Studies On Mycovirus Diversity Of Rhizoctonia Solani Of Graminaceous Crop And Its Relationship With Host Hypovirulence

Rhizoctonia solani Kuhn is a worldwide plant pathogenic fungus with a wide host range,which can cause grain blight of gramineous crops such as rice and maize,and seriously affect crop yield and quality.Mycoviruses exist widely in various fungal groups,including plant pathogenic fungi.Some mycoviruses can change the physiological and biochemical characteristics of host and cause low virulence phenotype of host,which has the potential of biocontrol against fungal diseases.At the same time,the interaction between mycoviruses and host can also be used to explore the pathogenic mechanism of pathogenic fungi.In this study,we analyzed the viral diversity of Rhizoctonia solani isolated from rice and maize,and explored the relationship between the presence of mycoviruses and host virulence differentiation.Specific research results are as follows:(1)By ds RNA extraction and high-throughput sequencing,the diversity of mycoviruses were analyzed.Thirty-three isolates were isolated from rice and maize in Hunan and Zhejiang provinces.Ds RNA extraction revealed that 32 strains contained ds RNA bands ranging from more than 1.5 kbp.The extracted ds RNAs were subjected to high-throughput sequencing,splicing,and BLASTx alignment analysis to obtain virus-associated contigs sequences.RT-PCR assays with specific primers were designed to validate the presence of38 viruses-associated contigs in these strains.Sequence analysis revealed that these contigs viral sequences belonged to 11 known viral families,such as Megabirnaviridae(2),Partitiviridae(7)Endornaviridae(5),Narnaviridae(1),Mitoviridae(6),Deltaflexiviridae(1),Fusariviridae(1),Megatotiviridae(2),Fusagraviridae(1),Rhabdoviridae(1)and unclassified Toti-like virus(2)and other unknown viral taxonomic units(9),of which 29 contigs were presumed to be new viral sequences,accounting for 76.3% The results indicated that there were abundant mycoviruses resources in R.solani.Comparing the culture characters,virulence and sclerotia-producing ability of 32 strains,it was found that the growth rate,sclerotia-producing ability and pathogenicity of virus-containing strains were differentiated,but the weak virulence of strains was not correlated with the sclerotia-producing ability.(2)The molecular characteristics and biological functions of eight viral genomes obtained from strain ZJXD1-1 were analyzed.The total RNA of strain ZJXD1-1 was extracted for high-throughput sequencing and combined with routine ds RNA sequencing.It was found that the strain contained Endornaviruses(2)and Mitoviruses(5),and Toti-like virus(1).They were named Rhizoctonia solani endornavirus 11(RsEV11),Rhizoctonia solani endornavirus 12(RsEV12),Rhizoctonia solani mitovirus 106(RsMV106)to Rhizoctonia solani mitovirus 110(RsMV110),Rhizoctonia solani toti-like virus 2(RsTLV2).RsEV12 contained a special 91 aa domain Peptidase C39(Peptidase family C39),which was first identification of this domain in Endornavirus.RsTLV2 is distantly related to the family Totiviridae in phylogenetic analysis,but is most similar to the animal virus Callinectes sapidus toti-like virus 1.The results showed that RsMV110,RsEV11,RsEV12 might enhance the virulence of the host fungus,and RsMV106 might reduce the virulence of host fungal.RsEV11 and RsEV12 are present or lost simultaneously and are not easily transmitted by mycelial contact.(3)It was proved that the multiple transmission of mycoviruses among strains caused virulence differentiation.Four strains,DLWK2-1-1,DLWK2-1-3,WNT2-1-1 and WNT2-1-2,were cultured in confrontation with ZJXD1-1,and it was found that ZJXD1-1obtained viruses from the confrontation strains,indicating that ZJXD1-1 had the ability to accommodate multiple viruses.The pathogenicity of the 8 derivative strains derived from ZJXD1-1 were significantly differentiated,and the pathogenicity of DWZ-6 was significantly reduced.By further confrontation culture,the virus carried in DWZ-6 was horizontally transferred to ZJXD1-1,and the pathogenicity of the descendant derivative strain was also decreased,which proved that the reduced pathogenicity of DWZ-6 was related to the obtained virus.In this study,the diversity of mycoviruses isolated from rice and maize in Hunan and Zhejiang provinces was analyzed,and the molecular characteristics and biological functions of the viruses in one of the strains were studied,and the effect of the transmission of fungal viruses between strains on the virulence differentiation of Rhizoctonia solani was investigated.This study provided potential mycoviruses resources for biological control of R.solani,and also provided theoretical basis for understanding virus evolution and virulence differentiation of R.solani population.