Characterization Of Chitinase From Chaetomium Spirale ND35 And Its Biocontrol Activity Against Botrytis Cinerea

Chaetomium spirale ND35 is a strain of endophytic biocontrol fungus isolated from the healthy Populus tomentosa, with great development potential as biocontrol agent (BCA)against a wide spectrum of phytopathogenic fungi.Gray mold caused by Botrytis cinerea, is one of important vegetable and fruits diseases in the world. It has developed quickly and became more harmful and serious since 1970s.At present, a several lines of evidents indicated that B. cinerea had developed severe drug-resistance because over use of chemical pesticides has serious negative and side effects on our society, as well as human health. Therefore biocontrol is a alternative to protect crops from gray instead of chemicals.We selected Bean-Botrytis cinerea as a model system, the objective of this study is to better understand the role of chitinases from C. spirale ND35 in biocontrolof gray mold, as well as its feasibility and the potential of application. The major results achived in detail are as follows:1. Dual culture on PDA plate and observation by microscope demonstrated that C. spirale ND35 was capable of strongly suppressing B. cinerea, the causal agent of gray mould. The crude extracts with chitinase activity inhibited conidial germination and mycelial growth of B. cinerea in vitro, as well as biocontrol of intact and detached leaves of bean against gray mould in greenhouse experiments.2. Cell wall preparations(CWPs) of several fungi including Valsa sordida, Saccharomyces cerevisiae, Rhizoctonia cereali and R. solani, as well as polysaccharides such as colloidal chitin and laminarin can induce chitinase by C. spirale ND35. The chininase activity induced by laminarin was maximun.The second one is colloidal chitin as carbon source.3. The crude extracts induced by colloidal chitin were obtained from C. spirale ND35 grown in SMCS liquid medium. One of the chitinases was purified by ammonium sulfate precipitation, electrophoretic homogeneit and DEAE Sepharose Fast Flow anion-exchange chromatography, and Phenyl Sepharose Fast Flow hydrophobic chromatography. Its molecular weight was ca 42 kDa analyzed by SDS-PAGE. Purification folds and yield were 10.03 and 7.10%, respectively.4. The purified chitinase Chit42 functioned optimally at 40℃and pH 5.5,and was stable within a broad range of pH 5-8.5 and below 30℃. The chitinase activity was inhibited by Hg²⁺,Fe³⁺,Zn²⁺ ,Cu²⁺ and Mg²⁺ and slightly stimulated by Na+. The Km and Vmax values for the chitinase, using colloidal chitin as substrate, were 1.72mg ml-1 and 21.18 U ml-1, respectively.5. The purified chitinase Chit42 and antibiotic from C. spirale ND35 showed synergisticlly inhibitory effects on conidial germination of B. cinerea.