Identification Of Mycoviruses In Alternaria Spp.causing Pear Black And Their Effect On Host Biological Characteristics

Pear black spot is caused by Alternaria spp.and is an important disease in pear production area in China.Based on the high-throughput sequencing data of pear black spot pathogen in our laboratory,we detected and analyzed mycoviruses types that carried by different Alternaria strains,observed the growth and pathogenesis of those strains,also conducted a systematic study on the molecular characteristics of the novel virus and the effect of different virus on the biological characteristics of its host.The aim was to identify the diversity of virus in Alternaria spp.,and provide novel viral resources for research on biological control technology of pear relate diseases.The obtained research results are as follow:1.Detection of mycoviruses in Alternaria and observation of growth and pathogenicity of the strains carrying virus.Through comparing and splicing 87 virusrelated contigs obtained from the mixed sample high-throughput sequencing of 78 strains of six species of Alternaria,we obtained sequences related to 21 viruses.The results of virus detection showed that the virus was detected in 22 strains of the remaining five species of Alternaria,except for A.longipes,with the detection rate of28.2%(22/78).Among those strains,there were 15,5 and 2 strains respectively carrying single virus,2 viruses and 4 viruses.The growth and pathogenicity of these strains with different viruses were observed.The results showed that the abnormal colony morphology,hyphal growth and weak pathogenicity in virus compound infection were more obvious than those in single infection.2.Complete genome sequence analysis of three mycoviruses in Alternaria.Systematic identification of three viruses detected from strain KEL-4-4(A.arborescens),two +ss RNA viruses: Alternaria arborescens ourmia-like virus(Aar OLV1)and Alternaria tenuissima ourmia-like virus 1(At OLV1),one-ss RNA virus: Alternaria arborescens negative-stranded RNA virus 1(Aa NSRV1).Aar OLV1 is a novel virus.Its genomic sequence is 2711 nt in length and contains only one ORF encoding Rd Rp.Multiple cyclic structures were predicted in the terminal non-coding region.Based on the phylogenetic analysis of the Rd Rp sequence,we found that Aar OLV1 belonged to Deltascleroulivirus of Botourmiaviridae,speculate it was a new member of the genus.The full-length sequence of At OLV1 was 2968 nt,with two open reading frames,The function of ORF1 was unknown,ORF2(507-2453 nt)encoded Rd Rp.The virus belonged to Magoulivirus of Botourmiaviridae,and was different from Cladosporium cladosporioides ourmia-like virus 2 in the same strain.Genome of Aa NSRV1 was 6479nt,and only encoding Rd Rp(91-6423 nt)was predicted.Phylogenetic analysis of Rd Rp sequences revealed that Aa NSRV1 was closely related to the Discoviridae virus of Bunyavirales,which might be a novel virus of the genus.3.Studies on different types of virus infection and their effects on host biological characteristics.The G-41 strain carrying only one virus: At OLV1,and the KEL-4-4strain carrying four viruses: At OLV1,Aar OLV1,Aa RV1 and Aa NSRV1.The two strain s were used to explore the transmission characteristics of those virus.The result showed that all four viruses could transmit vertically,but their virus transmission rates were different:At OLV1,Aa RV1,and Aa NSRV1 reach 100%,Aar OLV1 reached 81.25%;At OLV1、Aar OLV1 and Aa RV1 can horizontally transmitted to A.alternata strains AH-37 or KEL-9-2 and A.tenuissima strain CQ-1,while Aa NSRV1 is not.Through vitro RNA transfection could transmit Aar OLV1 and Aa RV1 into the strain HB-45(A.alternaria).When KEL-4-4 strain carrying four viruses only removed Aar OLV1,its growth and pathogenesis became slower and weaker,and the conidia production increased.After virus-free strain HB-45 was infected with Aar OLV1,its sporulation and pathogenicity were enhanced but its growth was slowed down.After removal of At OLV1,G-41 strain had enhanced dye accumulation,pathogenicity,and sensitivity to Na Cl,and conidia production had decreased.AH-37 infected with At OLV1 weakened its pigment precipitation and sensitivity to Na Cl,and increased conidia production.CQ-1 infected with At OLV1,Aar OLV1 and Aar OLV1 slowed down the growth and weakened the sensitivity to Na Cl and CR,when KEL-9-2 infected with At OLV1 and Aar OLV1 produced more conidia.