Isolation And Identification Of Pathogen Of Fraxinus Mandschurica Blight And Screening Of Antagonistic Fungi

Fraxinus mandschurica Rupr.is a kind of important timber and greening tree species in Northeast China,but in recent years,the frequent occurrence of diseases and insect pests has seriously threatened the forest resources of F.mandschurica.It has occurred in a large area in the area,causing the branches of F.mandschurica to die,causing great harm to the resources of F.mandschurica.At present,chemical pesticides are mostly used to prevent and control diseases,but the excessive use of pesticides has caused more and more serious problems of environmental pollution and ecological damage.Endophytes are ubiquitous in plants and distributed in various parts of plants.Plant endophytes are an important part of plant microbial ecosystems and can be used as new biocontrol factors,which can improve the disease resistance of host plants by synthesizing antibacterial active substances,inducing plant resistance and promoting plant growth,and have become plant disease organisms It has been paid much attention to the potential biocontrol bacteria in the control.In this paper,the pathogenic fungi were isolated from the diseased branches of F.mandschurica blight,and identified from the level of morphology and molecular biology,and then the isolated pathogenic fungi of F.mandschurica blight were used as aim strains to Identify and screen of stable antagonistic endophytic fungi from healthy F.mandschurica.nalysis of the effect of Aspergillus mandshurica dieback on the diversity of endophytic fungi using high-throughput technology.The cultivation characteristics of endophytic antagonistic fungi were studied and optimized,which provided a theoretical basis for the biological control of F.mandschurica blight by using endophytic fungi of F.mandschurica.The main results of this study are as follows.(1)Isolation and identification of the pathogenic fungi of F.mandschurica blightA total of 27 isolates were obtained from the surface-sterilized samples of F.mandschurica blight diseased branches,which were divided into 6 strains by morphological observation.The results of back-linking experiments showed that the hydroponic branches inoculated with strain FL1 showed obvious symptoms of branch blight,and were obtained again by tissue separation method.Molecular biological identification results showed that strain FL1 is Diaporthe cotoneastri.(2)Effects of F.mandschurica blight on the diversity of endophytic fungiA total of 507 ASVs were obtained through high-throughput sequencing technology.The species abundance before and after infection has changed greatly.The richness index(Chao1)and diversity index(Shanno,Simpson)of endophytic fungi increased significantly after infection,indicating that the blight of F.mandschurica had changed the community structure of endophytic fungi in F.mandschurica and improved the richness and diversity of endophytic fungi.(3)Isolation and identification of endophytic fungi from F.mandschurica114 isolates were obtained from the branch samples of Ashwagandha,which were identified by morphology and molecular biology.The identification results showed that except for two strains that could not be identified,112 isolates belonged to 21 genus,35 species,Includes 5 species of Alternaria,5 species of Fusarium,3 species of Didymella,2 species of Bjerkandera,2 species of Cladosporium,2 species of Dothiorella,2 species of Microdiplodia,Aureobasidium,Botryosphaeria,Coniochaeta,Coniothyrium,Diaporthe,Didymosphaeria,Diplodia,Leptosphaerulina,Microsphaeropsis,Nectria,Phomopsis,Nothophoma,Paraconiothyrium and Phaeoacremonium.(4)Screening of endophytic antagonistic fungiThe antagonistic endophytic fungi were screened by the confrontation culture method,and9 strains of endophytic antagonistic fungi were obtained.,which were Coniothyrium pyrinum,Aureobasidium sp.,Cladosporium anthropophilum,Coniochaeta velutina,Phaeoacremonium fraxinopennsylvanicum,Fusarium sp.,Didymella sp.,Nothophoma spiraea and Dothiorella sp..Among them,Coniothyrium pyrinum and Coniochaeta velutina had significant antagonistic effects on pathogenic fungi,and the antibacterial rates were 43.55% and 34.15%,respectively.(5)Optimization of culture conditions for endogenous antagonistic fungi.The optimal culture condition of Coniothyrium pyrinum is potato dextrose agar with pH=6,25℃,and light and dark alternating(12L/12D)conditions.The optimal culture condition of Coniochaeta velutina is CMD medium with natural pH,27℃,and light and dark alternating(12L/12D)conditions.