Identification Of The Pathogenic Fungi Of Pear Black Spot Disease From China And A DsRNA Virus In Them

Pear black spot disease is caused by the fungi of genus Alternaria,which is one of the important diseases in the main producing areas of pear.This disease seriously restricts the development of pear industry of China.However,there is no system identification of the pathogen species in China,and no mycovirus has been reported from the pathogen of pear black spot diseases.In this study,a large number of black spot disease samples were collected from the main producing areas of pear of China,and the pathogenic fungi were isolated and the morphological and molecular characteristions were identified,the pathogenicity were determined.A new mycovirus was isolated from SCFS-3 strain and the sequences were analyzed.The main research results are as follows:A total of 300 strains isolated from Chongqing,Jiangxi,Anhui,Hubei,Sichuan,Gansu,Zhejiang,Jilin,Shandong regions.The morphological observation revealed these strains of conidium chain length and branch number were different,can be divided into four types,the proportion of type?,type?,type ?,type ? were 72.6%,16.7%,6%,4.7%,respectively.Molecular identification results showed that rDNA-ITS and GAPDH failed to identify selected strain to species but only the genu Alternaria.Gene CAD and Alta can identify A.gaisen and A.longpies.Gene ATPase could be used to identify 6 species of genu Alternaria.128 strains identified by primers ATPase results showed 2 strains were A.malvae,3 strains were A.longpies,18 strains were A.citrimacularis,8 strains were A.tenuissima,4 strains were A.herbiphorbicola,18 strains were A.dumosa,and 72 strains were not identified as clear species,named Alternaria sp.Five species(A.malvae? A.longpies? A.citrimacularis? A.herbiphorbicola ? A.dumosa)were first found in pear black spot.The pathogenicity of 215 strains results were divided into three categories by K-value clustering analysis,the proportion of strong virulent strains,the medium virulent strains and weak virulent strains were 48%,22% and 30%,respectively.A new dsRNA virus was observed in strain SCFS-3,which was isolated from Chengdu,Sichuan Province.This dsRNA virus may be a new member of the Botybirnaviridae,this virus named Alternaria botybirnavirus 1(ABRV1),temporarily.Phylogenetic evolution analysis showed that ABRV1 has a close relationship with the reported mycoviruses SsBRV1 and BpRV1.The specific dsRNAs could be transmitted to the offspring vertically through the spores,and the conidia of the conidial progeny was 94%.ABRV1 can be transmited to the virus-free strain SCFS-50,and resulting the pathogenicity decreased.The virions of ABRV1 were spherical with a diameter of 40 nm,and the cleaved nucleic acid was consistent with the size of the dsRNA extracted from mycelium.SDS-PAGE electrophoresis of virus particles can detect three bands P80,P70,P60,the size were 80,70,60 KDa.P80 and P70 were encoded by ORF2,and P60 was encoded by ORF1.Phylogenetic evolution analysis showed that ABRV1 was a new member of Botybirnaviridae.