Heterokaryons and parasexual recombinants of Cryphonectria parasitica in two clonal populations in southeastern Europe

Evidence for parasexuality in natural populations of haploid fungi requires the demonstration of diploids or heterokaryons and recombinant genotypes in the absence of sex. We studied clonal populations of the chestnut blight fungus, Cryphonectria parasitica, in southeastern Europe and found evidence of parasexuality in two locations. In Osoj, Macedonia, we found one isolate (Os05-66) that had two alleles at six codominant loci, giving a haplotype that was a composite of two clones in this population. Six single-conidial isolates from Os05-66 had two alleles at some loci, suggesting partial diploidy or aneuploidy, and we found four recombinant haplotypes among single-conidial isolates from hyphal-tip isolates of the same isolate. In Teano, Italy, we found two heterokaryon isolates that were partial composites of two dominant clones. Single-conidial isolates from hyphal-tip isolates had recombinant haplotypes. These results provide evidence that is consistent with the hypothesis of parasexuality in C. parasitica in Europe, similar to an earlier report in a natural population in the USA.     

Multilocus PCR Assays Elucidate Vegetative Incompatibility Gene Profiles of Cryphonectria parasitica in the United States

Chestnut blight is a devastating disease of Castanea spp. Mycoviruses that reduce virulence (hypovirulence) of the causative agent, Cryphonectria parasitica, can be used to manage chestnut blight. However, vegetative incompatibility (vic) barriers that restrict anastomosis-mediated virus transmission hamper hypovirulence efficacy. In order to effectively determine the vegetative incompatibility genetic structure of C. parasitica field populations, we have designed PCR primer sets that selectively amplify and distinguish alleles for each of the six known diallelic C. parasitica vic genetic loci. PCR assay results were validated using a panel of 64 European tester strains with genetically determined vic genotypes. Analysis of 116 C. parasitica isolates collected from five locations in the eastern United States revealed 39 unique vic genotypes and generally good agreement between PCR and tester strain coculturing assays in terms of vic diversity and genotyping. However, incongruences were observed for isolates from multiple locations and suggested that the coculturing assay can overestimate diversity at the six known vic loci. The availability of molecular tools for rapid and precise vic genotyping significantly improves the ability to predict and evaluate the efficacy of hypovirulence and related management strategies.     

Heterokaryon incompatibility function of barrage-associated vegetative incompatibility genes (vic) in Cryphonectria parasitica

Six vegetative incompatibility (vic) loci have been identified in Cryphonectria parasitica based on barrage formation during mycelial interactions. We used hygromycin B- and benomyl-resistance as forcing markers in C. parasitica strains to test whether heteroallelism at each vic locus prevents heterokaryon formation following mycelial interactions. Paired strains that had allelic differences at any of vic1, 2, 3, 6 or 7 but not vic4 displayed heterokaryon incompatibility function, as recognized by slow growth or aberrant morphology. While clearly forming barrages in mycelial interactions, paired strains with different alleles at vic4 formed stable heterokaryons. With examples from other fungi, this inconsistency at vic4 suggests that barrage formation and heterokaryon incompatibility are not different manifestations of the same process. Rather, the evidence indicates that heterokaryon incompatibility represents a component of a vegetative incompatibility system that may also use cell-surface or extracellular factors to trigger programmed cell death to modulate nonself recognition in fungi.     

The Use of Duplication-Generating Rearrangements for Studying Heterokaryon Incompatibility Genes in Neurospora

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N. crassa are difficult because in commonly used stocks differences are present at several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.—Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had previously been established by conventional heterokaryon tests—het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplication-generating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.—The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.—The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests.     

Vegetative Incompatibility and the Mating-Type Locus in the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM

The genetic basis of vegetative incompatibility in the cellular slime mold, Dictyostelium discoideum, is elucidated. Vegetatively compatible haploid strains from parasexual diploids at a frequency of between 10^-6 and 10^-5, whereas "escaped" diploids are formed between vegetatively incompatible strains at a frequency of ~10^-8. There is probably only a single vegetative incompatibility site, which appears to be located at, or closely linked to, the mating-type locus. The nature of the vegetative incompatibility is deduced from parasexual diploid formation between wild isolates and tester strains of each mating type, examination of the frequency of formation of "escaped" diploids formed between vegetatively incompatible strains, and examination of the mating type and vegetative incompatibility of haploid segregants obtained from "escaped" diploids.     

Genetics of Vegetative Incompatibility in Cryphonectria parasitica

Vegetative incompatibility in the chestnut blight fungus, Cryphonectria parasitica, in Europe is controlled by six unlinked vic loci, each with two alleles. Four previously identified vic loci (vic1, vic2, vic3, and vic4) were polymorphic in European vegetative compatibility (vc) types. Two new loci, vic6 and vic7, also were identified among European vc types. In one cross, vic genes segregated independently at five loci, and 194 progeny were assigned to 32 vc types; none of these loci were linked. A total of 64 vc types were identified from all crosses. All 64 genotypes possible from six vic loci, each with two alleles (2⁶ = 64), were identified and assigned to vc types. Based on our model, vc types v-c 5 and v-c 10, which had been used in previous genetic studies, differ by only five vic genes. Future studies of vc types in C. parasitica can use knowledge of vic genotypes for analysis of population genetic structure based on vic allele frequencies and to determine the effect of each vic gene on virus transmission between vc types.     

Molecular characterization of vegetative incompatibility genes that restrict hypovirus transmission in the chestnut blight fungus Cryphonectria parasitica

Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.     

Heterokaryon incompatibility in fungi—more than just another way to die

In filamentous fungi heterokaryon (vegetative) compatibility is regulated by a number of different loci. Vegetative incompatibility is most often detected as the inability to form a prototrophic heterokaryon under forcing conditions, or as the formation of a barrage when two incompatible strains interact. Vegetative compatibility has been used as a multilocus phenotype in analysis of fungal populations. In some highly clonal populations the vegetative-compatibility phenotype is correlated with pathogenicity. The molecular basis for vegetative compatibility is not well understood. Fourhet loci have been cloned fromNeurospora crasset orPodospora anserina, inch but no two are alike and it is clear that thehet genes themselves do not encode the gene products that are directly responsible for cell death. We suggest that a broader view of vegetative compatibility would include genes that are responsible for prefusion, fusion, and postfusion activities. Postfusion activities could include the fungal apoptotic apparatus since microscopic observations of cell death resemble those in higher plants and animals.     

Mating System and Basidiospore Formation in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

Prototrophic strains recovered from crosses between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The progeny of most of these self-crosses were prototrophic, indicating that the nuclei of the original prototroph were wild-type recombinants rather than complementary heterokaryons and that the binucleate basidiospores of this organism are homokaryotic. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium. Colonies arising from single conidia of various wild-type strains were all capable of producing fruit bodies and basidiospores. In addition, single basidiospores from three wild-type strains all produced fruit bodies and basidiospores. Nonfruiting as well as fruiting isolates were obtained from single basidiospores of five other wild-type strains. Basidiospores from these fruiting isolates always yielded colonies that fruited, again indicating that the spores are homokaryotic. Nonfruiting isolates from the same strain did not produce basidiospores when allowed to form a heterokaryon, implying that these isolates do not represent mating types. All this evidence indicates that P. chrysosporium has a primary homothallic mating system. In addition to fruiting and nonfruiting phenotypes, basidiospores from strain OGC101, a derivative of ME-446, gave rise to colonies which did not grow on cellulose (Cel⁻). The fruiting, nonfruiting, and Cel⁻ phenotypes differed from each other and from the parental wild-type strain in a variety of characteristics, including growth, conidiation, and evolution of ¹⁴CO₂ from ¹⁴C-side chain-labeled lignin, indicating that strain OCG101 is a heterokaryon.     

Genetic diversification of the chestnut blight fungus Cryphonectria parasitica and its associated hypovirus in Germany

Chestnut blight in south-western Germany was first reported in 1992 and is since expanding in distribution. Here we investigated the invasion history of Cryphonectria parasitica and its associated hypovirus. For this, we characterized 284 isolates collected between 1992 and 2012 for hypovirulence, vegetative compatibility (vc), mating type, and microsatellite haplotype. A total of 27 haplotypes and 15 vc types were observed, although the C. parasitica population analyzed is currently dominated to 50 % by one haplotype and to 64 % by the vc type EU-2. Structure analysis indicated two divergent genetic pools. Over 66 % of the haplotypes belonged to a pool probably originating from northern Italy. Further diversification is expected due to ongoing sexual recombination, but also to new migration and additional introductions. Cryphonectria hypovirus 1 (CHV-1) was found in four of five C. parasitica populations from Baden-Württemberg. Genetic analysis of the 35 CHV-1 isolates obtained revealed that they all belong to the German subtype, although they have clearly diverged from the first German hypovirus isolated in 1992. Our study suggests that C. parasitica has been introduced into Germany several times from two different gene pools, whereas the hypovirus most probably has a single origin.     

Molecular genetic determinants of intraspecific polymorphism of the phytopathogenic fungus <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>

The review summarizes the current evidence on the phytopathogenic fungus Cryphonectria parasitica, which is a classic object for studying hypovirulence. Phenotypic manifestations of hypovirulence and the molecular mechanisms of action of the mycovirus Cryphonectria hypovirus (CHV) infecting the fungus are described in detail. Genetic determinants of vegetative incompatibility in C. parasitica (a phenomenon increasing polymorphism of the fungus and preventing CHV expansion) are considered. The data on C. parasitica polymorphism are correlated with the data on the distribution of different CHV species in the European, American, and Asian populations of the fungus.     

The Homologue of het-c of Neurospora crassa Lacks Vegetative Compatibility Function in Fusarium proliferatum

For two fungal strains to be vegetatively compatible and capable of forming a stable vegetative heterokaryon they must carry matching alleles at a series of loci variously termed het or vic genes. Cloned het/vic genes from Neurospora crassa and Podospora anserina have no obvious functional similarity and have various cellular functions. Our objective was to identify the homologue of the Neurospora het-c gene in Fusarium proliferatum and to determine if this gene has a vegetative compatibility function in this economically important and widely dispersed fungal pathogen. In F. proliferatum and five other closely related Fusarium species we found a few differences in the DNA sequence, but the changes were silent and did not alter the amino acid sequence of the resulting protein. Deleting the gene altered sexual fertility as the female parent, but it did not alter male fertility or existing vegetative compatibility interactions. Replacement of the allele-specific portion of the coding sequence with the sequence of an alternate allele in N. crassa did not result in a vegetative incompatibility response in transformed strains of F. proliferatum. Thus, the fphch gene in Fusarium appears unlikely to have the vegetative compatibility function associated with its homologue in N. crassa. These results suggest that the vegetative compatibility phenotype may result from convergent evolution. Thus, the genes involved in this process may need to be identified at the species level or at the level of a group of species and could prove to be attractive targets for the development of antifungal agents.     

Characterization of Fusarium oxysporum f.sp. cepae from Onion in Turkey Based on Vegetative Compatibility and rDNA RFLP Analysis

Seventy‐five isolates of Fusarium oxysporum f.sp. cepae, the causal agent of basal plate rot on onion, were obtained from seven provinces of Turkey. The isolates were characterized by vegetative compatibility grouping (VCGs) and restriction fragment length polymorphism (RFLP) analysis of the nuclear ribosomal DNA intergenic spacer region (IGS). Forty‐eight vegetative compatibility groups were found, each containing a single isolate. Only one isolate formed strong heterokaryons with the reference isolates of VCG 0423. Five isolates were heterokaryon self‐incompatible. Restriction fragment analysis with six different enzymes revealed 13 IGS types among 75 F. oxysporum isolates from Turkey as well as 16 reference isolates from Colorado, USA. The majority of single‐member VCGs produced identical RFLP banding patterns with minor deviations, considerably different from those of the reference VCG isolates. These results suggested that isolates of F. oxysporum f.sp. cepae in Turkey derived from distinct clonal lineages and mutations at one or more vegetative compatibility loci restrict heterokaryon formation.     

The location and analysis of two heterokaryon incompatibility (het) loci in strains of Aspergillus nidulans

The heterokaryon incompatibility system in Aspergillus nidulans has been investigated by parasexual methods. The use of complementary auxotrophs with a repeated serial transfer method or with a protoplast fusion technique has enabled heterokaryons and diploid strains to be recovered from heterokaryon incompatible combinations of strains. The effects of allelic interaction at heterokaryon incompatibility (het) loci on the morphologies of the heterokaryon and diploid colonies isolated are described. Parasexual analyses conducted among strains belonging to the heterokaryon compatibility groups, h-cGl and h-cB, and the two recombinant compatibility classes, have located the hetA and hetB genes to linkage groups V and VI respectively.     

Retardation of the Growth of Transplanted Apothecia: A Manifestation of Vegetative Incompatibility in Ascobolus stercorarius (Bull.) Schröt.

Bistis, G. N. 1994. Retardation of the growth of transplanted apothecia: A manifestation of vegetative incompatibility in Ascobolus stercorarius (Bull.) Schröt. Experimental Mycology 18, 103-110. Two loci, C and D, each with at least two alleles, C¹, C² and D¹, D², control a system of apothecium-transplant incompatibility in the fungus Ascobolus stercorarius. The recognition appears to occur between the sterile tissue of the transplanted apothecium and the vegetative hyphae of the recipient mycelium. When these carry similar C and D alleles the transplanted apothecium grows at a normal rate. If they carry different D alleles the apothecium grows at a subnormal rate. If they carry different C alleles (with like or different Ds) the apothecium grows at a sub-subnormal rate. The C locus, but not the D, also has a detectable effect in vegetative heterokaryons. Two auxotrophic mutants will complement only if they carry like C factors. The two mating-type alleles, A, a, also function as transplant and vegetative incompatibility factors.     

Control of mating type heterokaryon incompatibility by the tol gene in Neurospora crassa and N. tetrasperma.

The mating-type of Neurospora crassa (A and a) have a dual function: A and a individuals are required for sexual reproduction, but only strains of the same mating type will form a stable vegetative heterokaryon. Neurospora tetrasperma, in contrast, is a naturally occurring A+a heterokaryon. It was shown previously that the mating-type genes of both species are functionally the same and are not responsible for this difference in heterokaryon incompatibility. This suggests that a separate genetic system determines the heterokaryon incompatibility function of mating type. The mutant tolerant (tol) in N. crassa, unlinked to mating type, acts as a specific suppressor of A+a heterokaryon incompatibility. In the present study, the wild-type alleles at the tol locus were introgressed reciprocally, from N. crassa into N. tetrasperma and from N. tetrasperma into N. crassa, to investigate the action of these alleles in the A+a heterokaryon incompatibility systems of these species. The wild-type allele from N. tetrasperma (tolT) acts as a recessive suppressor of A+a heterokaryon incompatibility in N. crassa. Furthermore, the wild-type allele from N. crassa (tolC) causes A and a to become heterokaryon incompatible in N. tetrasperma, while having no effect on the sexual reproduction. Therefore, the tol gene plays a major role in determining the heterokaryon compatibility of mating type in these species: tolC is an active allele that causes incompatibility and tolT an inactive allele that suppresses incompatibility by its inactivity.     

Control of chestnut blight by the use of hypovirulent strains of the fungus Cryphonectria parasitica in northwestern Spain

Chestnut blight is controlled in Europe by using Cryphonectria hypovirus CHV1, a non-encapsulated RNA virus. The chestnut blight fungus, Cryphonectria parasitica, is weakened by the virus, and healing tissue growth occurs in the host tree. Transmission of this cytoplasmic hypovirus is restricted by the incompatibility system of the fungus, so that the hypovirus can be transmitted only between isolates of the same or closely related vegetative compatibility (vc) types. Hypovirulent isolates of C. parasitica (all of the French subtype CHV1-F1) from Castilla y León (NW Spain) were compared with virulent isolates in both laboratory (cut stems) and field inoculations (in two orchards in the province of León and one orchard in the province of Zamora). The tests were performed with the most common vc types in the region, EU1 and EU11. The cut stem assay revealed that the hypovirulent isolates of vc type EU1 did not reduce the growth of virulent cankers. By contrast, four hypovirulent strains H1, H4, H5 and H6 (all vc type EU11) reduced the growth of virulent isolates in the cut stem assay. Field tests showed that hypovirulent isolates of EU1 and EU11 were effective in reducing canker in both orchards in León with all treatments tested; however, in Zamora, where only EU11 was tested, all the treatments failed except H1, which was able to reduce growth of the canker eighteen months after the inoculation. The development of hypovirulence suggests that hypovirus subtype F1 is well adapted in the province of León. Both naturally extended and inoculated hypoviruses appear to have reduced the incidence of the canker, thus improving chestnut stands. However, the inoculations were not as effective in the orchards in Zamora. This indicates that the disease could be controlled in Castilla y León by inoculation of trees with hypovirulent strains, but that more tests should be done in provinces where the hypovirus is still not present.     

Multilocus self-recognition systems in fungi as a cause of trans-species polymorphism

Trans-species polymorphism, meaning the presence of alleles in different species that are more similar to each other than they are to alleles in the same species, has been found at loci associated with vegetative incompatibility in filamentous fungi. If individuals differ at one or more of these loci (termed het for heterokaryon), they cannot form stable heterokaryons after vegetative fusion. At the het-c locus in Neurospora crassa and related species there is clear evidence of trans-species polymorphism: three alleles have persisted for approximately 30 million years. We analyze a population genetic model of multilocus vegetative incompatibility and find the conditions under which trans-species polymorphism will occur. In the model, several unlinked loci determine the vegetative compatibility group (VCG) of an individual. Individuals of different VCGs fail to form productive heterokaryons, while those of the same VCG form viable heterokaryons. However, viable heterokaryon formation between individuals of the same VCG results in a loss in fitness, presumably via transfer of infectious agents by hyphal fusion or exploitation by aggressive genotypes. The result is a form of balancing selection on all loci affecting an individual's VCG. We analyze this model by making use of a Markov chain/strong selection, weak mutation (SSWM) approximation. We find that trans-species polymorphism of the type that has been found at the het-c locus is expected to occur only when the appearance of new incompatibility alleles is strongly constrained, because the rate of mutation to such alleles is very low, because the number of possible incompatibility alleles at each locus is restricted, or because the number of incompatibility loci is limited.     

Genetic Analyses of ENDOTHIA PARASITICA: Linkage Data for Four Single Genes and Three Vegetative Compatibility Types

The loci cre, met and ts segregate independently in Endothia parasitica. The phenotype brown (br) seems to be determined by an allele at or very near the cre locus. The vegetative compatibility types (v-c) 5 and 39 are determined by different alleles at a locus that is not linked to cre, met or ts. Analysis of two crosses of v-c 5 strains by v-c 10 strains provides evidence that these two v-c groups are different at 5 or more v-c loci.     

Genetics of self/nonself recognition in Serpula lacrymans

This study provides an analysis of the vegetative incompatibility system in Serpula lacrymans (Basidiomycota), a genetic system used to recognize nonself in fungi. Seventy-five worldwide isolates could be grouped into eight vegetative compatibility (VC) types, some of them distributed on different continents. Mating studies combined with vegetative incompatibility analyses revealed that the vegetative incompatibility response between isolates mainly could be explained by two biallelic vegetative incompatibility (vic) loci. The frequency distributions of the interpreted vic alleles do not seem to support the idea of frequency-dependent or balancing selection acting on the vic loci. We find little genetic variation at the vic loci and in one of the loci there was a significant heterozyote deficiency among strains in the overall material. The results may be explained by a recent worldwide dispersal of a few S. lacrymans isolates and, correspondingly, only a few vic alleles are being maintained in these populations.