Colonization Of B.axarquiensis In Xinjiang Cotton Research On Its Micro-ecological Effect

Verticillium wilt is a soil-borne vascular fungal disease caused by Verticillium dahliae Kleb.Verticillium wilt has seriously affected cotton yield and quality.In order to increase the income of cotton farmers in Xinjiang,long-term continuous cropping of cotton has led to the continuous increase in the soil-borne diseases of cotton verticillium wilt.At present,cotton verticillium wilt has become an important limiting factor restricting the high yield of cotton in Xinjiang.Therefore,cotton verticillium wilt disease is a common focus of cotton farmers.In our previous study,Bacillus axarquiensis TUBP1 was screened and found to be an antagonistic strain against V.dahliae with 43%biocontrol effect in the cotton field.In order to uncover the functional mechanism of B.axarquiensis against Verticillium wilt in cotton,the colonization of B.axarquiensis labeled with a green fluorescent protein(GFP)was investigated in cotton plants and the rhizosphere soil.And this paper studied the effect of TUBP1 with gfp gene marker on the microbial community structure of cotton rhizosphere soil and the effect on cotton yield and quality.Cotton Verticillium wilt incidence,soil biochemical properties and soil bacterial and fungal communities were analyzed.It lays a theoretical foundation for the further development of B.axarquiensis as a biocontrol agent.Firstly,a plasmid(pHT-315)containing gfp gene was successfully transformed into wild B.axarquiensis TUBP1 and labeled a green fluorescence by electroporation,which didn’t change the bioactivity in vitro.The surface of cotton seeds was disinfected and soaked in B.axarquiensis TUBP1-315gfp for 24 hours for seeding.After the leaves emerged,the leaves were sprayed with B.axarquiensis TUBP1-315gfp medium(10 ~8CFU/mL)and samples were taken every 7 days.(Soil,roots,stems,leaves),and qualitative and quantitative analysis with scanning electron microscope and fluorescence spectrophotometer.Observation with a confocal laser scanning microscope and a scanning electron microscope showed that GFP-labeled B.axarquiensis TUBP1 infected cotton roots and widely distributed in epidermis,cortical parenchyma,intercellular spaces,the xylem vessels,and pith cells as well as root hair cells through cracks formed at the lateral root junctions,followed by a slow migration from roots to stems and leaves.Quantitative fluorescence and flow cytometry(FACS)approaches showed a gradual decrease in the number of TUBP1-315gfp with increasing inoculation time.However,TUBP1-315gfp levels were detectable till 45 days after planting.In contrast,no fluorescence signal was detected in the non-inoculated groups.Therefore,GFP-labeled B.axarquiensis TUBP1 exhibited colonization in different parts of cotton plants from the rhizosphere soil.The colonization quantity was rhizosphere soil>root>stem>leaves.Secondly,after the cotton rhizosphere soil was inoculated with GFP-labeled B.axarquiensis,the soil total nitrogen(TN),available phosphorus(AP),and available potassium(AK)increased significantly compared with the control group,but when the inoculation concentration When the concentration was 10 ~8CFU/mL(T2)and the concentration was 10 ~100 CFU/mL(T3),there was no significant difference in the increase in soil total nitrogen(TN),available phosphorus(AP),and available potassium(AK).In addition,the soil polyphenol oxidase(PPO),polyphenol oxidase(ALPT),invertase(SC),urease(UE),and alkaline phosphatase(AKP)enzymes live in different growth stages All have increased to varying degrees,especially in sucrase(SC),urease(UE)increased significantly.The diversity index Chao and Shannon showed that after inoculation of biocontrol bacteria B.axarquiensis TUBP1-315gfp in the roots,according to the results of analysis of variance,the microbial community structure and diversity of rhizosphere soil in cotton seedling stage and budding stage changed significantly.The microbial community structure and diversity at the flowering stage and the bolling stage had little effect,indicating that inoculation of B.axarquiensis TUBP1-315gfp fungus had an effect on the diversity of early cotton microbial communities.Among them,the abundance of Bacillus varies greatly,and the number of Bacillus increases with the growth period of cotton,and the number of Bacillus in cotton seedling stage,budding stage and flowering stage is significantly increased compared with the control group.This study shows that the application of B.axarquiensis TUBP1-315gfp can change the microbial community structure of cotton rhizosphere soil.Finally,after the cotton roots were inoculated with GFP-labeled B.axarquiensis,the incidence of cotton verticillium wilt significantly decreased,especially during the cotton flowering stage and cotton bolling stage,the biocontrol effect was about 40%,and that without fluorescent label The biological control effect of wild strains(42.6%)is equivalent,and the control effect of cotton roots inoculated with GFP-labeled B.axarquiensis is 10 ~8 CFU/mL(T2)and 10 ~100 CFU/mL(T3)quite.In addition,the cotton yield and quality indicators have also been improved.At a concentration of 10 ~8 CFU/mL(T2),cotton yield can be increased by 40.6%.The cotton quality index has also been improved,including fiber length(FL),uniformity index(UI),fiber strength(STR),and short fiber index(SHI)compared with the control group.