Identification And Biological Analysis Of Alternaria Alternata Hypovirulent Strain Aa-YL3c Associated Virus

Alternaria leaf blotch disease,which primarily affects the foliage characterized as leaf spotting and dropping at early summer and circular,necrotic lesions with a light brown interior that later become surrounded by a darker purplish halo.The pathogen could also attack green woody twists,but it rarely infects the fruit.Alternaria leaf blotch disease,which caused by the ascomycetous fungus,Alternaria alternata f.sp.mali,wildly spread in China and resulted in destruction of apple trees.So far,there is no fungicide and cultural practices are available to efficiently control this disease.Thus,it is important and necessary to develop new techniques and methods to eradicate or efficient control the disease.In this study,we extensively screened for the mycoviruses that may be potential as biological control agents for alternaria leaf blotch disease.A hypovirulent strain of Alternaria alternata,Aa-YL3 c was isolated.Aa-YL3 c showed deficiency in pathogenicity and hyphal growth.We further confirmed that Aa-YL3 c contained double stranded viral RNAs.We identified the infecting mycovirus and investigated the molecular mechanisms of hypovirulence or morphological changes mediated by mycovirus.The results as follow:1.Identification of hypovirulent strain Aa-YL3 c.It was confirmed that Aa-YL3 c was a pathogenic fungus infecting apple leaves.The analysis of ribosomal DNA(r DNA)sequences of Aa-YL3 c which obtained by specific primers designed from conserved ascomycetous fungus,indicated that Aa-YL3 c is a strain of Alternaria alternate.2.Sequence analysis of hypovirulence associated mycovirus of Aa-YL3 c.Utilizing high-throughput sequencing,and rapid amplification of c DNA ends(RACE),the full length c DNA sequence of hypovirulence associated mycovirus of Aa-YL3 c,namely Alteraria alternate hypovirulence virus 1,(Aa HV1),was determined.The sequence analysis showed that Aa HV1 genome is 14101 ntlength and contains only one continuous open reading frame(Bangma et al.)encoding a polypeptide with 4277 amino acids.The molecular weight of deduced protein is about 478.22 k Da.Aa HV1 genome contains 491 nt and 1421 nt untranslated regions(UTR)at 5′ and 3′ terminus,respectively.Amino acids identities indicated that this protein contained RNA depend RNA polymerase(Rd Rp)conserved motifs,DEAD-like helicases superfamily domain(DEXDc)and DEAH-Box ATP dependent helicase Hrp B domains(DEAH).Phylogenetic analysis based on this Rd Rp domain showed that Aa HV1 shared some similarity with an insect infecting virus(Whuhan Insect Virus 14,Wh IV-14),which is a positive single stranded RNA virus.3.Biological characterization of Aa HV1,In order to characterize the Aa HV1,we examinedthe fungal virulence,hypha growth,virus transmission through hypha fusion and conidia.The Aa HV1 free strain of Aa-YL3 c was selected from single conidia cultures.Comparing to Aa HV1 free strain,the Aa HV1 infected strains showeda hypovirulence phenotype with slow growth hypha,and development a smaller lesion on apple leaves.Aa HV1 was able to transmit through hypha fusion and also able to spread through conidia with a relatively high efficiency(94%).However,Aa HV1 did not spread into a vegetative incompatibility(Jezic et al.)strains,papaya isolates of A.alternate.Aa HV1 did not cause hypovirulence symptom on papaya isolates of A.alternate.Our results suggest that Aa HV1 is a potential biocontrol agent against Alternaria leaf blotch disease and also expand our understanding on mycoviruses infecting plant pathogenic fungi.