Studies On The Biodiversity And Metabolite Bioactivities Of The Endophytic Fungi From Melia Azedarach

The production of antifungal bioactive compounds and new insecticidal drugs by microbial fermentation were studied in this paper, with the endophytic fungi of Melia azedarach isolated from Anhui Province, Jiangsu Province and Shanxi Province, based on the process of separation, purification, cultivation, identification and extraction process of bioactive compounds.290 strains of endophytic fungi isolated from the roots, branches, leaves, fruit, leafstalks and flowers of Melia azedarach collected from Anhui Agricultural University campus, Lingbi county of Anhui province, Wuxi city of Jiangsu Province, Xianyang city of Shanxi Province. After isolation and purification, morphological observation and microscopic identification, these strains belong to eleven genera, six families, five orders of a sub-phylum, displaying great diversity of the endophytic fungi in Melia azedarach.The diversity of endophytic fungal community of Melia azedarach varied with plant part and season , and the dominant group of them were Alternaria , accounting for 24.14% of the total isolated species. Because these strains have no obvious sporulation structure, they were confirmed by ITS-rDNA sequence analysis method. The sequence of KL233 was submitted to National Center for Biotechnology Information (NCBI), compared with DNA sequence in GeneBank by Blast process, and was confirmed that it may be Pyrenochaeta romeroi, or may be a new species. The ITS sequence identities of KL037 , compared with registered in GenBank of Phomopsis sp.,was 99%, from these data, it can be Phomopsis sp.The ITS sequence of KL259 , compared to Arthrinium sp. that was registered in GenBank, exhibited a similarity of, 100%. it can be identified as Arthrinium sp. The ITS sequence identities of KL017 , compared with registered in GenBank of Isaria farinosa, was 100%, identified as Isaria farinosa.The biologically active metabolite antagonist of sixty Melia azedarach representative endophytic strains was investigated, and the result showed that there were 48 (or 80.0%) endophytic fungi strains showed antifungal activities in the PDA media , and they posess the potentials to develope into biological control agents for plant diseases control. The fermentation product of strain KL233 had a high antifungal activity, showing the best activity against Helminthosporium maydis, Fusarium graminearum, Verticillum alboatrum, Glomerella cingulata. And, KL233 strain had good sunshine stability and storing stability, but a little worse in stability and pH stability. According to extraction effect of different solvents and the physical and chemical character tests of active components, an optimal extraction procedure was determined as follows: the liquid culture was condensed at 40℃to 1/5 volume of the broth, 3 times volume of 95% ethanol was added to remove large molecule impurities such as polysaccharide, Standing overnight, 5000rpm under 10min centrifugation and removal of sediment ,and then, supernatant was condensed and extracted with 2 times volume of ethylacetate twice. Finally, the extract was condensed to 1/10 volume of the ethylacetate phase. The concentrated substance derived was crude bioactive product.Pathogenic test of thirty Melia azedarach representative endophytic strains was done, the result showed KL017, KL025, KL031, KL042, KL062 and KL107 had better pathogenicity than others. The strain KL042 had the most highest pathogenicity. The spores of strain KL042 caused 83.33% mortalities after 2.5d and 100% mortalities after 5d against Dendrolimus punctatus. This strain was identified as Beauveria bassiana. It was reported for the first time from China that Beauveria bassiana was isolated from the plant.Bioassay test of pathogenic aciloty to Plagiodera versicolora. 9 representative endophytic strains was carried out, and the result showed KL017 metabolites caused 93.33% mortalities after 24h and 90.09% mortalities after 48h against Chrysomelidae, and the metabolites from strain KL017 had the higher active than others. The fermentation broth was droped to the Dendrolimus punctatus chest and back, the results of statistical analysis of toxicity showed, KL042, KL062 and KL017 metabolites had stronger toxicity , compared with the other significant difference between each treatment. KL042 metabolites caused 20.00% mortalities after 24h and 76.67% mortalities after 48h. KL042 metabolites had the higher toxicity than others.The extraction from fermentation broth of 120 endophytic fungi strains were preliminarily analysed with TLC method, and 7 strains which maybe produce AzedarachinToosendanin. But in comfirmed HPLC-MS test, only 1 strain named as KL062 from Lingbi county of Anhui province has AzedarachinToosendanin in the fermented metabolites. KL062 can be identified as Alternaria.