Biodiversity And Biological Characteristics Of Mycoviruses In Setosphaeria Turcica F.sp.sorghi

Sorghum leaf blight(SLB)caused by the pathogenic fungus Setosphaeria turcica(syn.Exserohilum turcicum)is an important fungal disease,which causes a serious reduction in quality and yield,resulting in significant economic losses.The current chemical approach has many disadvantages.Biological control,especially fungal viruses leading to low virulence,is becoming increasingly valuable in practice.Studies related to fungal viruses in S.turcica f.sp.sorghi have not been reported.Based on this,It is proposed to collect and isolate strains of SLB from Shennongjia,a concentrated sorghum producing area in Hubei Province,detect and analyze the diversity of fungal viruses in S.turcica f.sp.sorghi,determine the whole genome sequences and some biological properties of some common new viruses,analyze their molecular characteristics and evolutionary status,evaluate the influence of new viruses in S.turcica f.sp.sorghi,and explore the feasibility of using fungal viruses to control SLB.Firstly,327 strains of SLB were collected and isolated from Shennongjia forest area in Hubei Province.The double-stranded RNA(ds RNA)was extracted from the strains and 206 strains were found to contain ds RNA bands,accounting for 63%of the total number of strains.The ds RNA bands in the strains showed a rich diversity,ranging from 0.9-15 kb in size and from 1-7 bands in number.40 strains with representative ds RNA bands were selected for high-throughput sequencing.The sequencing results showed that 56 viruses were identified from 40 strains of SLB,including 10 ds RNA viruses,38 positive single-stranded RNA(+ss RNA)viruses,7 negtive single-stranded RNA(-ss RNA)viruses and 1 RT ss RNA virus.They belonged to 10 families.16 new viruses were included in the 56 strains:two polymycovirus;one chrysovirus;one endornavirus;one narnavirus;one mitovirus;five grapevine botourmiavirus;two ambiguivirus;two single-molecule negative-strand RNA viruses and one retrovirus.The above results indicate a rich diversity of fungal viruses among S.turcica f.sp.sorghi.Secondly,the full genome lengths of three viruses in S.turcica f.sp.sorghi were obtained by random cloning and rapid amplification of c DNA ends(RACE).Based on the molecular characteristics of these three viruses,they were named as Setosphaeria.turcica chrysovirus 1(St CV1),Setosphaeria turcica endornavirus 2(St EV2),and Setosphaeria turcica polymycovirus2(St Pm V2).The St CV1 genome consists of four fragments(ds RNA1-4)of 3647,2954,2861 and 2700 bp respectively.The putative protein encoded by ds RNA1 showed 80.50%identity to the RNA-dependent RNA polymerase(Rd Rp)of the most closely related virus,Alternaria alternata chrysovirus 1(Aa CV1),belonging to the Chrysoviridae.ds RNA2 encodes the coat protein(CP),while ds RNA3 and ds RNA4 encode proteins of unknown function,respectively.Phylogenetic analysis based on Rd RP proteins indicated that the viruses belonged to the Betachrysovirus.St EV2 has a genome about 11 kb in size,with a G+C content around 45%.Its encoded polyprotein has about 30%-40%amino acid sequence identical to that of some endogenous virus polyproteins.Based on the phylogenetic analysis of Rd RP protein,it can be classified into the genus Betaendornavirus in the family Endornaviridae.The genome of St Pm V2 consists of five fragments(ds RNA1-5)with a minimum of 965 bp and a maximum of 2,462 bp.The putative protein encoded by ds RNA1 is most closely related to Rd RP of Cladosporium cladosporioides virus 1,which belongs to the Polymycoviridae.with 64.52%homology.ds RNAs 2-4encode CP,methyltransferase(MTR)and proline-alanine-serine-rich protein(PASrp),and ds RNA5 encodes a protein of unknown function.Phylogenetic analysis based on the Rd RP protein indicates that St Pm V2 belongs to the genus Polymycovirus in the family Polymycoviridae.Thirdly,Setosphaeria turcica endornavirus 1(St EV1)from S.turcica f.sp.zeae and Bipolaris maydis endornavirus 1(Bm EV1)、Bipolaris maydis endornavirus 2(Bm EV2)from Bipolaris maydis,which are similar to St EV2,were determined.pairwise comparisons revealed that the genomes of the four endornaviruses shared59%-78%nucleotide sequence identities and the polyproteins of them showed 43%-87%amino acid sequence identities.St EV1 and Bm EV1,with a sequence identity of 78%at the full-genome level and 87%at the polyprotein level,may belong to the same species.Furthermore,we show that the four endornaviruses have a low cidence of about3-5%in S.turcica or B.maydis.At least for Bm EV1 and Bm EV2,the low incidence corresponds with their inability to transmit between hosts of different vegetative incompatibility groups.Finally,the original St CV1-infected and virus-cured strains were subjected to comparison with biological characteristics,including colony,mycelial and spore morphology;colony growth rate,sporulation ability,and virulence.Obvious differences in the colony morphology occurred between the original St CV1-infected and virus-cured strains after 7 days on PDA medium at 28℃.The colony morphology of the virus-cured strain formed a regular round and significantly higher mycelium density than that of the virus-infected strain.In contrast,the colony of the virus-infected strain exhibited an irregular configuration,which showed the abnormal mycelial and spore morphology.The colony growth rate of the virulent strain was(3.98±0.16)mm/d,much lower than that of the detoxified strain(11.17±0.41)mm/d.The sporulation capacity of the virulent strain was(1.79±0.15)×10~6 spores/ml,less than that of the detoxified strain(3.85±0.17)10~6spores/ml.There was also significant difference in the number of disease spots on each sorghum leave between the virus-infected strain strain and virus-cured strain,which were(6.17±0.79)and(16.67±1.63),respectively.St CV1 infection altered colony,mycelium and spore morphology,significantly reduced colony growth,spore production and virulence to the host fungus.This indicates that St CV1 is a hypovirus with potential for biocontrol.