engleski

Multilocus PCR assays reveal vegetative incompatibility gene profiles of Cryphonectria parasitica in Europe

The ascomycete fungus Cryphonectria parasitica (Murr.) Barr is the causal agent of chestnut blight, a devastating disease of Castanea sativa. Mycoviruses that reduce virulence of C. parasitica can be used as biocontrol agents of the chestnut blight. However, anastomosis- mediated virus transmission is hampered by vegetative (in)compatibility (vc) system involving at least six known di-allelic vic genetic loci. Traditionally, vic gene profile was determined by pairwise cultivation with 64 EU tester strains that represent all possible combinations of the vic alleles in Europe. Co- cultivation assay is a time-consuming task requiring well maintained in-house EU tester strains. Therefore, PCR-based vic genotyping was developed which, so far, was used only for characterization of C. parasitica populations in USA. The goals of this study were to validate and implement PCR-assay for routine characterization of European C. parasitica populations, with the emphasis on vic genetic structure of C. parasitica populations from Southern Europe. Analysis of 158 C. parasitica isolates collected from two locations in Croatia revealed 31 unique vic genotypes and generally good agreement between PCR, and tester strain co-culturing assays, in terms of vic diversity and genotype assignment. Unexpectedly, isolates that had two alleles at single vic loci (heteroallelism) were found, suggesting preservation of allogenic haploid nuclei in one mycelium. Interestingly, heteroallelism at vic4 was at least eight times more frequently observed, than the heteroallelism at vic1, vic2, vic6 or vic7. Isolates heteroallelic at vic4 were compatible with both EU testers having one or the other allele at this locus, while isolates heteroallelic at vic1, vic2, vic6 and vic7 were compatible with only one EU tester. Therefore, it was not always possible to differentiate between isolates’ EU types which differ in the vic4 allele (for example EU-1/EU-5, EU-2/EU-6, EU-4/EU-20 and EU-11/EU-13). In Buje and Hrvatska Kostajnica populations the most abundant vic genotype was EU-1 and/or EU-5. This study showed that PCR based vic genotyping could have a broad application in the research of C. parasitica, enabling quick and easy vc type assignation. High frequency of detected heteroallelism could increase horizontal transfer of mycoviruses in the populations of this pathogen.

Chryphonectria parasitica ; heteroallelism ; vegetative incompatibility

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