| Pear black spot disease(Alternaria spp.)is widespread in pear production regions of China,which seriously affects the quality of pear fruits and hinders the health development of pear industry.Presently,no efficient green control measures have been taken to control pear black spot disease.The applicatioin of mycovirus-mediated hypovirulent strain is an important biocontrol way.Therefore,it is particularly urgent to find mycoviruses in Alternaria spp.strains.In this study,the samples were collected and isolated from different pear varieties with pear black spot disease in Hubei province,which is to identify the pathogens of Alternaria spp.and analyze the infection status,classification and molecular characterization of mycoviruses in Alternaria spp.These findings aim to provide important molecular information for the classification,origin and genetic evolution of mycoviruses.On the other hand,it is to clarify the effects of mycoviruses on biological characteristics and pathogenicity of Alternaria spp.strains,and screen the hypovirulent strains,which provide candidate materials for the biological control of pear black spot disease in the field.The main results are as followings:1.The analysis of mycoviral types and infection status in Alternaria spp.strains:14 mycoviruses were obtained from 83 Alternaria spp.strains,including ds RNA and+ss RNA types,belonging to 8 families(Chrysoviridae,Partitiviridae,Curvulaviridae,Mitoviridae,Botourmiaviridae,Deltaflexiviridae,Fusariviridae and Togaviridae),which of 6 are new mycoviruses.The multiple mycoviral coinfection was common and prevalent,accounting for 78%(65/83),with each strain harboring different numbers of mycoviruses,ranging from two to seven in Alternaria spp.It was identified that the strains with different mycoviruses were classified into 4 species(A.alternata,A.gaisen,A.arborescens and A.tenuissima)and exhibited difference in pathogenic virulence and biological phenotype.The obtained results revealed the abundant mycoviruses in Alternaria spp.strains from Hubei province.2.Genomic molecular characteristic of three mycoviruses(Ag Df V1,Ag MV3 and Aa CV1)in Alternaria spp.strains: The Ag Df V1 full-length genome was 8,392 nt,excluding poly(A),which encoded a replication-associated polyprotein(containing Met and Rd Rp conserved domains)and 3 hypothetical proteins;The Ag MV3 genomic sequence is 2,160 nt,whose 5’ UTR is not obtained.It was identified that Ag Df V1 and Ag MV3 belonged to the new members in Deltaflexivirus and Mitovirus,respectively,according to the genome structure,homologous sequence similarity and phylogenetic tree analysis.In addition,it was confirmed that Aa CV1–HB isolates had the highest identity of ds RNAs aa sequences with those of Aa CV1–N18 isolate from Japan,which were clustered in the same branch of phylogenetic tree,revealing that there is no correlation of genetic evolution between Aa CV1 strains with host geographical origin.The obtained results revealed the molecular characteristics of Ag Df V1,Ag MV3 and Aa CV1 in Alternaria spp.and provided important molecular information for mycoviral classification,genetic evolution and screening of biocontrol agents.3.The effects of mycoviruses on hypovirulent strain HB–145: The derivative isolates from HB–145 with different mycoviruses were obtained by vertical transmission.The results indicated that the effect of different or mixed mycoviral infection on host was different.It identified that the derivative isolates with Aa Ol V1,Aa Ol V4 and Aa Ol V5 grew slowly and exhibited hypovirulence.In addition,the results of horizontal transmission showed that Ag Df V1 with several mycoviruses from HB-145 can be transmitted to HB-30 and HB-93 strains to inhibit the extension of lesions,respectively.It provides mycovirus-mediated attenuated strain as biocontrol agents and molecular information for exploration the interaction of mycoviruses and host.In conclusion,the abundant mycoviruses in Alternaria spp.were identified,the genomic molecular characteristics of mycoviruses were analyzed,and the potential mycoviruses with hypovirulence were explored.The findings provide candidate materials and theoretical foundation for the exploitation of mycoviruses as biocontrol agents and its effective application in the green biocontrol of fungal diseases. |
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