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.

Oligogalacturonides (OGs) are a bioactive carbohydrate derived from homogalacturonan. The OGs synthesized in this study significantly inhibited the mycelial growth of Rhizoctonia solani AG-4HGI in vitro, even at a low concentration (10 mg/L). The seed vigor test demonstrated that the application of 50 mg/L OGs to sugar beet seeds significantly increased average germination percentage, germination energy, germination index, and seedling vigor index. The same concentration of OGs also improved the seedling emergence percentage of sugar beet when seeds were sown in soil inoculated with D2 and D31 isolates, respectively. The lesion diameter on mature sugar beet roots caused by R. solani AG-4HGI isolates D2 and D31 also decreased by 40.60% and 39.86%, respectively, in sugar beets roots first treated with 50 mg/mL OGs in the wound site, relative to lesion size in untreated/pathogen inoculated wounds. Sugar beet roots treated with 50 mg/mL OGs prior to inoculation with the D2 isolate exhibited up-regulation of the defense-related genes glutathione peroxidase (GPX) and superoxide dismutase (SOD) by 2.4- and 1.6-fold, respectively, relative to control roots. Sugar beet roots treated with 50 mg/mL OGs prior to inoculation with D31 exhibited a 2.0- and 1.6-fold up-regulation of GPX and SOD, respectively, relative to the control. Our results indicate that OGs have the potential to be used for the protection of sugar beet against R. solani AG-4HGI.

Anastomosis groups (AGs) or subgroups of 244 Rhizoctonia isolates recovered from sugar beet roots with symptoms of root and crown rot were characterized to be AG-A, AG-K, AG-2-2IIIB, AG-2-2IV, AG-3 PT, AG-4HGI, AG-4HGII, and AG-4HGIII, with AG-4HGI (108 isolates, 44.26%) and AG-2-2IIIB (107 isolates, 43.85%) being predominate. Four unclassified mycoviruses and one hundred and one putative mycoviruses belonging to six families, namely Mitoviridae (60.00%), Narnaviridae (18.10%), Partitiviridae (7.62%), Benyviridae (4.76%), Hypoviridae (3.81%), and Botourmiaviridae (1.90%), were found to be present in these 244 Rhizoctonia isolates, most of which (88.57%) contained positive single-stranded RNA genome. The 244 Rhizoctonia isolates were all sensitive to flutolanil and thifluzamide, with average median effective concentration (EC₅₀) value of 0.3199 ± 0.0149 μg·mL⁻¹ and 0.1081 ± 0.0044 μg·mL⁻¹, respectively. Among the 244 isolates, except for 20 Rhizoctonia isolates (seven isolates of AG-A and AG-K, one isolate of AG-4HGI, and 12 isolates of AG-4HGII), 117 isolates of AG-2-2IIIB, AG-2-2IV, AG-3 PT, and AG-4HGIII, 107 isolates of AG-4HGI, and six isolates of AG-4HGII were sensitive to pencycuron, with average EC₅₀ value of 0.0339 ± 0.0012 μg·mL⁻¹. Correlation index (ρ) of cross-resistance level between flutolanil and thifluzamide, flutolanil and pencycuron, and thifluzamide and pencycuron was 0.398, 0.315, and 0.125, respectively. This is the first detailed study on AG identification, mycovirome analysis, and sensitivity to flutolanil, thifluzamide, and pencycuron of Rhizoctonia isolates associated with sugar beet root and crown rot.