Transfection of Diaporthe perjuncta with Diaporthe RNA virus

Diaporthe perjuncta is a pathogen of grapevines worldwide. A positive-strand RNA virus, Diaporthe RNA virus (DaRV), occurs in hypovirulent isolates of this fungus. A virus-free isolate from a South African grapevine was transfected with in vitro-transcribed positive strands of DaRV. Based on reverse transcription-PCR and partial sequence analysis, the transfected virus was identified as DaRV. The in vitro-transcribed RNA transcripts used to transfect fungal spheroplasts contained parts of the vector at their distal ends. These vector sequences were separated from the DaRV genome during replication in the new host. The transfected isolate had morphological features that differed from those of the isogenic virus-free strain, including production of a yellow pigment, a decreased growth rate, and lack of sporulation. An apple-based pathogenicity test did not reveal any differences in virulence between the virus-free and DaRV-transfected isolates. This study showed that virus-free fungal hosts can be successfully transfected with viruses other than the Cryphonectria parasitica hypovirus.