Six novel mycoviruses that collectively represent the mycovirome of Rhizoctonia solani anastomosis group (AG)-3 PT strain ZJ-2H, which causes potato black scurf, were identified through metatranscriptome sequencing and putatively designated as Rhizoctonia solani fusarivirus 4 [RsFV4, positive single-stranded RNA (+ssRNA)], Rhizoctonia solani fusarivirus 5 (RsFV5, +ssRNA), Rhizoctonia solani mitovirus 40 (RsMV40, +ssRNA), Rhizoctonia solani partitivirus 10 [RsPV10, double-stranded RNA (dsRNA)], Rhizoctonia solani partitivirus 11 (RsPV11, dsRNA), and Rhizoctonia solani RNA virus 11 (RsRV11, dsRNA). Whole genome sequences of RsFV4, RsMV40, RsPV10, RsPV11, and RsRV11, as well as a partial genome sequence of RsFV5, were obtained. The 3’- and 5’- untranslated regions of the five mycoviruses with complete genome sequences were folded into stable stem-loop or panhandle secondary structures. RsFV4 and RsFV5 are most closely related to Rhizoctonia solani fusarivirus 1 (RsFV1), however, the first open reading frame (ORF) of RsFV4 and RsFV5 encode a hypothetical protein that differs from the first ORF of RsFV1, which encodes a helicase. We confirmed that RsPV10 and RsPV11 assemble into the spherical virus particles (approximately 30 nm in diameter) that were extracted from strain ZJ-2H. This is the first report that +ssRNA and dsRNA viruses co-infect a single strain of R. solani AG-3 PT.

In this study, the diversity of putative mycoviruses present in 66 strains of binucleate Rhizoctonia (BNR, including anastomosis group (AG)-A, AG-Fa, AG-K, and AG-W) and 192 strains of multinucleate Rhizoctonia (MNR, including AG-1-IA, AG-2-1, AG-3 PT, AG-4HGI, AG-4HGII, AG-4HGIII, and AG-5), which are the causal agents of potato stem canker or black scurf, was studied using metatranscriptome sequencing. The number of contigs related to mycoviruses identified from BNR and MNR was 173 and 485, respectively. On average, each strain of BNR accommodated 2.62 putative mycoviruses, while each strain of MNR accommodated 2.53 putative mycoviruses. Putative mycoviruses detected in both BNR and MNR contained positive single-stranded RNA (+ssRNA), double-stranded RNA (dsRNA), and negative single-stranded RNA (-ssRNA) genomes, with +ssRNA genome being the prevalent nucleic acid type (82.08% in BNR and 75.46% in MNR). Except for 3 unclassified, 170 putative mycoviruses found in BNR belonged to 13 families; excluding 33 unclassified, 452 putative mycoviruses found in MNR belonged to 19 families. Through genome organization, multiple alignments, and phylogenetic analyses, 4 new parititviruses, 39 novel mitoviruses, and 4 new hypoviruses with nearly whole genome were detected in the 258 strains of BNR and MNR.