| Verticillium dahliae is a soil-borne plant pathogen that is responsible for Verticillium wilt disease. V. dahliae is reported to produce mycotoxin complex which is the main factor to induce host cell death. It has been proven that the key pathogenic factor is a kind of protein-lipopolysaccharide complex excreted by V. dahliae. Endophytic bacteria exist in plants all their life. The internal tissues of plants provide a relatively uniform and protected environment for endophytic bacteria. As biocontrol agents, endophytic bacteria have potential for producing a wide variety of anti-microbial metabolites, and theirs induction of systemic resistance.In this dissertation work, the mycotoxin of V. dahliae and endophytic bacteria were used as a toxin-microb interaction model system to identify and screen attenuated endophytic bacteria. Then we initially probed the mechanism of attenuated. The main results were as followed:211strains of endophytic bacteria were isolated from cotton plants by surface sterilization method.211stains of endophytic bacteria plus19strains of laboratory pre-separation of endophytic bacteria were used for screening. Adopting growth confrontation method and toxinic attenuated on cotton sprouts method to screen12strains of endophytic bacteria. In the toxinic attenuated on cotton sprouts experiment, we obtained strains of DP10, CCM9, XJL12, and SZ5with which high antibacterial activity and stability of attenuated effect.Establishing the system of co-cultured between stains of DP10, CCM9, SZ5, and crude mycotoxin, and detecting the effect of toxinic attenuated on cotton seedling. Extracellular substances could completely attenuate the wilt activity of toxinic protein after the bacterial suspension of DP10and VD toxin co-cultured for48h. The bacterial suspension of CCM9, SZ5, and VD toxin co-cultured for60h could also completely attenuate the wilt activity of toxinic protein.The next experiment was potted cotton seedlings bacterization with attenuated bacteria. Evaluation of attenuated effect was initiated40d after inoculating with bacteria. For evaluation, the growth parameters (plant height and dry weight of cotton plants) were used for data collection. From the result we know that the best bacteria of attenuated effect were endophytic bacteria SZ5and DP10. What’s more, it also indicated that3strains of DP10, CCM9, and SZ5could survive in the cotton plants last for40d and play a sustainable attenuated effect.The protein was purified by ammonium sulfate precipitation, Sephadex G-75fractionation, and SDS-PAGE. The common point of three endophytic bacteria was that they lacked of26kD protein. It is conjectured that26kD protein was one of the actively toxinic proteins. The40kD protein of VD toxin was significantly attenuated after co-culturing with DP10. So,40kD protein indeed involved the activity of wilt. Studying on the mechanism underlying attenuated effect of SZ5and CCM9are the next goals of further research.In addition, four endophytic bacteria with high attenuated activity were identified by physiology-biochemistry analysis and16SrDNA sequence analysis. DP10and CCM9were identified to be Bacillus subtilis, while XJL12was identified to be B. subtilis or B. tequilensis, and SZ5was identified to be B. cereus or B.thuringiensis.Our study demonstrates the potential of some Bacillus isolated from cotton plant and3known strains of DP10, CCM9, and SZ5as effective biocontrol agents against defoliating V. dahliae in natural environment. To our best knowledge, this is the first report showing attenuated endophytic bacteria existence and mycotoxin of V. dahliae could be attenuated in vitro. The study will also provide a practical and promising alternative for controlling Verticillium wilt of cotton under field conditions in the near future.
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