Oyster mushroom spherical virus (OMSV) is a positive-sense single-stranded RNA mycovirus which is associated with a devastating oyster mushroom die-back disease. However, little is known about its diversity, and the effects of OMSV infection on its fungal host are not well understood. In this study, we determined the nearly complete nucleotide sequence of OMSV isolated from cultivated oyster mushrooms in China. Sequence analysis suggested that the virus represents a new strain of OMSV (referred to here as OMSV-Ch). A GenBank BLAST search of the genomic sequences demonstrated that the OMSV-Ch had the highest identity (74.9%) with the OMSV from Korea (OMSV-Kr). At the amino acid–sequence level, these two strains shared 84.1% identity in putative replication protein (RP) and 94.1% identity in coat protein (CP). Phylogenetic analysis based on RP showed that OMSV-Ch clustered with OMSV-Kr, closely related to Tymoviridae. Phylogenetic analysis based on both the RP and CP showed that OMSV had a distant clade relationship with tymoviruses, marafiviruses, and maculaviruses. We obtained the OMSV-Ch-free Pleurotus ostreatus strain via single hyphal tip cultures combined with high-temperature treatment. Preliminary studies indicate that OMSV-Ch can significantly inhibit mycelial growth, cause malformations of the fruiting bodies, and reduce the yield of P. ostreatus. Co-cultivation resulted in horizontal transmission of the OMSV-Ch to a virus-cured strain. The findings of our study contribute to the prevention and control of mycoviral diseases in the future.

Mycoviruses are viruses that specifically infect and replicate in fungi. Several mycoviruses have been previously reported in Pleurotus ostreatus, including the oyster mushroom spherical virus (OMSV), oyster mushroom isometric virus (OMIV), Pleurotus ostreatus spherical virus (POSV), and Pleurotus ostreatus virus 1 (PoV1). This study was designed to develop a multiplex RT-PCR for simultaneous detection and differentiation of the four P. ostreatus mycoviruses. Four pairs of primers were designed from conserved regions based on the reported sequences and the multiplex RT-PCR products were 672 bp for OMSV, 540 bp for OMIV, 310 bp for POSV, and 200 bp for PoV1. The optimal annealing temperature of the multiplex RT-PCR was 62 °C and the detection limits of the plasmids were 100 fg for OMSV and OMIV and 1 pg for POSV and PoV1. This technique was successfully applied for the detection of OMSV, OMIV, and POSV from different P. ostreatus strains and the plasmid containing the PoV1 sequence. This methodology can serve as a powerful diagnostic tool for the survey of the incidence and epidemiology of the four P. ostreatus mycoviruses, further contributing to the prevention and treatment of mycoviral diseases in P. ostreatus.