The complete sequence and genome organization of a novel Endornavirus from the hypovirulent strain GD-2 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight, were identified using a deep sequencing approach and it was tentatively named as Rhizoctonia solani endornavirus 1 (RsEV1). It was composed of only one segment that was 19,936 bp in length and was found to be the longest endornavirus genome that has been reported so far. The RsEV1 genome contained two open reading frames (ORFs): ORF1 and ORF2. ORF1 contained a glycosyltransferase 1 domain and a conserved RNA-dependent RNA polymerase domain, whereas ORF2 encoded a conserved hypothetical protein. Phylogenetic analysis revealed that RsEV1 was phylogenetically a new endogenous RNA virus. A horizontal transmission experiment indicated that RsEV1 could be transmitted from the host fungal strain GD-2 to a virulent strain GD-118P and resulted in hypovirulence in the derivative isogenic strain GD-118P-V1. Metabolomic analysis showed that 32 metabolites were differentially expressed between GD-118P and its isogenic hypovirulent strain GD-118P-V1. The differential metabolites were mainly classified as organic acids, amino acids, carbohydrates, and the intermediate products of energy metabolism. Pathway annotation revealed that these 32 metabolites were mainly involved in pentose and glucuronate interconversions and glyoxylate, dicarboxylate, starch, and sucrose metabolism, and so on. Taken together, our results showed that RsEV1 is a novel Endornavirus, and the infection of virulent strain GD-118P by RsEV1 caused metabolic disorders and resulted in hypovirulence. The results of this study lay a foundation for the biocontrol of rice sheath blight caused by R. solani AG1-IA.

Rice sheath blight, caused by the soilborne fungus Rhizoctonia solani, causes severe yield losses worldwide. Elucidation of the pathogenic mechanism of R. solani is highly desired. However, the lack of a stable genetic transformation system has made it challenging to examine genes' functions in this fungus. Here, we present functional validation of pathogenicity genes in the rice sheath blight pathogen R. solani by a newly established tobacco rattle virus (TRV)–host‐induced gene silencing (HIGS) system using the virulent R. solani AG‐1 IA strain GD‐118. RNA interference constructs of 33 candidate pathogenicity genes were infiltrated into Nicotiana benthamiana leaves with the TRV‐HIGS system. Of these constructs, 29 resulted in a significant reduction in necrosis caused by GD‐118 infection. For further validation of one of the positive genes, trehalose‐6‐phosphate phosphatase (Rstps2), stable rice transformants harbouring the double‐stranded RNA (dsRNA) construct for Rstps2 were created. The transformants exhibited reduced gene expression of Rstps2, virulence, and trehalose accumulation in GD‐118. We showed that the dsRNA for Rstps2 was taken up by GD‐118 mycelia and sclerotial differentiation of GD‐118 was inhibited. These findings offer gene identification opportunities for the rice sheath blight pathogen and a theoretical basis for controlling this disease by spray‐induced gene silencing.

The complete genome of a novel double-stranded (ds) RNA mycovirus, named as Rhizoctonia solani partitivirus 5 (RsPV5), isolated from rice sheath blight fungus R. solani AG-1 IA strain C24, was sequenced and analysed. RsPV5 consists of two segments, dsRNA-1 (1899 nucleotides) and dsRNA-2 (1787 nucleotides). DsRNA-1 has an open reading frame (ORF) 1 that potentially codes for a protein of 584 amino acid (aa) containing the conserved motifs of a RNA-dependent RNA polymerase (RdRp), and dsRNA-2 also contains a ORF 2, encoding a putative capsid protein (CP) of 513 aa. Phylogenetic analysis revealed that RsPV5 clustered together with six other viruses in an independent clade of the genus Alphapartitivirus, indicating that RsPV5 was a new member of the genus Alphapartitivirus, within the family Partitiviridae.

Rhizoctonia solani AG-1 IA is one of the most important fungal pathogens that causes severe yield losses in rice-growing regions worldwide. Catechol (1, 2-benzenediol) is a natural catecholamine which can induce melanization of fungi and contribute to enhanced virulence and fungal resistance to diverse environments. In this study, the effects of catechol on growth, activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), glutathione S-transferase (GSH-ST), glutathione peroxidase (GSH-PX) and glutathione reductase (GR), as well as expression of melanin biosynthesis genes in R. solani AG-1 IA, were evaluated by culturing the fungus in catechol-containing nutrient media. The results showed that the activities of CAT, POD, SOD, GSH-ST, GSH-PX and GR were decreased in the presence of 12.5–50 μg mL⁻¹ catechol, while GSH-PX activity was significantly increased (by 310%) in the presence of 50 µg mL⁻¹ catechol. The growth rates of R. solani AG-1 IA on potato dextrose agar (PDA) containing catechol were significantly decreased, whereas the pathogenicity to rice of R. solani AG-1 IA cultured on PDA containing different concentrations of catechol was enhanced when compared with the catechol-free control. Quantitative RT-PCR results showed that the expression of four melanin biosynthesis genes, i.e. protein tyrosine phosphatase (Ptp), chorismate mutase (Cm), peroxidase (Per) and phenol hydroxylase (Ph), was significantly down-regulated in R. solani AG-1 IA cultured in catechol-containing PDB at 100 µg mL⁻¹. These results suggest that catechol was utilized by the fungus to synthesize melanin, and it could also induce the activities of antioxidant enzymes and melanization in R. solani AG-1 IA. Our results provide strong evidence to support the view that melanin contributes to virulence directly through fungal antioxidant defence systems in which melanization is induced by oxidative stress.

Here, we describe a novel double-stranded (ds) RNA mycovirus designated Rhizoctonia solani dsRNA virus 5 (RsRV5) from strain D122 of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight. The RsRV5 genome consists of two segments of dsRNA (dsRNA-1, 1894 bp and dsRNA-2, 1755 bp), each possessing a single open reading frame (ORF). Sequence alignments and phylogenetic analyses showed that RsRV5 is a new member of the genus Gammapartitivirus in the family Partitiviridae. Transmission electron microscope (TEM) images revealed that RsRV5 has isometric viral particles with a diameter of approximately 20 nm. The mycovirus RsRV5 was successfully removed from strain D122 by using the protoplast regeneration technique, thus resulting in derivative isogenic RsRV5-cured strain D122-P being obtained. RsRV5-cured strain D122-P possessed the traits of accelerated mycelial growth rate, increased sclerotia production and enhanced pathogenicity to rice leaves compared with wild type RsRV5-infection strain D122. Transcriptome analysis showed that three genes were differentially expressed between two isogenic strains, D122 and D122-P. These findings provided new insights into the molecular mechanism of the interaction between RsRV5 and its host, D122 of R. solani AG-1 IA.