Differentiation Of Virulence And Identification Of Mycoviruses In Alternaria Spp.causing Pear Black Spot

Pear black spot is an important disease in main pear production areas in China,resulting in serious precocious defoliation and fruit decay.And it has caused serious losses to the economic benefits of pear orchards in the major pear-cultivation areas.The results of previous experiments in our laboratory confirmed that Alternaria tenuissima and A.alternata were the main pathogens causing pear black spot.In this study,we further established a rapid test method about the pathogenicity of the two dominant pathogens in the laboratory,to analyze their differentiation status of pathogenicity.We analyzed the diversity of mycoviruses carried by the Alternaria spp.through high-throughput sequencing,and systematically identified the novel viruses.The main results were as follows:1.Establishment the methods of pathogenicity test about Alternaria spp.in the laboratory.The mycelial plugs and conidial suspensions of the representative strain HB-36(A.tenuissima)and CQ-31(A.alternata)were used as inoculums,and the leaves(Pyrus pyrifolia cv.Cuiguan),twigs(P.pyrifolia cv.Cuiguan)and fruits(P.bretschneideri cv.Huangguan)of pear were used as detached host materials to compare the pathogenicity difference.The results showed that,the formation of the lesion was more stable with the mycelial plugs inoculation than the conidial suspension inoculation under the same wound treatment on leaves,and expansion of the lesion was faster;the average diameters of lesions were all larger under conidial suspensions inoculation than mycelial plugs on inoculated fruits,except for the CQ-31 strain;and there were no lesions produced under mycelial plugs inoculation on the twigs.The comprehensive results show that the pathogenicity comparison using fully expanded healthy leaves as the detached inoculation material have the best homogeneity,with the method of inoculating mycelial plugs by puncturing 3 needles,it is suitable to compare the pathogenicity of pear black spot pathogen.2.Pathogenicity differentiation analysis about dominant pathogens of pear black spot.We tested the virulence of 146 isolates of Alternaria strains(A.tenuissima 72 strains and A.alternata 74 strains)collected from Chongqing,Hubei,Sichuan,Anhui,Gansu,Shaanxi,Jiangxi,Yunnan and Shandong provinces by laboratory test methods and compared the average diameter of the lesion using SAS software.The results showed that,there were differences in pathogenicity types and distribution proportion among isolates obtained from different regions,pear species,and pear tissues.Among them,there were17 strains with strong virulence,110 strains with middle virulence,and 19 strains with weak virulence,accounting for 12%,75% and 13% of the tested strains,respectively.3.Diversity analysis of mycoviruses in Alternaria spp.and identification of novel viruses.Metatranscriptomics analysis of 78 strains in six species of Alternaria collected from 10 pear production areas by using high-throughput sequencing technology,we found24 mycoviruses in total.Among them,there were 11 species of +ss RNA viruses,8 species of ds RNA viruses,and 5 species of-ss RNA viruses.The phylogenetic tree analysis based on the typical GDD conserved domain of Rd Rp of the obtained sequence showed that 13 viruses could be divided into 6 known families(Botourmiaviridae,Narnaviridae,Deltaflexiviridae,Partitiviridae,Chrysoviridae,and Mymonaviridae).Two –ss RNA viruses had been identified in strain HB-15,which we namely Alternaria sclerotimonavirus 1(ASV1)and Alternaria negative-stranded RNA virus 1(ANSRV1).The full length of ASV1 genome was 9164 nt,the genome contained five open reading frames(ORFs Ⅰ-Ⅴ),which was linearly arranged.The ORF Ⅱ encoded the viral nucleocapsid protein(N)and the ORF Ⅳ encoded the viral replicase(Rd Rp).The virus protein had a domain related to the structure of m RNA capping based on the conserved domain prediction.Phylogenetic analysis revealed that it was clustered into the members of Sclerotimonavirus of Mymonaviridae.The virus particles were filamentous observed by transmission electron microscope,about 15 nm in diameter and 400 nm-1500 nm in length.Another +ss RNA virus had been found in strain SC-8,temporarily named Alternaria tenuissima deltaflexvirus 1(At DFV1).The genome was 8434 nt in full length and contained four open reading frames(ORFs Ⅰ-Ⅳ).ORF Ⅰ encoded an Rd Rp contained a conserved methyltransferase domain.Phylogenetic analysis revealed that the virus belonged to Deltaflexiviridae.The virus-carrying strain SC-8 was weak in virulence and the mycelial tips were curved,compared to the virus-free strains.The virus particles were spherical with a diameter of 15 nm approximately.This study clarified the pathogenicity differentiation status of the two dominant populations of pear black spot disease in my country and the diversity of mycoviruses carried by the Alternaria spp.,at the same time,the two new mycoviruses discovered were systematically identified.It provides relevant technical methods and new information for further research and pear black spot disease control.