Análise de micoviroses em Pseudocercospora griseola, agente causal da mancha angular do feijoeiro

Abstract

Angular spot, caused by the fungus Pseudocercospora griseola (Sacc.) Crous & U. Braun, is one of the most important diseases of common bean (Phaseolus vulgaris L.). The presence of double-stranded RNA (dsRNA) mycoviruses has been observed in this pathogen. Therefore, the objective of this work was to analyze different isolates stored in mycotheca, and also, recently collected isolates of P. griseola regarding sporulation, detection of double-stranded RNA mycoviruses and their effects on colony morphology, sporulation and virulence of the pathogen. Furthermore, four isolates were cured, that is, isogenic isolates without the presence of the virus were obtained. 29 isolates of P. griseola were used, nine of which were stored in a library and 20 were collected in an experimental bean field. The isolates were cultivated in PDA medium and later in GPYE liquid medium to obtain the mycelial mass. Total RNA extraction followed the TRIzol Reagent protocol, and was subsequently followed by treatment with the S1 endonuclease enzyme to obtain dsRNA and subjected to agarose gel electrophoresis to identify the presence or absence of dsRNA. Isogenic, virus-free isolates were obtained through monosporic culture grown in PDA culture medium with cycloheximide. The isolates showed different levels of sporulation capacity at the times evaluated and of virulence when inoculated in the bean cultivars Rosinha, BRS Horizonte and the MAIII-16159 lineage. The presence of dsRNA was observed in 69% of the isolates evaluated. Application of cycloheximide for viral clearance was successful in 75% of isolates, resulting in the elimination of low molecular weight dsRNA. Isogenic isolates did not show differences in colony pigmentation, but showed lower mycelial density when compared to wild-type isolates. In the absence of dsRNA fragments after viral clearance, isolate 9 (race 63.63) showed less sporulation. dsRNA had a negative effect on the virulence of P. griseola isolates, this being the first report of hypovirulence in this pathogen.