Genome Organizations and Functional Analyses of a Novel Gammapartitivirus from Rhizoctonia solani AG-1 IA Strain D122.

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.

Functional validation of pathogenicity genes in rice sheath blight pathogen Rhizoctonia solani by a novel host-induced gene silencing system.

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.

Transcriptome analysis reveals molecular mechanisms of sclerotial development in the rice sheath blight pathogen Rhizoctonia solani AG1-IA.

Rhizoctonia solani AG1-IA is a soil-borne necrotrophic pathogen that causes devastating rice sheath blight disease in rice-growing regions worldwide. Sclerotia play an important role in the life cycle of R. solani AG1-IA. In this study, RNA sequencing was used to investigate the transcriptomic dynamics of sclerotial development (SD) of R. solani AG1-IA. Gene ontology and pathway enrichment analyses using the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to investigate the functions and pathways of differentially expressed genes (DEGs). Six cDNA libraries were generated, and more than 300 million clean reads were obtained and assembled into 15,100 unigenes. In total, 12,575 differentially expressed genes were identified and 34.62% (4353) were significantly differentially expressed with a FDR ≤ 0.01 and |log2Ratio| ≥ 1, which were enriched into eight profiles using Short Time-series Expression Miner. Furthermore, KEGG and gene ontology analyses suggest the DEGs were significantly enriched in several biological processes and pathways, including binding and catalytic functions, biosynthesis of ribosomes, and other biological functions. Further annotation of the DEGs using the Clusters of Orthologous Groups (COG) database found most DEGs were involved in amino acid transport and metabolism, as well as energy production and conversion. Furthermore, DEGs relevant to SD of R. solani AG1-IA were involved in secondary metabolite biosynthesis, melanin biosynthesis, ubiquitin processes, autophagy, and reactive oxygen species metabolism. The gene expression profiles of 10 randomly selected DEGs were validated by quantitative real-time reverse transcription PCR and were consistent with the dynamics in transcript abundance identified by RNA sequencing. The data provide a high-resolution map of gene expression during SD, a key process contributing to the pathogenicity of this devastating pathogen. In addition, this study provides a useful resource for further studies on the genomics of R. solani AG1-IA and other Rhizoctonia species.

Molecular Characterization of a Novel Endornavirus Conferring Hypovirulence in Rice Sheath Blight Fungus Rhizoctonia solani AG-1 IA Strain GD-2.

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.

Complete Nucleotide Sequence of a Partitivirus from Rhizoctonia solani AG-1 IA Strain C24.

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.

ROS and trehalose regulate sclerotial development in Rhizoctonia solani AG-1 IA.

Rhizoctonia solani AG-1 IA is the causal agent of rice sheath blight (RSB) and causes severe economic losses in rice-growing regions around the world. The sclerotia play an important role in the disease cycle of RSB. In this study, we report the effects of reactive oxygen species (ROS) and trehalose on the sclerotial development of R. solani AG-1 IA. Correlation was found between the level of ROS in R. solani AG-1 IA and sclerotial development. Moreover, we have shown the change of ROS-related enzymatic activities and oxidative burst occurs at the sclerotial initial stage. Six genes related to the ROS scavenging system were quantified in different sclerotial development stages by using quantitative RT-PCR technique, thereby confirming differential gene expression. Fluorescence microscopy analysis of ROS content in mycelia revealed that ROS were predominantly produced at the hyphal branches during the sclerotial initial stage. Furthermore, exogenous trehalose had a significant inhibitory effect on the activities of ROS-related enzymes and oxidative burst and led to a reduction in sclerotial dry weight. Taken together, the findings suggest that ROS has a promoting effect on the development of sclerotia, whereas trehalose serves as an inhibiting factor to sclerotial development in R. solani AG-1 IA.

Characterization of a novel dsRNA mycovirus isolated from strain A105 of Rhizoctonia solani AG-1 IA.

Rhizoctonia solani dsRNA virus 3 (RsRV3), a novel mycovirus, was isolated from the rice sheath blight pathogen Rhizoctonia solani AG-1 IA strain A105. The RsRV3 genome consists of two segments of dsRNA (dsRNA1, 1,890 bp and dsRNA2, 1,811 bp). DsRNA1 has a single open reading frame (ORF) with a putative conserved RNA-dependent RNA polymerase (RdRp) domain, and dsRNA2 comprises a single ORF, predicted to encode a coat protein. Purified viral particles of RsRV3 were isometric and measured approximately 20 nm in diameter by negative-stain transmission electron microscope (TEM). Phylogenetic analyses indicated that RsRV3 is highly similar to viruses taxonomically classified in the genus Alphapartitivirus, family Partitiviridae. Taken together, the integrative analyses of viral genomic organization, amino acid sequence alignments and phylogenetic analyses clearly demonstrate that the RsRV3 virus isolated from R. solani AG-1 IA strain A105 is classifiable as a new member of the genus Alphapartitivirus, family Partitiviridae.

Two distinct classes of protein related to GTB and RRM are critical in the sclerotial metamorphosis process of Rhizoctonia solani AG-1 IA.

Sheath blight of rice, caused by Rhizoctonia solani Kühn AG-1 IA [teleomorph: Thanatephorus cucumeris (Frank) Donk], is one of the major diseases of rice (Oryza sativa L.) worldwide. Sclerotia produced by R. solani AG-1 IA are crucial for their survival in adverse environments and further dissemination when environmental conditions become conducive. Differentially expressed genes during three stages of sclerotial metamorphosis of R. solani AG-1 IA were investigated by utilizing complementary DNA amplified fragment length polymorphism (cDNA-AFLP) technique. A total of 258 transcript derived fragments (TDFs) were obtained and sequenced, among which 253 TDFs were annotated with known functions through BLASTX by searching the GenBank database and 19 annotated TDFs were assigned into 19 secondary metabolic pathways through searching the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY database. Moreover, the results of quantitative real-time PCR (qRT-PCR) analysis showed that the expression patterns of eight representative annotated TDFs were positively correlated with sclerotial metamorphosis. Sequence annotation of TDFs showed homology similarities to several genes encoding for proteins belonging to the glycosyltransferases B (GTB) and RNA recognition motif (RRM) superfamily and to other development-related proteins. Taken together, it is concluded that the members of the GTB and RRM superfamilies and several new genes involved in proteolytic process identified in this study might serve as the scavengers of free radicals and reactive oxygen species (ROS) and thus play an important role in the sclerotial metamorphosis process of R. solani AG-1 IA.

Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani

Background Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is one of the most important pathogens of rice (Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani, five methods, including E.Z.N.A.™ Fungal RNA Kit, sodium dodecyl sulfate (SDS)-sodium borate, SDS-polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study. Results The electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS-sodium borate, SDS-PVP and E.Z.N.A.™ Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment. Conclusion It is concluded that SDS-sodium borate and SDS-PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A.™ Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield.

Coupling of anodic biooxidation and cathodic bioelectro-Fenton for enhanced swine wastewater treatment.

A novel bioelectrochemical reactor with anodic biooxidation coupled to cathodic bioelectro-Fenton was developed for the enhanced treatment of highly concentrated organic wastewater. Using swine wastewater as a model, the anode-cathode coupled system was demonstrated to be both efficient and energy-saving. Without any external energy supply to the system, BOD(5), COD, NH(3)-N and TOC in the wastewater could be greatly reduced at both 1.1g COD L(-1)d(-1) and 4.6g COD L(-1)d(-1) of OLR, with the overall removal rates ranging from 62.2% to 95.7%. Simultaneously, electricity was generated at around 3-8 Wm(-3) of maximum output power density. Based on electron balance calculation, 60-65% of all the electrons produced from anodic biooxidation were consumed in the cathodic bioelectro-Fenton process. This coupled system has a potential for enhanced treatment of high strength wastewater and provides a new way for efficient utilization of the electron generated from biooxidation of organic matters.