Diversity Of Mycoviruses In Diaporthe Spp.and Identification Of Three Mycoviruses

Pear shoot canker caused by Diaporthe spp.is an important trunk disease.In recent years,it occurred and became more serious in pear producing areas,which seriously affected the development of industry.Recently,the research and development of biological control technology and products has been a hot spot.It has been proved that mycoviruses are potential biological control resources.In this study,the virus species diversity of Diaporthe spp.was studied by HTS combined with detection using Diaporthe spp.isolated and identified from samples of pear shoot canker in the laboratory.And the newly identified three Diaporthe spp.virus systemic features of the molecular biology research.The purpose of this study was to clarify the diversity of mycoviruses in Diaporthe spp.and to provide new useful information for probing into the origin and evolution of mycoviruses.The main research results are as follows:1.Diversity of virus species in Diaporthe spp.HTS was performed on 25 strains of pear shoot canker from 9 provinces(municipalities),which belonged to 10 different species of Diaporthe spp.The results showed that there were 9 viruses from 4 families in the mixed samples of these strains,8 of which were novel mycoviruses.There were 5+ss RNA viruses,3-ss RNA viruses and 1 ds RNA virus.Phylogenetic analysis based on Rd Rp sequences showed that 5 +ss RNA viruses belonged to Mitoviridae and Virgaviridae,3-ss RNA viruses belonged to Bunyavirales,and 1 ds RNA virus belonged to Quadriviridae.Further detection by RT-PCR showed that 9 of the above 25 strains carried viruses,among which 1 strain carried 2 +ss RNA viruses,and the other 8 strains carried 1 virus each.2.Molecular biological characteristics of three newly discovered Diaporthe spp.viruses.(1)A novel-ss RNA virus was identified from D.padina strain DK160 originating from Jiangxi Province and was temporarily named Diaporthe negative stranded RNA virus 1(DNs RV1).The genome length of DNs RV1 is 9945 nt and contains an open reading frame encoding Rd Rp.The virus belongs to a newly proposed family of Bunyavirales,the Mybuviridae.DNs RV1 can be transmitted stably by conidia and between Diaporthe strains of different species.The results of stand-off culture showed that DNs RV1 had no significant effect on the host phenotype.(2)A novel +ss RNA virus was identified from D.hongkongensis strain DK114 originating from Fujian Province,and was tentatively named Diaporthe virga-like virus 1(Dv LV1).The total length of the genome of Dv LV1 is 11441 nt,which contains an open reading frame,encodes one helicase and one Rd Rp conserved domain.The virus belongs to the Virgaviridae,but cannot belong to any known genus.It is suggested to establish a new genus of virus in the Virgaviridae with this virus and its close relatives.(3)A novel ds RNA virus has been identified from D.caryae strain DK380 from Jiangsu Province and tentatively named Diaporthe disease-associated mycovirus(Dd AMV).Dd AMV contains four ds RNA genomes,the total length of RNA1-3 genomes is 4828 bp,3628 bp and 4185 bp,and the known length of RNA4 genome is 2133 bp.Dd AMV virions are spherical with an average diameter of 41.7 nm.Based on the phylogenetic analysis of Rd Rp amino acid sequences,Dd AMV is closely related to the Quadriviridae,in which the known member of the family Rn QV1 is clustered in one branch,while Dd AMV is clustered in another branch with some viruses that have not been clearly assigned to the family.