Isolation,Screening And Fermentation Optimization Of Endophytic Fungi Produing Anti-TMV Active Substances From Ailanthus Altissima

Plant virus disease is a kind of important disease that damage to the agricultural production,so far there is no proven chemical agent that control plant virus disease completely.Because of the rich diversity of species and metabolite,plant endophytic fungi have become important resource for anti-virals research and development.Base on the theory that endophytic fungi can produce same or similar bioactive substances as or to what their host plants produce,in this experiment,endophytic fungi isolated from Ailanthus altissima was studied by screening and identifying anti-TMV active strain,and Optimizing its liquid fermentation conditions,in order to lay the foun-dation for development and application of anti-virals.107 endophytic fungi were isolated from the roots,branches and leaves of Ailan-thus altissima.Among these fungi,30 strains were isolated from roots,27 strains isolated from branches,51 from leaves.The isolation results show that the distribution of endophytic fungi in leaves is more than roots and branches.Antiviral activity of Ailanthus altissima endophytic fungi against TMV replicat-ion was screened by leaf discs assay,the results show that most of the endophytic fun-gi have antiviral activity against TMV replication,the inhibitory TMV replication rate of 4 endophytic fungi were above 80%among these,fermenting the 4 endophytic fugi again,and then measuring the inhibition ratio of their crude extractings from fermen-ted liquid against TMV replication in different concentration and against TMV infect-ion at the concentration of 1 mg/mL,the result indicate that only AL14 strain has higher inhibition ratio against TMV infection and replication,and more stable,this indicate that it has potential application value.Based on the traditional identification method of morphology,the strain AL14 was identified by ITS sequence analysis,its classification status is:Imperfecti fungi,Hyphomycetes,Moniliales,Dematiaceae,Alternaria,Alternaria alternate.Through single factor test and orthogonal experiment,optimize the culture medium and ferm-entation conditions of strain AL14,the results were as follow:the optimal solid culture medium is Sabouraud's dextrose agar,the optimal liquid fermentation culture medium is:on the basis of the potato juice medium,add 4.0%glucose,1.0%ammonium sulfate,CaCO30.3%and FeSO4 0.02%.The optimum fermentation conditions is:fermentation temperature 28? the initial PH 7.5,5%AL14 strain,9d,120 r/min.