Deep sequencing of single Pleurotus ostreatus strain revealed mixed infection of seven RNA viruses

BACKGROUND: As a globally cultivated edible fungus, Pleurotus ostreatus is highly nutritious and rich in proteins, vitamins, minerals, and amino acids. However, viral infections cause significant losses in quality and yield during cultivation. In our survey, weakened growth and malformed fruiting bodies were observed in Sanbaoxiang Agricultural Technology Park, which is located in Tongzhou District of Beijing, but the associated viruses were not clear. RESULTS: The mycelia were isolated, purified and used for ribosomal RNA-depleted total RNA deep sequencing. A total of 118,973,697 clean reads were obtained and subsequently assembled into 53,864 contigs. BLAST analysis indicated that most contigs originated from the host P. ostreatus; however several contigs showed high amino acid sequence identities to known mycoviruses. Further complete genome sequencing with RT‒PCR and rapid amplification of cDNA ends for the terminal sequences revealed that mixed infection with seven RNA viruses was associated with the observed symptoms: four reported viruses, including oyster mushroom spherical virus, pleurotus ostreatus deltaflexivirus 2, pleurotus ostreatus alpha-like virus, and pleurotus ostreatus ambivirus 2, and three new viruses tentatively named pleurotus ostreatus deltaflexivirus 3, pleurotus ostreatus ambivirus 3 and pleurotus ostreatus lentinuvirus 1. The sequence characterization, genomic structure and phylogenetic relationships of these new viruses with other known viruses were also characterized. Further virus distribution patterns were explored with RT-PCR analysis of different strains isolated from different locations. CONCLUSIONS: In this study, a mixed infection with seven RNA viruses from a P. ostreatus strain was identified via deep sequencing, and their genomes were characterized. These findings advance our understanding of the diversity of viruses infecting P. ostreatus and provide critical insights for further research on virus‒host interactions and the development of viral elimination strategies.