1 Heredity 86 (2001) 134±143 Received 6 June 2000, accepted 21 August 2000 The mating system of the fungus Cryphonectria parasitica: sel®ng and self-incompatibility ROBERT E. MARRA* & MICHAEL G. MILGROOM Department of Plant Pathology, 334 Plant Sciences Building, Cornell University, Ithaca NY 14853, U.S.A. Although the genetic components of mating systems in fungi are well understood as laboratory phenomena, surprisingly little is known about their function in nature or about their role in determining mating patterns and population genetic structure. Our study of the mating system of the haploid ascomycete fungus, Cryphonectria parasitica, resulted in the following. (1) Laboratory crosses among 20 isolates, chosen randomly from North America and China, resolved into two incompatibility groups (occurring on both continents), con®rming that C. parasitica has a diallelic, bipolar sexual self-incompatibility system, typical of other self-incompatible Ascomycetes, in which mating is only successful between isolates of opposite mating type. (2) PCR-based markers for mating-type alleles correlated perfectly with mating-type phenotypes of individual isolates. (3) Three genotypes, isolated from natural populations in Virginia and West Virginia, were inoculated onto chestnut trees in two sites in West Virginia and were con®rmed to have self-fertilized and outcrossed in both sites. (4) Ten isolates, of a total of over 200 assayed, were con®rmed to have self-fertilized in the laboratory, albeit at very low frequency. Five of these 10 isolates were ramets of a single genet, suggesting a genetic basis underlying the proclivity to self-fertilize in the laboratory. (5) Self- fertilization could not be induced in the laboratory with exudates (ostensibly containing pheromones) from isolates of opposite mating type. These results demonstrate that, a sexual self-incompatibility system notwithstanding, self-fertilization occurs under both laboratory and ®eld conditions in C. parasitica. The disparity between observations of frequent sel®ng in nature and rare sel®ng in the laboratory suggests that the mating system is under ecological as well as genetic control. Keywords: chestnut blight, Cryphonectria parasitica, fungus, mixed mating, self-fertilization, self- incompatibility. Introduction cinereus), spatial and temporal dynamics of fungal mating systems in nature remain relatively unstudied. Because of the near ubiquity of hermaphroditism in In those species where genetically determined self- plants and fungi, the phenomena of self-compatibility incompatibility has been identi®ed, it ranges from and self-incompatibility are central to studies of plant simple one-locus, two-allele systems to complex two- and fungal mating systems. However, in contrast to locus, multiallelic systems (Alexopoulos et al., 1996; more than a century of empirical and theoretical work p. 501). In the Ascomycota, sexual self-incompatibility is addressing plant mating systems (reviewed in Barrett, under the control of a single diallelic mating-type locus 1988; Barrett & Eckert, 1990; Jarne & Charlesworth, (MAT), which controls the fertilization process, and 1993; Richards, 1997), the majority of fungal mating subsequent steps in mating, as a regulator of diusable systems has yet to be investigated. Although a great deal and MAT-speci®c pheromones and receptors (Kues & of laboratory work has been focused on the mating Casselton, 1992). Ascomycete self-incompatibility is systems of a few fungi (e.g. Saccharomyces cerevisiae, considered `bipolar'; that is, mating only occurs between Neurospora crassa, Podospora anserina, and Coprinus individuals that have opposite alleles, or idiomorphs (MAT-1 and MAT-2) at this locus (Coppin et al., 1997; Kronstad & Staben, 1997). *Correspondence and present address: Department of Microbiology, With the exception of a few studies of ascomycete and Jones Building Box 3020, Duke University Medical Center, Durham basidiomycete species (Raper, 1966; Rayner & Turton, NC 27710, U.S.A., Tel: 919 684 9096, E-mail: [email protected] 134 Ó 2001 The Genetics Society of Great Britain. SELF-INCOMPATIBILITY IN CRYPHONECTRIA PARASITICA 135 1982; Ainsworth, 1987; Ennos & Swales, 1987; Leucht- were (1) to demonstrate and describe the self- mann & Clay, 1989; Sharland & Rayner, 1989a; incompatibility system in C. parasitica, and (2) to Sharland & Rayner, 1989b; Grin et al., 1992; Kohli determine if self-fertilization can in fact occur in the & Kohn, 1992), little is known about the manner or ®eld and laboratory. degree to which the mating systems of fungi, as characterized in the laboratory, determine genetic Study system structure in natural populations. The importance of studying fungal mating systems in both nature and the Cryphonectria parasitica, the ascomycete fungus that laboratory is demonstrated well by research on the causes chestnut blight, was introduced into North ascomycete Cryphonectria parasitica, a fungus patho- America from Japan around 1900 (Milgroom et al., genic on chestnut trees (Castanea dentata) in eastern 1996), and spread rapidly across the natural range of the North American forests. Mixed mating has been found American chestnut (Castanea dentata), which currently in ®ve populations in eastern North America, with persists as sprouts that regenerate from surviving root outcrossing rates (t) ranging from 0.68 to 0.73 (Marra, systems (reviewed in Anagnostakis, 1987). 1998; Milgroom et al., 1993). The life cycle of the typical ®lamentous ascomycete is The mating system of C. parasitica has so far eluded reviewed in Nelson (1996). C. parasitica infects chestnut simple classi®cation: both sel®ng and self-incompatibil- trees and forms cankers that produce two types of ity have been claimed, but neither has been suciently spores: asexual (conidia) and sexual (ascospores). documented. Signi®cant amounts of self-fertilization Conidia may function either as vegetative propagules occur in nature (see above), yet C. parasitica does not or as male gametes in sexual reproduction. Fertilization appear to self-fertilize readily in the laboratory (Marra, in C. parasitica (as well as many other ascomycetes) 1998; this study). A previous report of sel®ng in the occurs when a conidium and a receptive hypha (the laboratory (Puhalla & Anagnostakis, 1971) cannot be female reproductive structure) fuse to form a dikaryon considered conclusive, because controls for fertilization (n + n). Synchronous nuclear divisions result in a by contaminating male gametes (conidia) were not used, cluster of dikaryotic hyphae within the developing an essential control for demonstrating sel®ng unambig- fruit-body. Each terminal n + n cell (ascus initial) uously (Whitehouse, 1949). In marked contrast to the within the fruit-body undergoes karyogamy, resulting evidence for self-fertility in the ®eld, it has been in the brief diploid (2n) phase. Therefore, a single suggested (Anagnostakis, 1988) that C. parasitica is fertilization results in a fruitbody containing many 2n sexually self-incompatible, and recent work suggests cells. Each 2n nucleus undergoes meiosis, followed by a that C. parasitica may have a self-incompatibility locus single mitosis, resulting in eight ascospores per mature (MAT) similar to those of other self-incompatible ascus. In C. parasitica, and all other members of the ascomycetes (Marra & Milgroom, 1999). Class Pyrenomycetes, asci are borne inside a ¯ask- One explanation for this apparent contradiction is shaped fruit-body, the perithecium. Dispersal occurs that what appears to be sel®ng in nature is not actually when ascospores (there may be thousands per perithe- sel®ng, but biparental inbreeding to a degree that is cium) are forcibly ejected through an opening in the beyond the resolution of available markers. In this case, neck of the perithecium, which protrudes from the absence of marker segregation may not be diagnostic of surface of the canker. Each haploid ascospore is self-fertilization, but rather due to `eective' or `appar- dispersed by air currents, and is capable of infecting a ent' sel®ng (Hedrick, 1990). Spatial aggregation of tree and developing into a new canker, completing the genotypes (Milgroom & Lipari, 1995b), and biparental fungus's life cycle. inbreeding (Marra & Milgroom, 1994; Marra, 1998) suggest that some populations may actually comprise Materials and methods relatively restricted genetic neighbourhoods (Wright, 1946). These alternative explanations for lack of Strains and isolates used marker segregation require that the hypothesis that self-fertilization occurs in nature be tested rigorously. We used a combination of ®eld and laboratory-derived The possibility that a self-incompatible fungus is isolates of C. parasitica in this study. Unless otherwise capable of self-fertilization warrants further examina- speci®ed, ®eld isolates were collected from Mt. Lake, tion, and is the motivation for our interest in establish- Virginia (Milgroom et al., 1993); Danby, New York; ing the genetics underlying self-incompatibility in the Depot Hill, New York (Milgroom & Lipari, 1995a); and mating system in C. parasitica. However, because Parsons, West Virginia (Marra, 1998). Field isolates were neither self-fertilization nor self-incompatibility has obtained from bark samples (approx. 4 cm2) of chest- been suciently documented, our goals in this paper nut trees with visible blight cankers. New laboratory Ó The Genetics Society of Great Britain, Heredity, 86, 134±143. 136 R. E. MARRA & M. G. MILGROOM strains originated as ascospores