| Rhizoctonia solani Kǜhn(R.solani)is a plant pathogenic fungus with a wide host range and geographic distribution,among which R.solani anastomosis group 3(AG-3)and AG-4 cause leaf spot and damping off in many crops.Mycoviruses are viruses that can replicate and proliferate in fungi.Most mycoviruses do not show any symptoms to their hosts,but some can cause low virulence in their hosts,thus becoming potential biocontrol agents against plant fungal diseases.In this study,we analyzed the diversity of viruses isolated from tobacco,potato,bean,and amaranth plants in Yunnan Province belonging to AG-3 and AG-4.We investigated the molecular characteristics and functions of two new viruses and two low virulence partitiviruses.The results of the study were as follows:A total of 114 strains of R.solani AG-3 and AG-4 were sequenced and analyzed by 24 groups of mixed samples in high throughput sequence.A total of 125 contigs annotated to viral information were obtained for the two anastomosis groups,with the absolute numerical dominance of viruses of Mitoviridae(33)and Partitiviridae(19),accounting for 26.4% and 15.2% of the total number of contigs,respectively.Among them,26 viruses obtained nearly complete genomes,Mitoviridae(10),Fusariviridae(6),Endornaviridae(3),Hypoviridae(1)and Partitiviridae(6),and 12 of these viral sequences belonged to new viruses,indicating viral diversity in this two anastomosis groups.The complete genome sequences of Rhizoctonia solani fusarivirus 6(Rs FV6)and Rhizoctonia solani partitivirus BS5(Rs PV-BS5)were obtained by isolating of the AG-3 of R.solani.The Rs FV6 had a single-stranded positive-sense RNA(+ss RNA)genome of 6141 nucleotides containing two open reading frames(ORFs)and a poly A tail.ORF1 encoded a large polypeptide of 1,862 amino acids(aa)with conserved RNA-dependent RNA polymerase(Rd Rp)and helicase(Hel)domains.ORF2 encoded a putative 167 aa protein of unknown function.BLASTp searched revealed that the ORF1-encoded polypeptide showed the highest sequence similarity(70.67% identity)to that of Rhizoctonia solani fusarivirus 3(Rs FV3)which was isolated from R.solani AG-2-2LP.Multiple sequence alignment and phylogenetic analysis based on Rd Rp and Hel indicated that Rs FV6 could be a novel member of the genus Alphafusarivirus,in the family Fusariviridae.The whole genome of Rs PV-BS5 consisted of two ds RNAs,ds RNA-1 was 2580 bp and contained a large ORF1 encoding Rd Rp protein with a length of730 aa;ds RNA-2 was 2444 bp and contained a small ORF2 encoding CP protein with a length of 683 aa.Multiple alignment and phylogenetic analysis of Rs PV-BS5,which was a new strain of the species Rhizoctonia solani virus 717(Rhs V-717)isolated from R.solani AG-3.Transmission electron microscopy(TEM)images showed that Rs PV-BS5 had virus particles with a diameter of about 40 nm.Rs PV-BS5 had the potential to slow down the growth of mycelium of AG-3 TB and AG-3 PT.Moreover,it also reduced the spore production of AG-3 TB and reduced the host pathogenicity.In terms of transmission,Rs PV-BS5 could be transmitted horizontally and vertically,but not in vitro or across kingdom.By measuring the toxin extraction and phosphorus exosmosis concentration of strain BS5 with Rs PV-BS5 and YBS-5 without Rs PV-BS5,Rs PV-BS5 could reduce phosphorus exosmosis,consequently,it reduced the pathogenicity of the host to tobacco.In addition,transcriptome sequencing of strains 06-2-15 V and 06-2-15 showed the presence of a large number of differentially expressed genes that are mainly involved in secondary metabolite anabolism,peptide synthesis and metabolism,including up-regulated expression of a large number of genes for methyltransferases and cytochrome P450 enzymes,which inhibit the growth of R.solani,down-regulated expression of genes related to cell wall degrading enzymes,which resolved the mechanism of reduced pathogenicity.The complete genome sequences of Rhizoctonia solani mitovirus 106(Rs MV-106)and Rhizoctonia solani partitivirus HX2C(Rs PV-HX2C)was obtained by isolation of the AG-4 of R.solani.The full genome of Rs MV-106 was 2794 nt.There was an ORF encoding Rd Rp protein with a molecular mass of 95.76 k Da.AUG was the start codon and UAAUAG were the two continuous stop codons.Similar to other previously reported mitoviruses,two potential stem-loop structures in the 5’-and 3’-UTRs of Rs MV-106 were predicted,with initial ΔG values of-17.0 kcal/mol and-8.80 kcal/mol,respectively.Phylogenetic analysis showed that Rs MV-106 was a novel member of the genus Duamitovirus,in the family Mitoviridae.By constructing an infectious clone of Rs MV-106,the mycelial growth rate of the regenerated strain after transfection was facilitated by Rs MV-106 infection of R.solani.In addition,Rs MV-106 could be transmitted horizontally,but other modes of transmission were unknown.The whole genome of Rs PV-HX2 C consisted of two ds RNAs.Ds RNA-1 was 1988 bp and contained a large ORF1 encoding Rd Rp protein with a length of 623 aa;ds RNA-2 was 1788 bp and contained a small ORF2 encoding CP protein with a length of 489 aa.The two ds RNA ends were analyzed by comparison and the 5’-UTR showed a highly conserved expression,while the 3’-UTR showed an interrupted poly A tail.After multiple alignment and phylogenetic analysis Rs PV-HX2 C,which was a new strain of the species Rhizoctonia solani partitivirus 2(Rs PV2)isolated from R.solani AG-4.The viral particles of Rs PV-HX2 C were spherical in shape with a diameter of about 26 nm.In terms of mode of transmission,Rs PV-HX2 C could be transmitted horizontally,while other modes of transmission were unknown.Rs PV-HX2 C had potential for biocontrol because it slowed down the mycelial growth of the host fungus,leading to a reduction in the pathogenicity of R.solani.In this study,we analyzed the mycovirus diversity of AG-3 and AG-4,expanded the virus resources of R.solani,and we studied the two partitiviruses with biocontrol potential to provide a strategy for some diseases caused by R.solani.It is likely to be used for disease of tobacco target spot and potato black scurf in the future. |
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