Vegetative compatibility groups among

Vegetative compatibility groups among nonpathogenic root-colonizing strains of Fusarium oxysporum J. C. CORRELLAND J. E. PUHALLA Department of Plant Pathology, University of California, Berkeley, CA, U.S.A. 94720 AND

R. W. SCHNEIDER Department of Plant Pathology and Crop Physiology, Louisiana State University, Baton Rouge, LA, U.S.A. 70803

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Received November 27, 1985

J. C., J. E. PUHALLA, and R. W. SCHNEIDER. 1986. Vegetative compatibility groups among nonpathogenic rootCORRELL, colonizing strains of Fusarium oxysporum. Can. J . Bot. 64: 2358-2361. A total of 110 isolates of Fusarium oxysporum were collected from apparently healthy celery roots from the major celery production areas of California. All isolates used in this study were nonpathogenic on celery. Nitrate-nonutilizing mutants (nit mutants) were used to test for vegetative compatibility among the isolates. Twenty-eight isolates were randomly selected from the collection and used as tester isolates. Complementary nit mutants, arbitrarily designated nitA and nitB, were recovered from each of the 28 tester isolates and paired in all combinations to test for vegetative compatibility among the isolates. Fourteen distinct vegetative compatibility groups, designated VCG2001 through VCG2014, were recovered. The remaining 82 isolates were then tested for vegetative compatibility with these 14 vegetative compatibility groups. Fifty of the 110 isolates belong to one of the 14 vegetative compatibility groups. Two vegetative compatibility groups, VCG2001 and VCG2002, were found in the highest frequency and from the most fields. The data suggest that some vegetative compatibility groups found colonizing celery roots may be much more common than others. Vegetative compatibility may be a way to identify and characterize strains of nonpathogenic F. oxysporum. CORRELL, J. C., J. E. PUHALLA et R. W. SCHNEIDER. 1986. Vegetative compatibility groups among nonpathogenic rootcolonizing strains of Fusarium oxysporum. Can. J . Bot. 64: 2358-2361. Les auteurs ont rCcoltC 110 souches de Fusarium oxysporum a partir de racines de cCleris apparemment en santC, venant des principales regions de production de cCleri en Califomie. Aucune des souches utilisCes dans cette Ctude ne s'est avCrCe pathogbne pour le cCleri. Des mutants incapables d'utiliser le nitrate (nit mutants) ont CtC utilisCs pour tester la compatibilitC vCgCtative entre les souches. Vingt-huit souches ont CtC choisies au hazard 2 partir de la collection et utiliskes pour les essais. Des mutants complCmentaires nit, arbitrairement appelCs nitA et nitB, ont CtC obtenus de chacune des 28 souches "test" et ont CtC paires selon toutes les combinaisons pour Cvaluer la compatibilitC vegetative parmi les souches. Quatorze groupes distincts de compatibilitC vCgCtative ont CtC obtenus et numCrotCs de VCG2001 2 VCG2014. Les 82 autres souches ont alors CtC essayCes pour dCceler leur compatibilitC avec les 14 groupes distincts de compatibilitC vegetative. Cinquante des 110 souches appartiennent i une des 14 groupes distincts de compatibilitC vCgCtative. Deux groupes de compatibilitC vCgCtative, VCG2001 et VCG2002, sont apparus avec la plus grande friquence et i partir de la plupart des champs. Les ksultats suggbrent que certains groupes de compatibilitk vCgCtative trouvCs sur les racines de cCleri seraient beaucoup plus communs que d'autres. La compatibilitC vCgCtative apparait cornme un moyen qui permettrait d'identifier et de caractkriser des souches non pathogbnes de F. oxysporum. [Traduit par la revue]

Introduction Fusarium oxysporum is a ubiquitous soil-borne species that can be found in cultivated and noncultivated soils (18, 22, 30). In cultivated soils, F. oxysporum often makes up a major portion of the fungal flora (14, 17, 22). Meyer (15) showed that the F. oxysporum population reached 80-90% of the total fungal community in the rhizosphere of several agricultural crops. A great deal of diversity exists within F. oxysporum. The pathogenic strains of the species have received the most attention because they are vascular wilt pathogens of economically important agricultural crops (19). These strains have been divided into formae speciales (and races) based on their ability to cause disease on a particular host or group of hosts (5). Currently, there are in excess of 122 formae speciales and races of F. oxysporum (4). However, many strains of F. oxysporum do not cause disease on any known host. These strains have been referred to as saprophytes (29, 31), avirulent formae speciales (lo), and nonpathogens (2,21,28). In this paper, these strains will be referred to, collectively, as nonpathogens. In the past, the nonpathogenic strains of E oxysporum have received relatively little attention. More recently, however, Printed in Canada I lmprimt au Canada

several workers have shown that these nonpathogenic strains may reduce disease incidence and (or) severity of certain Fusarium vascular wilt diseases (10, 11, 12, 13, 21) and that they may contribute to the biological suppressiveness of certain soils to vascular wilt diseases (1, 2, 16, 27,28). However, the ecological significance of the nonpathogenic component of F. oxysporum populations under field conditions is poorly understood. In light of the differences in virulence and host specificity displayed by the pathogenic strains of F. oxysporum, we would expect at least as much variability among the nonpathogenic strains of the species. However, these nonpathogens, for the most part, have been considered a single ecological entity, no doubt because of our inability to distinguish strains within a given population. Although some workers have recognized certain "clonal types" within groups of both pathogenic and nonpathogenic strains, most strains are morphologically indistinguishable (14, 18, 29, 32). If indeed there are different strains of nonpathogenic F. oxysporum, we need to be able to differentiate these strains if we are to test for ecological differences among them. Puhalla (24) generated nitrate-nonutilizing mutants (nit

2359

CORRELL ET AL.

TABLE 1. Nonpathogenic isolates of Fusarium oxysporum from celery

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\.

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.)

i

- -1

I

L .J

Field

County

A B C E F G H I J X

Ventura Santa Barbara San Luis Obispo Ventura Ventura Ventura Monterey San Diego San Diego Alameda

Total

No. of No. of tester isolates strainsa 21 12 7 8 21 6 10 4 10 11

3 3 0 0 1 0 3 1 3 0

110

14

OTester straln, vegehtlve cornpat~b~l~ty group

FIG. 1. Location of fields sampled in southern California.

mutants) from pathogenic strains of F. oxysporum without the use of a mutagen. Correll et al. also generated nit mutants from strains of F. oxysporum nonpathogenic on celery (7). These nit mutants grow as thin expansive colonies on a minimal agar medium (MM) (24) that has sodium nitrate (NaNO,) as the sole nitrogen source. When certain nit mutants are paired on MM, they complement one another (24); that is, dense aerial mycelium develops where the two thin nit mutant colonies come in contact. This complementation is an indicator of vegetative compatibility; pairings between vegetatively incompatible strains remain thin. Two complementary nit mutants from a particular strain can then be used to test for vegetative compatibility with other isolates of F. oxysporum. All isolates which are vegetatively compatible are said to belong to the same vegetative compatibility group (VCG). Vegetative compatibility, which is the result of hyphal fusion (anastomosis) between two fungal strains, has been shown to be under the control of many gene loci (3, 23, 26). Only strains with the same allele at each of these loci are compatible. This suggests that strains within a VCG are genetically similar; there is substantial evidence in support of this. Puhalla (25) divided pathogenic strains of F. oxysporum into groups (VCGs) on the basis of vegetative compatibility and suggested that there was a correlation between these groups and formae speciales. Correll et al. (7) have shown that vegetative compatibility is a useful criterion for identifying F. oxysporum f. sp. apii race 2 among isolates of F. oxysporum recovered from celery roots. Specific races of F. oxysporum f. sp. pisi also have been differentiated on the basis of vegetative compatibility (8). In this study, F. oxysporum strains colonizing celery roots, which were previously described as nonpathogens (7), were classified on the basis of vegetative compatibility. Vegetative compatibility was evaluated as a means of identifying and characterizing strains within several nonpathogenic F. oxysporum populations. A preliminary report has been published (6).

Materials and methods Fungal strains A total of 110 nonpathogenic F: oxysporum isolates colonizing celery (Apium graveolens L. var. dulce (Miller) Pers.) roots were collected from 10 fields throughout the celery-growing regions of

California (Fig. 1 and Table 1) (7). In addition to being avirulent on celery, nonpathogens typically have a large colony size on sorbose medium (7) and are vegetatively incompatibile with the F: oxysporum f. sp. apii race 2 pathogen (7). Therefore, designation of these strains as nonpathogens on celery is not based solely on virulence reactions. Test for vegetative compatibility Nitrate-nonutilizing (nit) mutants were selected by the method of Puhalla (24). Two nit mutants were paired by placing them 1.5 cm apart in the center of a 9-cm Petri plate containing MM. The pairings 2°C) in the dark and were incubated at room temperature (23 scored for complementation 7 and 14 days later. Development of dense aerial mycelium where the two thin nit mutant colonies came in contact indicated vegetative compatibility. Complementary nit mutants, arbitrarily designated nitA and nitB, were generated within each of 28 randomly selected isolates by using the method described above. The two nit mutants from each tester strain were then paired in all combinations to determine the number of VCGs present among the original 28 tester strains. Fourteen distinct VCGs were recovered from the original 28 isolates. At least one nit mutant was then generated from each of the remaining 82 nonpathogenic isolates of F. oxysporum in the sample. These nit mutants were then paired with the two complementary nit mutants of each of the 14 tester strains. All pairings were repeated at least once, and all pairings that scored positive for complementation were repeated at least three times.

+

Results and discussion Fourteen distinct vegetative compatibility groups were identified among 28 nonpathogenic isolates of F. oxysporum. These 14 VCGs, designated VCG2001 through VCG2014, had very similar colony morphologies when grown under standard conditions (20) on potato dextrose agar. Twenty-two (27%) of the remaining 82 isolates tested belonged to one of these 14 VCGs. VCG2001 and VCG2002 were found in the highest frequency and in the most fields (Table 2). Sixty isolates were vegetatively incompatible with all 14 of the designated testers. In no case was an isolate found to belong to more than one VCG. Croft and Jinks divided the wild population of Aspergillus nidulans into a number of independent subpopulations (or VCGs) on the basis of vegetative compatibility (9). Isolates within a VCG had similar characteristics such as colony size and antibiotic production. The plant pathogen Verticillium dahliae also has been divided into VCGs and there was a tendency for isolates from the same host to be in the same

CAN. J. BOT. VOL. 64, 1986

TABLE2. Vegetative compatibility groups among nonpathogenic Fusarium oxysporum isolates, source of VCG, and number of isolates per field

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VCG

Source of tester

No. of isolates from each field location A

B

C

E

F

G

H

I

J

X

Total per VCG

"-, none found.

VCG (25). Isolates of the vascular wilt pathogen F. oxysporum f. sp. apii race 2, from a broad geographical area, also were vegetatively compatible and thus in the same VCG (7). Although strains within a number of VCGs are genetically similar, preliminary evidence indicates that there is genetic heterogeneity within a VCG of E oxysporum f. sp. pisi (8). Puhalla found 16 VCGs among 21 pathogenic isolates of Fusarium oxysporum (24). There were 14 VCGs among 28 randomly selected nonpathogenic isolates in the current study. These data would suggest that there may be a greater VCG diversity within Fusarium oxysporum than has been demonstrated in other fungal species (3, 9, 25). In the studies with Aspergillus nidulans and Verticillium dahliae, there apparently was a nonrandom geographical distribution of VCGs. Our data suggest that the distribution of the VCGs found in the collection of nonpathogenic strains of F. oxysporum from celery roots also was nonrandom; several VCGs were far more common than others. For example, VCG2001 and VCG2002 were found to have a wide distribution among the 10 fields sampled. Moreover, several isolates belonging to these two VCGs were found in individual fields. Twenty-six percent of all the F. oxysporum isolates collected belong to these two VCGs. The fact that these two VCGs were recovered so frequently from such a limited sample would suggest that there may be some selection pressure favoring these two groups. Without sexual reproduction, and therefore exchange of genetic material, the strains of F. oxysporum which were vegetatively incompatible may have become genetically isolated subpopulations (9). If this were the case, we would expect to find biological and ecological differences between VCGs in the various nonpathogenic F. oxysporum populations. Indeed, differences in the rate of celery root colonization have been observed between two of these VCGs of E oxysporum obtained from celery roots (D. J. Jacobson and J. C. Correll, unpublished). Toussoun (3 I), as early as 1963, suggested that such differences may exist. In light of the ongoing work with soils suppressive to Fusarium vascular wilt diseases, differentiating strains among the nonpathogenic F. oxysporum isolates is very important. Alabouvette et al. (I), Schneider (28), and others (2, 10, 11, 12, 13, 21) have shown that nonpathogenic strains of F. oxy-

sporum can reduce disease incidence and severity of several vascular wilt diseases incited by various formae speciales of F. oxysporum. Little is known, however, about the ecological significance and the population dynamics of the nonpathogenic strains of F. oxysporum, particularly at the subspecies level. It is likely that the isolates used in these studies belong to different VCGs. Vegetative compatibility and VCGs are naturally occumng genetic markers. They could provide a means of identifying and characterizing the various subpopulations of the nonpathogenic F. oxysporum. If natural markers, such as VCG, prove useful in identifying nonpathogenic strains of F. oxysporum, some intriguing questions could be addressed concerning the ecology of this cosmopolitan species. 1. ALABOUVETTE, C., Y. COUTEADIER, and J. LOUVET.1984. Studies on disease suppressiveness of soils. X. Comparison of the fungal microflora colonizing the roots of muskmelons growing in a wilt-suppressive and a wilt-conducive soil. Agronomie (Paris), 4(8): 135- 140. 2. ALABOUVETTE, C., F. ROUXEL, and J. LOUVET.1975. Characteristics of Fusarium wilt-suppressive soils and prospects for their utilization in biological control. In Soil borne plant pathogens. Edited by W. Gams. Academic Press, New York. pp. 165 - 182. 3. ANAGNOSTAKIS, S. L. 1977. Vegetative incompatibility in Endothia parasitica. Exp. Mycol. 1: 306 -3 16. G. M., and J. K. ARMSTRONG. 1981. Formae spe4. ARMSTRONG, ciales and races of Fusarium oxysporum causing wilt diseases. In Fusarium: diseases, biology, and taxonomy. Edited by P. E. Nelson, T. A. Toussoun, and R. J . Cook. Pennsylvania University Press, University Park, PA. pp. 391 -399. 5. BOOTH,C. 1971. The genus Fusarium. Commonwealth Mycological Institute, Kew , Surrey, England. J. C., J. E. PUHALLA, and R. W. SCHNEIDER. 1985. 6. CORRELL, Distinct vegetative compatibility groups (populations) of Fusarium oxysporum colonizing celery roots from California. Phytopathology, 75: 1347. (Abstr.) J. C., J. E. PUHALLA, and R. W. SCHNEIDER. 1986. 7. CORRELL, Identification of Fusarium oxysporum f. sp. apii on the basis of virulence, colony size, and vegetative compatibility. Phytopathology, 76: 396 -400. J. C., J. E. PUHALLA, R. W. SCHNEIDER, and J. M. 8. CORRELL,

CORRELL ET AL.

9.

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12. 13. 14. 15. 16. 17. 18. 19.

20. 21.

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