = -0.176.


48

Table 3.6. Rate of infection by the orange mutant (ORA-22) in the presence of nonpathogenic isolates of £. oxysporum for members of VCG 2061.


O.RA-22 NONPATHOGENS

CAL-51 4.23 4.66CAL-64 2.38 1.92CAL-33 2.15 2.17CAL-56 2.02 1.12ORA-22 1.74 0.06CAL-29 1.59 1.88CAL-6 1.32 3.22OG-37 1.17 1.95CAL-47 1.07 1.83CAL-69 1.02 2.81CAL-44 0.88 2.16CAL-32 0.57 1.28CONTROL 0.04 0.24

LSDA 2.16 2.29

ALeast significant difference ( P = 0.05 ), R2 - -0.36.


49

LUCC

2.5

zg 1.5oLUL l.

? 1LU>H

0.5

laIfnmirTv

MEANS

E3 VCG 2035 , 0.15 a

CD VCG 2028 , 0.19 ab

0 VCG 2050 , 0.36 b

□ VCG 2061 , 0.96 c

T-Ttco(DO)(Oh~h'0 )^toj «Db~c\icoo>r^.cv^ftocoo)co T-eoinini-coincor>.’5i- (DOJcoaii-cv^co^tcoo) in cocoinc\)_,co^-<£>■<*« pjCoojojcocv^-t-ojojt-^ in^co^-T-i-TtT-r-r- ^■_JT-cvinco'5rcoT-ciiio_ ^ _____ C/5 CO CO CO CO CO CO CO CO—I CO CO T T T f i J J I T —!1—•—Ii ^

o a o o o °°o o o o ° ^ o o o o o 8 o o oo o°< <o< << oooooooooooC X T 'O O O

ISOLATES

Figure 3.2. Rate of infection by an orange mutant (ORA-22) of Fusarium oxysporum f. sp. lycopersici in the presence of nonpathogenic isolates of E. oxysporum in all VCGs.


50

Discussion

Cook and Baker (3.8) listed three general approaches to biological

control of plant pathogens: (i) control of pathogen inoculum, (ii)

protection against infection, and (iii) cross-protection or induced resistance.

With diseases caused by £. oxysporum. two models have been proposed;

enhanced resistance of the host induced by nonpathogenic Fusarium spp.

(3.4, 3.6, 3.18, 3.22, 3.32), and competition between pathogenic and

nonpathogenic strains of £. oxysporum for niches or limiting substrates as a

means for biological control (3.1, 3.27).

Using a pigmented mutant of £. oxysporum. Schneider (3.27)

demonstrated considerable variability in competitive infection within a

nonpathogenic population collected from symptomless celery roots. Some

of the nonpathogenic, parasitic strains of £. oxysporum predisposed celery

root to infection by the pathogenic forma, £. fl. f. sp. apii. while others

competitively suppressed infection. On the basis of a mathematical

analysis, he theorized that these nonpathogens competed with the

pathogen for the same ecological niche-namely, the infection court.

Garibaldi et al (3.12) determined the ability of £. oxysporum and E. solani to

compete with pathogenic Fusaria and speculated that effective strains were

able to rapidly follow root growth, preceding the pathogen in occupation of

infection sites. Finally, Paulitz, Park and Baker (3.24) documented very

large differences in biocontrol ability in their collection of nonpathogenic


51

strains of E. oxysporum. While the above cited researchers showed that

there is variability in competitive infection ability within populations of

nonpathogens, they did not determine if this trait is associated with

discrete populations or if the trait is randomly distributed in the population

of nonpathogens.

Results from competitive root infection tests performed with

nonpathogenic strains of E. oxysporum from tomato, regardless of VCG, in

sterilized soils showed a range of effectiveness from no effect to

suppression and enhancement of root infection by the pathogen, E.

oxysporum f. sp. lycopersici. However, when categorized by VCG, there

were significant differences in competitive infection ability among these

clonal populations. In VCGs 2028, 2035, and 2050, the majority of isolates

acted similarly and suppressed infection by the pathogen. Members of VCG

2061 were not significantly different from the orange mutant control and

did not significantly suppress or enhance the rate of infection. This VCG

may be thought of as neutral. Vegetatively compatible strains are thought

to represent clonal populations and members of these VCG do act as

discrete units in their effect on infection of tomato roots by the orange

mutant. This theory will be tested by isozyme analysis to determine actual

genetic relatedness. These results suggest that competitive infection ability

is subject to selection pressures. Otherwise, one would expect to see this

trait randomly distributed across all VCGs. Clearly, these strains establish a


52

parasitic relationship with the host, and it is to be expected that the host

would exert a significant selective force on the populations that are capable

of establishing such a relationship. In addition, if there is host specificity

within the nonpathogenic population, it would be expected to contribute to

differences in competitive infection abilities among VCGs. Because the role

of nonpathogens has been unrecognized and undefined in the past, little is

known of the effects of breeding tomato for resistance to Fusarium w ilt on

evolution of the nonpathogenic population. Future research should examine

the role of host genotype and crop rotation on the development of the

pathogenic and nonpathogenic populations using VCG as a genetic marker.

A possible evolutionary scenario is that competitive infection ability

provides a selective advantage to those strains that are able to parasitize

roots of specific plant species. These strains would be competing for a

specific niche, and selection pressure exerted by the host would favor

those strains that are most competitively fit. These strains would share

genetic traits associated with competitive infection ability and thus would

be more closely related than those that are less competitively fit. Those

strains that lost one or more of these traits would eventually be lost from

the population. The question arises as to why strains were recovered that

have substantially less competitive infection ability from tomato roots. It is

possible that these isolates would be more competitively f it on other hosts,

but, as has been established for pathogenic formae (3.2, 3.7, 3.13, 3.14,


53

3.15), they are able to colonize other hosts as well. This scenario parallels

that which is already established for the pathogenic formae in which host

specificity is confined to unique VCGs (3.10).


54


3.1. Alabouvette, C., Y. Couteaudier, and J. Louvet. 1984. Recherches sur la resultant de sols aux maladies, IX Dynamiques des populations du Fusarium spp. et de Fusarium oxysporum f. sp. meionis dans un sol resultant et dans un sol sensible aux fusarioses vasculaires. Agronomie 4:729-733.

3.2. Armstrong, G. M. and J. K. Armstrong. 1948. Nonsusceptible hosts as carriers of w ilt fusaria. Phytopathology 38:808-826.

3.3. Armstrong, G. M. and J. K. Armstrong. 1981. Formae speciales and races of Fusarium oxysporum causing w ilt diseases. Pages 391 - 399 in Fusarium: Diseases, Biology and Taxonomy. P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. Pennsylvania State University Press, University Park.

3.4. Baker, R., P. Hanchey, and S. D. Dottarar. 1978. Protection of carnation against Fusarium stem rot by fungi. Phytopathology 68:1495-1501.

3.5. Beckman, C. H. 1987. The Nature of Wilt Diseases in Plants. American Phytopathological Society, St. Paul. 175 pp.

3.6. Biles, C. L., and R. D. Martyn. 1989. Local and systemic resistance induced in watermelons by formae speciales of Fusarium oxysporum. Phytopathology 79:856-860.

3.7. Burgess, L. W. 1981. General ecology of the Fusaria. Pages 225- 235 in Fusarium: Diseases, Biology and Taxonomy. P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. Pennsylvania State University Press, University Park.

3.8. Cook, R. J. and K. F. Baker. 1983. The Nature and Practice of Biological Control of Plant Pathogens. The American Phytopathological Society, St. Paul. 539 pp.

3.9. Correll, J. C., C. J. R., Klittich, and J. F. Leslie. 1987. Nitrate nonutilizing mutants of Fusarium oxysporum and their use in vegetative incompatibility tests. Phytopathology 77:1640-1646.


55

3.10. Elias, K. S. 1989. Vegetative compatibility groups of Fusarium oxysporum f. sp. lycopersici and nonpathogenic strains of £. oxysporum and a isozyme analysis of pathogenic populations. Ph.D. dissertation, Louisiana State University, Baton Rouge. 93 pp.

3.11. Elias, K. S., R. W. Schneider, and M. M. Lear. 1991. Analysis of vegetative compatibility groups in nonpathogenic populations of Fusarium oxysporum isolated from symptomless tomato roots. Can. J. Bot. 69:2089-2094.

3.12. Garibaldi, A., L. Gugliemone, and M. L. Gulino. 1990. Rhizosphere competence of antagonistic Fusaria isolated from suppressive soils. Symbiosis 9:401-404.

3.13. Gordon, T. R., D. Okamoto, and D. J. Jacobson. 1989.Colonization of muskmelon and nonsusceptible crops by Fusarium oxysporum f. sp. melonis and other species of Fusarium. Phytopathology 79: 1095-1100.

3.14. Hendrix, D. L., and L. W. Nielsen. 1958. Invasion and infection by crops other than the forma suscept by Fusarium oxysporum f. sp. batatas and other formae. Phytopathology 48: 224-228.

3.15. Katan, J. 1971. Symptomless carriers of the tomato Fusarium w ilt pathogen. Phytopathology 61: 1213-1217.

3.16. Katan, T., and Katan, J. 1988. Vegetative compatibility grouping of Fusarium oxysporum f. sp. vasinfectum from tissue and the rhizosphere of cotton plants. Phytopathology 78:852-855.

3.17. Komada, H. 1975. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev. Plant Protection Res. 8:114-124.

3.18. Larkin, R. P., D. L. Hopkins, and F. N. Martin. 1993. Effect of successive watermelon plantings on Fusarium oxysporum and other microorganisms in soils suppressive and conducive to Fusarium w ilt of watermelon. Phytopathology 83:1097-1105.

3.19. Mandeel, Q., and R. Baker. 1991. Mechanisms involved in biological control of Fusarium w ilt of cucumber with strains of Fusarium oxysporum. Phytopathology 81: 462-469.


3.20. Nelson, P. E., T. A. Toussoun, and W. F. 0. Marasas. 1983. Fusarium Species: An Illustrated Manual of Identification. Pennsylvania State University Press, University Park. 193 pp.

3.21. Newman, E. I. 1966. A method of estimating the total length of root in a sample. J. Appl. Ecol. 3:139-145.

3.22. Ogawa, K. and H. Komada. 1985. Biological control of Fusarium w ilt with cross-protection by nonpathogenic Fusarium oxysporum. Pages 121-123 in: Ecology and Management of Soilborne Plant Pathogens . C. A. Parker, A. D. Rovira, K. J. Moore and P. T. W. Wong, eds. The American Phytopathological Society, St. Paul.

3.23. Perkins, D. D. 1962. Preservation of Neurospora stock cultures with anhydrous silica gel. Can J. Microbiol. 8:591-594.

3.24. Paulitz, T. C., C. S. Park, and R. Baker. 1987. Biological control of Fusarium wilt of cucumber with nonpathogenic isolates of Fusarium oxysporum. Can. J. Microbiol. 33:349-353.


3.26. SAS Institute Inc. 1987. SAS/STAT Guide for Personal Computers, Version 6 Edition. J. Chris Parker, ed. SAS Institute Inc., Cary.1028 pp.

3.27. Schneider, R. W. 1984. Effects of nonpathogenic strains of Fusarium oxysporum on celery root infection by Fusarium oxysporum f. sp. apii and a novel use of the Lineweaver-Burk double reciprocal plot technique. Phytopathology 74:646-653.

3.28. Stoner, M. F. 1981. Ecology of Fusarium in noncultivated soils. Pages 276-286 in: Fusarium: Diseases, Biology and Taxonomy. P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. The Pennsylvania State University Press, University Park.

3.29. Torssell, W. R., J. E. Begg, C. W. Rose, and G. F. Byrne. 1968. Stand morphology of Townsville lucerne (Stylosanthes humilis). Seasonal growth and root development. Aust. J. Exp. Agric. Anim. Hub. 8:532-543.


57

3.30. Toussoun, T. A. 1975. Fusarium-suppressive soils. Pages 145-151 in: Biology and Control of Soil-Borne Plant Pathogens. G. W.Bruehl, ed. American Phytopathological Society, St. Paul.

3.31. Williams, P. H. 1981. Fusarium yellows. Pages 124-129 in: Screening Crucifers for Multiple Disease Resistance. P. H. Williams, ed. University of Wisconsin Press, Madison.

3.32. Wymore, L. A., and R. Baker. 1982. Factors affecting crossprotection in control of Fusarium w ilt of tomato. Plant Dis. 6:908- 910.


CHAPTER 4

ISOZYME ANALYSIS OF SELECTED VEGETATIVE COMPATIBILITY GROUPS OF NONPATHOGENIC FUSARIUM OXYSPORUM

58


59

Introduction

Puhalla (4.20) developed a technique to assign fungi to vegetative

compatibility (heterokaryon) groups (VCG) using nitrate metabolism (nit)

mutants. In Gibberella fujikuroi (Fusarium moniliforme) (4.5, 4.16, 4.21), it

has been shown that a minimum of ten yic loci control vegetative

compatibility. It is generally assumed that £. oxysporum has a set of

vegetative incompatibility loci which are similar to <£. fujikuroi (4.21). In

order for tw o isolates to be vegetatively compatible, alleles at each of the

vie loci must be identical (4.16, 4.21). Puhalla and Spieth (4.21) showed,

using sexual crosses between vegetatively incompatible strains of Q..

fujikuroi. that yic genes are not expressed in the sexual phase, and they

theorized that vegetative incompatibility is independent of the factors

governing sexual reproduction.

Correll, Puhalla and Schneider (4.6) first utilized VCG analysis to

examine a collection of £. £. f. sp. apii. Since then, VCG analysis has been

used to delimit populations of many formae speciales of £. oxysporum (4.2,

4.6, 4.8, 4.10, 4.12, 4.13, 4.15, 4.19, 4.20), but few populations of

nonpathogenic £. oxysporum have been studied (4.7, 4.9). VCG analysis

provides categorical evidence for genetic relatedness but does not address

genetic diversity in a quantitative manner. That is, if two populations are

vegetatively compatible, additional experimental analysis would have to be

conducted in order to establish how diverse the two populations are.


60

Analysis of enzyme polymorphisms, as defined by isozyme

electrophoresis, can be used to estimate the amount of genetic variation

among organisms. Having established that competitive infection ability is

associated with specific VCGs, the objective of this study was to estimate

genetic diversity within and among these clonal populations using starch

gel electrophoresis.


Fungal strains. Isolates used for isozyme analysis were members of the

following VCGs: 2028, 2035, 2050, and 2061 (Table 4.1). One isolate of

E. oxysporum (OG-15), one isolate of FPL (LSU-2), and the orange mutant

(ORA-22) were included as standards in all assays.

Tissue preparation. Each fungal strain was grown in four 250-ml

Erlenmeyer flasks containing 100 ml each of potato dextrose broth for 4-5

days on an orbital shaker at room temperature. Mycelial mats were

centrifuged at 5,000 rpm at 4 C in sterilized centrifuge bottles, and the

supernatant was discarded. One hundred ml of sterile, distilled water was

added to the mycelial pellet, and the suspension was shaken and

centrifuged again at 5,000 rpm at 4 C. The mycelial pellet was placed in a

sealed plastic petri dish and frozen at - 70 C overnight before lyophilization.

Protein extraction. Lyophilized mycelium was ground to a fine powder with

an ice cold mortar and pestle. A suspension of 0.6 g of the mycelial

powder and 2.25 ml of 0.05M Tris-HCI (pH 7.1) was mixed in a small


61

centrifuge tube and incubated for 15 min on ice. Samples were centrifuged

for 27 min at 18,000 rpm at 4 C. The supernatant was withdrawn from the

tube using a Pasteur pipette without disturbing the lipid layer at the top of

the tube or the pellet at the bottom. The supernatant was dispensed into

microfuge tubes in 100 //I aliquots and stored at -70 C. Enough extract

was prepared for 10 electrophoretic runs. Extra mycelial powder was

stored in small vials at -70 C.

Electrophoresis. Ten-//I aliquots of the extracts were absorbed onto filter

paper wicks (Whatman No. 3). The wicks were loaded onto a 15 cm by 20

cm horizontal potato starch gel (12% w/v), and the extracts were subjected

to electrophoresis at 50mAmp for 4 to 6 hr depending on the buffer system

used (Appendix B). Gels were cooled with ice packs for the duration of the

run. After completion of electrophoresis, six 1.5 mm-thick horizontal slabs

were cut from each gel, each slab was treated with a specific enzyme

visualization reagent, and fixed using a 5:5:1 glacial acetic acid/methyl

alcohol/water solution (4.11, 4.17, 4.22, 4.23) (Table 4.2). Up to 15

samples, including one standard isolate with known banding patterns, were

analyzed on each gel slab. All isolates were analyzed at least tw ice in

separate gel runs.


62

Table 4.1 Strains of Fusarium oxysporum used in the present study and their designation by vegetative compatibility group.

STRAIN ORIGIN

VCG 2028

CH-51 * Franklin ParishCH-5 Franklin ParishCH-35 Franklin ParishCH-43 Franklin ParishCH-45 Franklin ParishCAL-11 Ouachita ParishCAL-13 Ouachita ParishCAL-14 Ouachita ParishCAL-17 Ouachita ParishCAL-18 Ouachita Parish

VCG 2035

TOM-32* Caddo ParishTOM-13 Caddo ParishTOM-14 Caddo ParishTOM-23 Caddo ParishTOM-29 Caddo ParishTOM-38 Caddo ParishTOM-44 Caddo ParishTOM-46 Caddo ParishTOM-51 Caddo ParishTOM-59 Caddo ParishCAL-2 Ouachita Parish

'Denotes tester strain


63

Table 4.1 (cont.)

STRAIN ORIGIN

VCG 2050

CS3-27* Plaquemines ParishCS3-6 Plaquemines ParishCS3-14 Plaquemines ParishCS3-19 Plaquemines ParishCS3-22 Plaquemines ParishCS3-26 Plaquemines ParishCS3-28 Plaquemines ParishCS3-37 Plaquemines ParishCS3-39 Plaquemines ParishCS3-42 Plaquemines ParishCS3-43 Plaquemines ParishCAL-23 Ouachita Parish

VCG 2061 -

CAL-64* Ouachita ParishCAL-6 Ouachita ParishCAL-29 Ouachita ParishCAL-32 Ouachita ParishCAL-33 Ouachita ParishCAL-44 Ouachita ParishCAL-47 Ouachita ParishCAL-51 Ouachita ParishCAL-56 Ouachita ParishCAL-69 Ouachita ParishOG-37 West Carroll Parish

'Denotes tester strain


64

Table 4.2. Enzymes, abbreviations, Enzyme Commission (E.C.) numbers, and buffer systems used in this study.

Enzyme E. C. Number

Buffer

Aspartate amino transferase (AAT) 2.6.1.1 AAEsterase (EST) 3.1.1.1 CFumarase (FUM) 4.2.1.2 CGlucose-6-phosphate dehydrogenase (G6PDH) 1.1.1.1.49 CGlucose phosphate isomerase (GPI) 5.3.1.9 ALactate dehydrogenase (LDH) 1.1.1.27 CPhosphoglucomutase (PGM) 2.7.5.1 A

AA = amine citrate, pH 6.1; C = Tris-citrate/lithium borate, pH 8.5 (Appendix B).


Analysis. The intensity and electrophoretic mobility, or the distance of

migration from the origin, were recorded for all bands. Bands on the gels

were numbered in the order of the fastest to slowest migration. A

composite of all bands for each isolate was tabulated, and these data were

used to calculate simple matching coefficients between isolates according

to the equation:

(+ +) + (- -)

Sm = -------------------------------

( + + ) + (--) + ( + -) + (- + )

where Sm = simple matching coefficient; + + = positive matches;

- - = negative matches; and (+ -) and (- + ) = mismatches. The

coefficients were then used to construct a similarity matrix (4.1) among the

electrophoretic phenotypes (EPs). Principal components analysis (PCA)

was employed to resolve phylogenetic groups. The computer program

NTSYS-pc, ver. 1.60 (Exeter Software, Setauket, NY) was used for these

analyses.

Results

Electrophoresis and analysis. Since optimal gel, electrode buffers and pH

had been previously determined for each enzyme assayed (4.8),

electrophoresis and data collection proceeded smoothly. Most of the

enzymes observed had multiple bands which indicate different loci, and/or


66

alleles (Table 4.3). Results from the present study cannot be used to

distinguish alleles from loci because sexual crosses cannot be made.

The thirty-six isolates of FPL which make up VCGs 2028, 2035,

2050, and 2061 were assigned to 39 electrophoretic phenotypes (EP).

Principle components analysis (PCA) of isozyme polymorphisms in this

nonpathogenic population was performed, and the results showed that each

VCG was unique and clearly separated from the other VCGs. All

nonpathogenic VCGs were separated from the race 1 pathogenic isolates

used as controls (Figure 4.1).

Discussion

Nonpathogenic populations of £. oxysporum have not been

characterized because of problems in identification and separation. Elias,

Schneider and Lear (4.9) conducted a large survey and VCG analysis of

nonpathogenic isolates of £. oxysporum from tomato collected from 8

locations in Louisiana. Of 317 nonpathogenic isolates from symptomless

tomato roots, 186 isolates were in 48 multiple member VCGs and 131

isolates belonged to single member VCGs. Twenty one VCGs contained

members from more than one collection site. The nonpathogenic

population was found to be quite diverse.

Competitive infection test results show that there is an association

between competitive effectiveness or ineffectiveness and VCG. All VCGs

used in the present study contained members from two collection sites


67

Table 4.3. Putative distribution for 15 putative enzyme loci in 36 isolates of Fusarium oxysporum isolated from symptomless tomato roots.

LocusA 1 2 3 4

AAT/GOT-1 3.0(10)B 2.5(20)AAT/GOT-2 0.7(10) 0.5(20)PGM-1 3.4(10) 3.0(10)PGM-2 1.3(10) 1.2(10)GPI-1 3.4(10) 2.8(19) 1.7(10)GPI-2 0.8(10)G6PDH 4.2(10) 3.3(10) 3.0(10) 2.5(9)FUM 3.4(10) 2.4(10)LDH-1 5.5(10) 2.6(10) 2.1(10) 1.9(9)LDH-2 3.3(10) 1.8(10) 1.7(9)LDH-3 2.7(10)EST-1 6.2(10) 4.6(13) 3.7(9)EST-2 3.3(10) 2.7(10) 2.1(9)EST-3 1.3(10) 1.1(10) 0.7(9)EST-4 0.8(13)

Abbreviations for enzyme loci according to Richardson (4.22). BDistance from origin (cm) (number of isolates).


68

CM

j_O

" oo

Li_

0.8

0.6

0.4

0.2

0.0

- 0.2 •

-0 .4

0.6

- 0.8

Pathogens VCG 2028 VCG 2035 VCG 2050 VGG 2061

1.0 -0 .5 0.0 0.5

Factor 11.0 1.5

Figure 4.1. Principal components analysis of isozyme polymorphisms in nonpathogenic populations of Fusarium oxysporum. Numerals indicate the number of isolates in each cluster. Several symbols are representative of more than one isolate.


69

(Table 4.1). Each population was clearly separated from the pathogenic

standards and was unique. These results provide strong evidence that the

genetic traits associated with competitive infection, beneficial, neutral or

detrimental, are confined to genetically isolated populations of

nonpathogenic £. oxysporum as delimited by VCG in much the same way

as pathogenicity is confined to specific VCGs (4.14, 4.16, 4.20).

Nonpathogens are possibly subject to the same selection pressures that

pathogens. Puhalla and Spieth (4.21) suggested that, in fungi, if the

sexual cycle is lost or ceases to occur, yic genotypes could be viewed as

separate "vegetative compatibility groups" each with a chance combination

of alleles from the last round of sexual reproduction. Any newly arising

variability would be confined to the VCG in which it originates. Normal

selection pressure would lead to the loss of some VCGs. Caten (4.3)

reported that isolates which belong to the same VCG are near isogenic and

probably clonally related, even though they may have been isolated

hundreds or thousands of miles apart.

Future research should include ecological studies to determine the

role of nonpathogenic Fusaria in agricultural systems. Attention should be

shifted to determining the population biology of nonpathogens. Now that

we are aware that the nonpathogenic population of Fusarium oxysporum is

composed of unique subpopulations, more research is needed to investigate

how they interact with the pathogen and host population and affect disease


70

development. Long-term greenhouse experiments or outdoor microplot

experiments are needed to determine what effect the environment and /or

the combination of the host and environment have on competitive infection

ability of nonpathogenic £. oxysporum isolates and the development of host

specificity. Long-term greenhouse experiments using susceptible tomato

varieties in rotation with commonly grown crops should determine the

effect of crop rotation on VCG stability. In addition, colonization levels of

nonpathogens in soil in the absence of the pathogen should be monitored.


71


4.1. Atlas, R. M., and R. Bartha. 1981. Microbial Ecology:Fundamentals and Applications. Addison-Wesley Publishing Co., Reading. 560 pp.

4.2. Bosland, P. W., and P. H. Williams. 1987. An evaluation of Fusarium oxysporum from crucifers based on pathogenicity, isozyme polymorphism, vegetative compatibility, and geographic origin. Can. J. Bot. 65:2067-2073.

4.3. Caten, C. E. 1987. Genetic integration of fungal life styles. Pages215-229 in: Evolutionary biology of the fungi. A. D. M. Rayner, C.M. Brasier, and David Moore, eds. Cambridge University,Cambridge. 465 pp.

4.4. Cornell, J. C. 1991. The relationship between formae speciales,races and vegetative compatibility groups in Fusarium oxysporum.Phytopathology 81:10061-1064.

4.5. Cornell, J. C., C. J. R. Klittich, and J. F. Leslie. 1989. Heterokaryon self-incompatibility in Gibbfi.rella fujikuroi (Fusarium moniliforme). Mycol. Res. 93:21-27.

4.6. Correll, J. C., J. E. Puhalla, and R. W. Schneider. 1986. Identification of Fusarium oxysporum f. sp. apii on the basis of colony size, virulence, and vegetative compatibility. Phytopathology 76:396-400.

4.7. Correll, J. C., J. E. Puhalla , and R. W. Schneider. 1986. Vegetative compatibility groups among nonpathogenic root-colonizing strains of Fusarium oxvsporum. Can. J. Bot. 64:2358-2361.

4.8. Elias, K. S. 1989. Vegetative compatibility groups of Fusarium oxysporum f. sp. lycopersici and nonpathogenic strains of £. oxysporum and a isozyme analysis of pathogenic populations. Ph.D. dissertation, Louisiana State University, Baton Rouge. 93 pp.

4.9. Elias, K. S., R. W. Schneider, and M. M. Lear. 1991. Analysis of vegetative compatibility groups in nonpathogenic populations of Fusarium oxysporum isolated from symptomless tomato roots. Can.J. Bot. 69:2089-2094.


6

72

4.10. Elmer, W. H., and C. T. Stephens. 1989. Classification of Fusarium oxysporum f. sp. asparagi into vegetative compatibility groups. Phytopathology 79:88-93.

4.11. Harris, H., and D. A. Hopkinson. 1976. Handbook of enzyme electrophoresis in human genetics. North-Holland Bionedical Press, Amsterdam.

4.12. Jacobson, D. J., and T. R. Gordon. 1988. Vegetative compatibility and self-incompatibility within Fusarium oxysporum f . sp. melonis. Phytopathology 78:668-672.

4.13. Katan, T., and Katan, J. 1988. Vegetative compatibility grouping of Fusarium oxysporum f . sp. vasinfectum from tissue and the rhizosphere of cotton plants. Phytopathology 78:852-855.

4.14. Kistler, H. C., and E. A. Momol. 1990. Molecular genetics of plant pathogenic Fusarium oxysporum. Pages 49-54 in : Fusarium Wilt of Banana. R. C. Ploetz, ed. APS Press, St. Paul.

4.15. Larkin, R. P., D. L. Hopkins , and F. N. Martin. 1990. Vegetative compatibility within Fusarium oxysporum f. sp. niveum and its relationship to virulence, aggressiveness and race. Can. J. Microbiol. 36:352-358.

4.16. Leslie, J. F. 1990. Genetic exchange within sexual and asexual populations of the genus Fusarium. Pages 37-48 in: Fusarium Wilt of Banana. R. C. Ploetz, ed. APS Press, St. Paul.

4.17. Micales, J. A., M. R. Bonde, and G. L. Petterson. 1986. The use of isozyme analysis in fungal taxonomy and genetics. Mycotaxon 27:405-449.

4.18. Perkins, D. D., and B. C. Turner. 1988. Neurospora from natural populations: toward the population biology of a haploid eukaryote. Exper. Mycol. 12:91-131.

4.19. Ploetz, R. C., and J. C. Correll. 1988. Vegetative compatibility among races of Fusarium oxvsporum f. sp. cubense. Plant Dis. 72:325-328.



73

4.21. Puhalla, J. E., and P. T. Spieth. 1985. A comparison of heterokaryosis and vegetative incompatibility among varieties of GibbeteJIa fujikuroi (Fusarium moniliforme). Exp. Mycol. 9:39-47.

4.22. Richardson, B. J., P. R. Braverstock, and M. Adams. 1986. Allozyme electrophoresis. Academic Press, New York. 410 pp.

4.23. Shaw, C. R., and R. Prasad. 1970. Starch gel electrophoresis of enzymes - a compilation of recipes. Biochem. Genet. 4:297-320.


APPENDIX A.

MEDIA RECIPES

74


Carnation Leaf Agar (CLA) (Nelson et al, 1983)

Carnation leaf agar is prepared by sprinkling treated (oven dried, dessicated with propylene oxide) carnation leaf tissue on freshly poured 2% water agar plates. The plates should be incubated at room temperature.

Czapek’s Solution (Tuite. 1969)

To 1 L of glass distilled water add:

NaNOg 2.0 g

K2HP04 1.0 g

MgS04-7H20 0.5 g

KCI 0.5 g

FeS04-7H20 10.0 mg (1 ml stock solution)*

Sucrose 30.0 g

Decant 100 ml solution into 250-ml Erlenmeyer flasks. Sterilize by autoclaving for 15 min. *Stock solution is made by adding 1 g to 100 ml water. This equals .01 g/ml or 10 mg/ml.


Komada's Medium (Komada, 1975)

KH2P04 1.0 g

KCI 0.5 g

MgS04-7H20 0.5 g

FeNa EDTA 0.01 g

D - galactose 2.0 g

L - asparagine 2.0 g

Na2B40 7 1.0 g

PCNB 1.0 g(dissolve in 95% ETOH for 1 hr) orTerraclor 1.35 g

Oxgall (Bile Bovine) 0.5 g

Agar 15.0g

In separate flasks, sterilize 250 ml distilled water plus agar and 750 ml water. Autoclave for 15 min. Immediately after removal, add remaining ingredients to the flask containing the water and agar and bring to 1 L with additional sterilized H20.When cool add the following ingredients:

10% phosphoric acid 7.5 ml

Tergitol 0.5 ml

Tetracycline 0.3 g

Streptomycin 0.3 g

Pour into sterile petri dishes and cool.


Minimal Medium (MM) (Puhalla, 1985)

To 1 L of glass-distilled water add:

Sucrose 30.0 g

NaNOg 2.0 g

KH2P04 1.0 g

MgS04-7H20 0.5 g

KCI 0.5 g

FeS04-7H20 0.1 g

Trace elements so l.** 0.2 g

Bacto agar 20.0 g

* * Trace elements solution

To 95 ml of distilled water add:

Citric acid 5.0 g

ZnS04-7H20 5.0 g

Fe(NH4)2(S04)2-6H20 1.0 g

CuS04-5H20 0.25 g

MnS04-H20 50.0 mg

H3B04 50.0 mg

NaMo04-2H20 50.0 mg


78

For the other nitrogen source media (Correll et al, 1987), substitute one of these five nitrogen sources for nitrate as in MM described above:

1. Nitrate medium - use MM

2. Nitrite medium - add 0.5 g/L NaN02

3. Hypoxanthine medium - add 0.2 g/L hypoxanthine

4. Ammonium medium - add 1.0 g/L ammonium tartrate

5. Uric acid medium - add 0.2 g/L uric acid

Potato Dextrose Agar (PDA)

1. Cook 200 g peeled potatoes in 1 L of glass-distilled water for 15 min in the autoclave.

2. Strain the potatoes from broth using cheesecloth.

3. Bring to 1 L with glass-distilled water and add:

Dextrose 20.0 gBacto Agar 17.0 g

4. Autoclave for 15 min, cool and pour into sterile petri dishes.

Potato Dextrose Broth (PDB)

1. Cook 200 g peeled, sliced potatoes in 1 L of glass-distilled water for 15 min in the autoclave.

2. Strain the potatoes from the broth using cheesecloth.

3. Bring to 1 L with glass-distilled water and add:

Dextrose 18.0 g

4. Decant into 250-ml Erlenmeyer flasks, seal, and autoclave for 15 min.


I

79

Potato Sucrose Medium (KPS) (Puhalla, 1985)

1. Cook 200 g peeled, sliced potatoes in I L of glass-distilled water for15 min in the autoclave.

2. Strain the potatoes from broth using cheesecloth.

3. Bring to 1 L w ith glass-distilled water and add:

4. Autoclave for 15 min, cool, and pour into sterile petri dishes.

Sucrose Bacto Agar KCI03

20.0 g20.0 g15.0 g


80

Literature Cited in Appendix A

1. Correll, J. C., C. J. R. Klittich, and J. F. Leslie. 1987. Nitratenonutilizing mutants of Fusarium oxvsporum and their use in vegetative compatibility tests. Phytopathology 77:1640-1646.

2. Komada, H. 1975. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Rev. Plant Protection Res. 8:114-124.

3. Nelson, P. E., T. A. Toussoun, and W. F. 0 . Marasas. 1983.Fusarium Species: An Illustrated Manual of Identification. Pennsylvania State University Press, University Park. 193 pp.

4. Puhalla, J. E. 1985. Classification of strains of Fusariumoxysporum on the basis of vegetative compatibility. Can. J. Bot. 63:179-183.

5. Tuite, J. 1969. Plant Pathological Methods: Fungi and Bacteria. Burgess Publishing Co., Minnesota. 239 pp.


APPENDIX B.

BUFFER SYSTEMS AND ENZYME STAINING SYSTEMS

81


Buffer Systems

A. Amine-citrate Buffer (pH 6.1)

Electrode buffer

.04M citrate (C-0759) N-(3-amino-propyl)-morpholine (A-9028)

Adjust pH of citrate to 6.1 with morpholine.

fiaLbuffer

1:20 dilution of the electrode buffer.

Run at 50mAmp for 4 hours.

B. Tris-citrate/lithium-borate Buffer

Electrode buffer

.06M lithium hydroxide (L-4256),3M boric acid (B-0252)

Adjust to pH 8.1.

Gel buffer

.03M Tris (T-1503)

.005M citrate (C-0759)1 % electrode buffer

Adjust to pH 8.5.

Run at 50 mAmp for 6 hours.

Fixer

5:5:1 glacial acetic acid/ methyl alcohol/water


83

Enzyme Staining Systems

Aspartate Aminotransferase (AAT/GOT)

0.1 M Tris-HCI (pH 8.0) 40.0 ml

Stock solution: 2.5 ml10 g Aspartate (A-9006)5 g alpha ketoglutyrate (K-1750)

in 100 ml H20 plus 25 ml 5N NaOH to pH 7.0

Pyridoxal-5-P04> dry (P-9255) 5.0 mg

FastBlue BB Salt (100 mg/ml) (F-0250) 0.75 ml

Incubate at room temperature for 15 min prior to adding FB. Incubate at 37 C for 30-60 min, rinse and fix.

Esterase (EST)

.2M monobasic sodium phosphate adjusted to pH 5.8 with .2M dibasic sodium phosphate 40.0 ml

a Naphthyl Acetate 7.5 mg (N-7000) 1.0 ml.15 g in 20 ml (10 ml acetone & 10 ml water)

P Naphthyl Acetate 7.5 mg (N-6875) 1.0 ml

1-propanol 2.5 ml

FastBlue RR Salt (50mg/ml) (F-0500) 1.0 ml

Incubate for 30-60 min at 37 C, rinse and fix.


Fumarase (FUM)

0.1MTris-HCI 40.0 ml

1M Fumaric Acid, pH 7.0 with NaOH 5.0 ml

Malate dehydrogenase 100 units

NAD 10 mg (10mg/ml) (N-7004) 1.0 ml

MTT (5 mg/ml) 1.0 ml

PMS (1 mg/ml) 1.0 ml


g iu c Q s e - 6 - p h o s D h a te d e h y d ro g e n a s e (G6PDH)

0.1M Tris-HCI, pH 8.0 40.0 ml

Glucose-6-phosphate, dry (G-7250) 100 mg

1M MgCI2 50 mg (100 mg/ml) 0.5 ml

NADP 10 mg (5 mg/ml) 2.0 ml

MTT 5 mg (5 mg/ml) 1.0 ml

PMS 2 mg (1 mg/ml) 2.0 ml



85

Glucose phosphate isomerase (GPI)

0.1 M Tris-HCI, pH 8.0 15.0 ml

Fructose-6-Phosphate 12 mg/ml (F-3627) 1.0 ml

Glucose-6-phosphate dehydrogenase 2.5 ml(G6PDH) 25 units (10 units/ml) (G-8878)


1MMgCI2 0.5 ml



Incubate for 15-30 min at 37 C, bands appear quickly. Do not overstain. Rinse and fix.

Lactate Dehydrogenase (LDH)


1M Lactate, pH 7.0 5.0 ml

NAD 10 mg (10 mg/ml) 1.0 ml





Phosphoglucomutase (PGM)


Glucose-1-phosphate (G-1-P) 1.0 ml(100 mg/ml) (G-7000)

Glucose-1,6 diphosphate (.2 mg/ml) (G-7137) 1.0 ml

G6PDH 25 units (10 units/ml) 2.5 ml

1M MgCI2 1.0 ml


MTT 5 mg (5 mg/ml) 1.0 ml

PMS 1 mg (1 mg/ml) 1.0 ml

Agar overlay. Add 20 ml molten agar just prior to pouring over gel. Incubate for 30-60 min at 37 C and fix.


Literature Cited in Appendix B

1. Harris, H., and D. A. Hopkinson. 1976. Handbook of Enzyme Electrophoresis in Human Genetics. North-Holland Biomedical Press, Amsterdam.

2. Micales, J. A., M. R. Bonde, and G. L. Petterson. 1986. The use of isozyme analysis in fungal taxonomy and genetics. Mycotaxon 27:405-449.


VITA

Monica Michelle Lear was born on May 30, 1959 in New Orleans,

Louisiana. She received a B. S. in Horticulture in May, 1982 and a M. S. in

Plant Health in August 1986 from Louisiana State University. In August,

1986, she enrolled in the Ph.D. program in the Department of Plant

Pathology and Crop Physiology. In April, 1991, she was employed as

Assistant Director of Horticulture and Quarantine Programs/Louisiana

Horticulture Commission at the Louisiana Department of Agriculture and

Forestry.

88


DOCTORAL E X A M IN A T IO N AND D IS S E R T A T IO N REPORT

Candidate: Monica Michelle Lear

Major Field: Plant Health

Title of Dissertation: Interactions Between Fusarium oxysporum f . sp.lycopersici and Nonpathogenic Strains o f Fusarium oxysporum

Approved:

Major Bfofessor and chairman

EXAMINING COMMITTEE:

q w - hLyQ £.C l%6. <p.

W l . C .

n.

Date of Examination:

November 3, 1993


Documents

Interactions Between Fusarium Oxysporum F. Sp. Lycopersici