Isolation And Identification Of Nematicidal Compound And Analysis Of Bioactive Compounds Gene Cluster Of Snef5

Snef5isolated in this study was identified as S. chinense based on morphological characteristics and molecular phylogenetic analysis. The filtrate of fermentation fluid of Snef5is tested for nematicidal activities against4targeted nematode species and showed a high level of nematicidal activity. The experiments are studied systematically on bioactive compounds and structural analysis of metabolites by chromatography and nuclear magnetic resonance (NMR) methods, and its safety to plants have been evaluated. SSH was used in research on analysis of bioactive compounds gene cluster of Snef5. It is the first time to report that S. chinense can be used as a biological control agent against plant parasitic nematodes. And there are some innovative results as follow:1. Identification and the test of bioactivity to nematodes of Snef5:The Snef5was further identified as Simplicillium chinense, by morphological characteristics, sequence determination of ITS, β-tubulin gene and18S rDNA, molecular phylogenetic analysis. The filtrate of fermentation fluid of Snef5showed a high level of nematicidal activity to Meloidogyne incognita J2, Heterodera glycines J2, Aphelenchoides besseyi and Caenarhabditis sp. There are some advantage of Snef5that are high dilution limit points and stability.2. The evaluation of safety:The high concentration filtrate of fermentation fluid (Dilute with1x and5x) of Snef5showed some inhibition to the seeds of tomato L402and cucumber Lvlong7, but it was not obvious to soybean L15. The cucumber Lvlong7plant treated by Snef5indicated some sensitive of growth. By contrast it is safety to the plant of tomato L402, soybean L15, ryegrass and wheat. But Antheraea pernyi pupaes can be infected by Snef5.3. Separation, purification and structure determined of nematicidal metabolites of fungus strain Snef5: The nematicidal metabolites of Snef5is a strongly polar and faintly acid soluble components. It possessed light, thermal, acid and base stability. The three of pure nematicidal compound are extracted from fermentation fluid, separated and purified by ion exchange chromatography, TLC, silica column chromatography, Sephadex LH-20chromatography, and HPLC. It showed that among the three compound1,2were furanose amino and3was glucose according to them NMR data and amino acids test. And it need be futher research.4. The successful construction of the mutant:After many UV, DES and ATMT mutagenetic treatments, and the selections from a lot of mutants. We acquired8high toxic strains and3low toxic strains. Finally high stable high toxic strain UV-17, UV-18and low toxic strains UV-15, T-25have been screened after five generation.5. SSH is used in research on analysis of bioactive compounds gene cluster of Snef5:Forward and reverse SSH libraries were contructed by using RNA from Snef5-UV-17and Snef5-T-25strains. By comparing sequences in the GenBank, there are10function-known EST, such as two EST with function of nitrogen, phosphate, sulfur metabolism from Metarhizium acridum, one EST with function of transcript antisense to ribosomal RNA from Pinus sylvestris and Heterobasidion annosum mixed EST library, five EST with function of hypothetical protein from Metarhizium acridum, one EST with function of hypothetical protein from Trichoderma longibrachiatum and one EST with function of bleomycin hydrolase from Metarhizium acridum. And it need be futher research.