A Study On The Endophytes Communities And Biocontrolling Of Downy Mildew Infected Grapevine Leaves

Downy mildew and powdery mildew are two of the most serious grapevine fungal diseases,and it is undoubtedly an effective way to explore disease biocontrolling endophytes in grapevines.In this study,the diversity distributions of endophytes in grapevine leaf tissues（Vitis.Vinifera L.× V.labrusca L.,cultivar: Rose Honey（RH））with different degrees of downy mildew symptoms were analyzed by High-throughput sequencing technology.It was found that the distribution abundance of an endophytic fungus（Aureobasidium sp.）positively correlated with the degrees of downy mildew symptoms in grapevine leaf tissues,and the relative abundance of this endophytic fungus was the highest in the severely infected tissue,suggested the fungal endophytes may be a potential grapevine pathogen.Then we isolated the endophytic fungus HA-18 by culture method.And then the pathogenicity of a culture-based method isolated endophytic fungal strain HA-18（Aureobasidium sp.）and its potential biological controlling approaches were systematically studied,and the main results as follows:1.In total 406 endophytic fungal features were detected,which belonging to187 species,145 genera and 4 phyla.And meanwhile,382 endophytic bacterial features were detected,which belonging to 193 species,179 genera and 17 phyla.The endophytic communities in grapevine leaf tissue samples showed a gradient differences in abundances and diversities along with the degrees of downy meldew symptoms.The similar degrees of disease symptoms appeared higher similarity of endophytes composition in leaf tissues.In total 163 strains and 47 OTU cultivable endophytic fungi were isolated from grapevine leaf tissues with different degrees of downy mildew symptoms,which belonging to 24 genera.The highest isolation rate and diversity index of endophytic fungi were observed in disease spots within severe downy mildew infected leaves.And the diversity index increased along with the increased degrees of disease symptoms,with a few exceptions.2.Based the results of ITS sequencing,the distribution abundances of an endophytic fungus（Aureobasidium sp.）in grapevine leaf tissues were observed the significant and positive correlation with the degree of downy mildew infection,while negatively correlated with the abundances of the powdery mildew pathogen Erysiphe sp.in the grapevine leaf tissues.And the relative abundance of this endophytic fungus was the highest in the severely infected tissue.This suggested the endophytic fungus may be a potential grapevine pathogen.And the pathogenicity of a culture-based isolated endophytic fungal strain HA-18（Aureobasidium sp.）was proved the pathogenicity to the RH leaves by the leaf disc method and aseptic seedling method.3.Endophytic fungi with antagonism effect against HA-18 were screened out via confrontation culture method.In total 24 endophytic fungal strains were tested,and all of them showed different degrees of antagonism against the pathogen HA-18.HB-15（Alternaria alternata）,HB-21（uncultured fungus）and HA-51（Nigrospora sphaerica）showed the best antagonistic effects,and the inhibition rates to the growth of the pathogen were 52.63 %,52.63 % and 60.53 % respectively.We futher verified the alleviation functions of these endophytic fungi to the pathogenicity of HA-18 in grapevine aseptic leaves,by inoculation of them before or after the HA-18 infection.And the inoculation of antagonistic fungal strains HA-51（Nigrospora sphaerica）after the infection of pathogen HA-18 had the best biocontrol effect.4.The relative abundance of Aureobasidium sp.and Erysiphe sp.appeared an negative correlation,that is,the higher relative abundance of Erysiphe sp.along with the lower relative abundance of Aureobasidium sp.in grapevine leaf tissues.And Aureobasidium sp.may become biocontrol agents to protect the grapevine from powdery mildew.The treatments with HA-18,MA-1（uncultured fungus）,normal saline and sterile water and found that all these treatments could alleviate the symptoms of grape powdery mildew.MA-1 appeared the most effective,followed by normal saline and HA-18.This implicated that the pathogen HA-18 also had certain degrees of effect on grape powdery mildew biocontrolling.