Screening,Identification And Genetic Modification Of Biocontrol Bacteriaagainst Verticillium Wilt In Cotton

Verticillium wilt seriously restricts the sustainable development of China’s cotton industry.Antagonistic bacteria have a great development potential in controlling Verticillium wilt.In this study,a bacterial strain named Bacillus sp HT-7 with good control effect on Verticillium wilt of cotton was isolated from the root soil of Verticillium wilt-resistant cotton(Hai 7124).It was proved that HT-7 had a strong antagonistic effect on the pathogen of Verticillium wilt of cotton according to the confrontation experiments in plate,the coculture experiments by mixing spores with fermentation broth and the potted experiments.It was found that the main antagonistic factors of strain HT-7 were protein substances by detecting the thermal stability of the antimicrobial metabolites.A virulent protein was further isolated from strain HT-7,which had high homology with known beta-glucanase.It was heterologous expressed in E.coli BL21,and the recombinant enzyme could significantly inhibit the growth of V.dahliae,indicating that beta-glucanase was an important antagonistic virulence factor.The beta-glucanase gene in strain HT-7 was overexpressed by gene recombinant technology,and the overexpressed strain was constructed to achieve better disease resistance activities.The main results of this study are as follows:1.The screening and identification of antagonistic Bacillus.Fifty-six strains of Bacillus sp.were successfully isolated from the root soil in V.dahlia-resistant cotton(Hai 7124)based on the heat resistance of Bacillus.There were nine strains of Bacillus sp.showing strong inhibitory effect through the plate confrontation test.Among which,the strain HT-7 had the best antagonistic effect against V.dahlia.The flatbed confrontation experiment showed that the inhibition rate of HT-7 on the pathogen of Verticillium wilt of cotton was 40%.The 16 S rRNA sequence of the HT-7 showed 99.86% similarity with the strain Bacillus vanillea.The strain HT-7 was deemed to Bacillus genus according to morphological and 16 S rRNA sequence identification.The antibacterial mechanism was further explored.It was found that the spore germination inhibition rate of HT-7 against V.dahliae was 68% when the strain HT-7 fermentation broth was mixed with the spore of V.dahliae for 12 h.Pot experiments showed that strain HT-7 could significantly improve the resistance of upland cotton to V.dahliae.The incidence and disease index of the experimental group decreased by 60% and by 82% compared with the control group,respectively.2.The identification of strain HT-7 virulence factors.It was found that the main antagonists were thermolabile substances through the thermostability test of extracellular metabolites of strain HT-7.The main virulence factor in strain HT-7 was determined to be a proteinaceous substance.The mass spectrometric identification of potential active proteins revealed that the amino acid sequence was 95.99% homologous to the glycosyl hydrolase family protein in B.velezensis(WP-038460387)and 97.94% homology with the beta-glucanase in B.amyloliquefaciens(AAO43052).Therefore,it is speculated that the main virulence factor in the HT-7 strain was beta-glucanase.The growth curve of the strain HT-7 and the activity of beta-glucanase were measured.The results showed that the activities of beta-glucanase of the strain HT-7 reached a plateau and a maximum of 20 U/mL when shaking culture for 16 h and 60 h,respectively.The mixed spore test was carried out on the bacterial liquid in different periods of culture.The results showed that the fermentation broth had the strongest anti-fungal effects when the strain was cultured in shaking flask for 60 h.It was concluded that beta-glucanase was the main virulence protein of strain HT-7.3.Cloning,expression,purification and antifungal activity of beta-glucanase gene.The beta-glucanase gene fragment was 732 bp in size and encodes 243 amino acids.It was successfully expressed in E.coli DH5α,detected by SDS-PAGE and purified via metal chelate affinity chromatography of Ni-NTA medium.The recombinant beta-glucanase showed effectively inhibiting activities against the growth of V.dahliae with Inhibition rate up to 22.5%.Meanwhile,the purified beta-glucanase had a significant inhibitory effect on the germination of V.dahliae spores,and the activity was measured to be 23.5 U/mL,which further confirmed that beta-glucanase in strain HT-7 plays a major role on the control of cotton V.dahliae.4.The construction of the overexpressed engineering strain and detection of its anti-pathogenic activity.In this study,the overexpression engineering strain CT-7 was constructed using molecular biology technology.The beta-glucanase gene was overexpressed in wild-strain HT-7 to enhance the disease resistance.Western hybridization results showed that the amount of beta-glucanase secreted by the overexpressing strain was 2.8 times higher than that of the original strain.The plate-to-sputum experiment showed that the diameter of the inhibition zone of the overexpressing strain increased to 65% versus 40% in the wild strain.Moreover,the fermentation broth of CT-7 had strong inhibition on the spore germination of V.dahliae.The plate experiment showed that the inhibition zone diameter of the CT-7 fermentation broth was 22 mm and the inhibition rate was increased by 57%.In the pot experiment,the disease index of the overexpression test group reduced by 88%,the incidence rate reduced by 70%,and the control effect was as high as 91% compared with the wild strain HT-7 group.It was indicated that the overexpressed strain CT-7 could be used as a biocontrol agent to control Verticillium wilt of cotton.