Multilocus PCR assay reveals high diversity of vegetative compatibility types in populations of Cryphonectria parasitica in Croatia

Abstract

The ascomycete fungus Cryphonectria parasitica, causal agent of chestnut blight, is probably one of the best known invasive fungal pathogens in forests of Europe and North America. Mycovirus that reduces virulence of C. parasitica can be used as a biocontrol agent of the chestnut blight. However, anastomosis-mediated virus transmission is limited by a vegetative (in)compatibility (vc) system involving at least six known diallelic vic genetic loci. This study looked at vegetative compatibility (vc) diversity in two populations of C. parasitica in Croatia. For that purpose, a PCR assay was validated and implemented using already known/published and newly designed primers for amplification of six known vic loci. The vc genotypes determined by PCR for 158 C. parasitica isolates investigated in this study were in complete agreement with the vc genotypes determined by pairwise co-culturing of the same isolates, revealing the specificity and accuracy of the PCR-based molecular vic genotyping assay. Twenty-six unique vc genotypes were found among 158 isolates, and 19 vc types per population, which makes Croatian C. parasitica populations among the most diverse in Europe regarding the number of vc types and genetic diversity. Low values of multilocus linkage disequilibrium suggest sexual reproduction as a major contributor to high C. parasitica genetic diversity in studied populations.

High vegetative compatibility diversity of Cryphonectria parasitica infecting sweet chestnut (Castanea sativa) in Britain indicates multiple pathogen introductions

“…In particular, EU1 and EU2 dominate in northern Italy, southern France, Switzerland, eastern Spain and Croatia (Robin & Heiniger, ; Krstin et al ., ), whilst EU12 has been found in many European countries but is particularly frequent in eastern Europe (Robin & Heiniger, ). EU14, the dominant VCG in the England outbreaks, also has a wide distribution in Europe with records from Bosnia‐Herzegovina (Trestic et al ., ), Turkey (Akilli et al ., ), southwestern France and northern Spain (Robin et al ., ), Germany (Peters et al ., ) and Croatia (Mlinarec et al ., ). In contrast, EU65 has only been found in southwestern France, northern Spain and Germany (Robin et al ., ; Peters et al ., ).…”

“…However, genetic variation in C. parasitica , as a product of sexual recombination, limits spread of hypovirus between individuals (Scibilia & Shain, ; Liu & Milgroom, ). Population diversity can be measured in a number of ways, but an accepted approach for C. parasitica is to evaluate the number of vegetative compatibility groups (VCGs) in populations and subpopulations (Mlinarec et al ., ). Vegetative incompatibility in C. parasitica is controlled by at least six vegetative incompatibility ( vic ) loci, with two alleles at each locus (Cortesi & Milgroom, ).…”

The potential diversity of vegetative compatibility types in the population of Cryphonectria parasitica in Turkey

“…Six single ascospore isolates that gave ambiguous reaction in the co-culturing assay were successfully vic genotyped in the PCR assay. PCR-based vic genotyping has been previously studied in North America and Europe (Choi et al, 2012;Mlinarec et al, 2018;Peters et al, 2014;Short et al, 2015). Short et al (2015) have distinguished the 12 known alleles of the six vic loci of C. parasitica by using multiple PCR primer F I G U R E 2 Gel electrophoresis of amplicons produced by the six PCR assays using EU-1 and EU-12 vc type strains of Cryphonectria parasitica for 10 alleles.…”

“…(2015) and Mlinarec et al. (2018) were used. DNA was extracted by using quick genomic DNA miniprep developed for C. parasitica (Milgroom et al., 2008).…”

“…For vic genotyping, the parent isolates, K5-5 and A-2490, the single ascospores isolates representing each of the 14 vc types F I G U R E 1 In vitro cross between the dominant vc types (EU-1 and EU-12) of Cryphonectria parasitica in Turkey (a) A sexual pairing of the isolates K5-5 (EU-1)) and N-2490 (EU-12) on the autoclaved chestnut wood (P1:Parent 1; P2:Parent 2; Br:Barrage formation) (b) Developing perithecia on the chestnut wood (Pr: a perithecial neck) (c) Asci running from perithecia stained with acid fuchsin, were subjected to PCR. In the PCR assay, a number of specific primers targeting each of the six vic loci which have been previously reported by Short et al (2015) and Mlinarec et al (2018) were used. DNA was extracted by using quick genomic DNA miniprep developed for C. parasitica (Milgroom et al, 2008).…”