Construction Of Antagonistic Multiple Bacteria Against Verticillium Wilt And Exploration On Their Active Factors

Cotton Verticillium wilt is a soil-borne disease caused by Verticillium dahliae Kleb.It is one of the main factors restricting cotton production in China.Finding an efficient and environmentally friendly way to prevent and control Verticillium wilt is the primary task for the sustainable development of the cotton industry.In the process of cultivating V.dahliae in the laboratory,our research group found that the cultured V.dahliae plate was full of bacteria and the fungus was completely swallowed.Therefore,it is preliminarily speculated that the plate contains bacteria that can inhibit the fungus.In order to verify this hypothesis,six strains of bacteria were isolated and purified from the plate by streaking separation method,and they were numbered T1,T2,T3,T4,T5,and T6,respectively.The antifungal activities of these six strains were tested through plate confrontation test,inverted plate test,spore mixed fermentation broth co-culture and pot experiments.The results of the plate confrontation experiment showed that the antibacterial rates of the T1-T6 strains were 11.6%,28%,6.7%,10.7%,58%,and 75%,respectively.Among which,the T6 strain with the highest activity is further explored for its activity factors using GC-MS analysis and activity tracking test.The results showed that the antagonistic virulence factors of T6 strain were mainly volatile substances.The results of GC-MS and pure product verification experiments showed that styrene produced by T6 strains was an important antagonistic virulence factor.The antifungal mechanism of styrene at the molecular level was explored via transcriptome and quantitative PCR analysis.The results confirmed that expression level of the genes related to transportation and degradation of V.dahliae changed after styrene induction,thus inhibiting the growth of the fungus V.dahliae.Finally,the composite microbial system with different antifungal properties were constructed according to the results of the analysis of the antifungal activity factors of each strain,the six strains isolated above and Enterobacteriaceae sp.V1 screened in our previous study,which showed comparativelly strong antifungal activities.The inhibitory activities of the composite microbes were confirmed by plate confrontation experiment and pot experiment to achieve different strains with synergistic complementation and enhance the effects of the resistance against Verticillium wilt.The main results of this study are as follows:(1)Isolation,screening and identification of antagonistic strains.Six strains of bacteria were isolated from the contaminated plate of V.dahliae.Through morphological identification and 16S r RNA sequence alignment analysis,they were identified as Staphylococcus sp.T1,Bacillus sp.T2,Microbacterium sp.T3,Stenotrophomonas sp.T4,Bordetella sp.T5 and Bacillus sp.T6,respectively.The inhibition rates of the fermentation broth of T1?T6 on the spore germination of V.dahliae were 7.18%,27.54%,0,11.78%,43.25%and 67.15%,respectively.Pot experiments showed that the incidence rate of T1?T6 respectively decreased by 10.32%,20.89%,3.77%,3.45%,58.89%and 59.11%compared with the control group,and the disease index decreased by 10.90%,43%,11.96%,6.85%,79.30%and 79.30%,respectively.Strains T5 and T6 could significantly improve the resistance of the upland cotton TM-1 to Verticillium wilt.The T2 strain increased the resistance of TM-1 to Verticillium wilt,while the T1,T3,and T4 strains cannot improve the resistance of upland cotton TM-1 to Verticillium wilt.(2)Preliminary study on the active factors of T6 strain.It was found that T6 strain could inhibit the activity of V.dahliae without contact,so it was speculated that the active factor of the bacteria might be volatile compounds.The gas produced by T6 strain was collected and sent to Qingdao Kechuang quality testing Co.,Ltd.for GC-MS analysis.A total of 46 kinds of gas molecules produced by T6 strain were obtained.Among which,the gas with similarity greater than 90 and relative content higher than 1%was screened out for 12 pure gas products,and 11 of them were verified.The pure product verification experiment confirmed that Styrene produced by the T6 strain was its main virulence factor.(3)Preliminary study on the antagonistic mechanism of T6 strain.Four groups of samples of V.dahliae were prepared with the fungus induced by styrene(99%)for 2 hours,2.5 hours,3 hours and non-induced.The samples were sent to Shanghai Meiji Biomedical Technology Co.,Ltd.for transcriptome sequencing analysis.The transcriptome data showed that 10812 genes were detected,of which 3367 genes changed the expression levels.When styrene was induced for 2 h,2.5 h and 3 h,the expression levels of 907,1156,and 1304 genes in V.dahlia changed,respectively..Among them,72 genes were down regulated in the three sets of groups induced by three time periods,including 2 chitinase genes,2 protease genes,5 transport-related genes and 33 hypothetical protein genes.There were 247 genes up-regulated in three sample groups,including 4 hydrolase genes,8dehydrogenase genes,11 reductase genes,17 genes related to transport and transfer,and142 of these genes.A hypothetical protein gene.(4)According to theup-regulation amplitude and function of expression,four potential antimicrobial related genes were screened out.They are VDAG?02838 gene,which is related to the BTB domain and has functions of degradation transcription regulation,chromatin remodeling and protein degradation;VDAG?09554 gene,which is related to Dna J domain,plays an important role in protein translation,folding,unfolding,translocation and degradation;VDAG?04557 gene,a gene related to lysozyme,is widely present in nature and has antifungal and antibacterial effects;VDAG?08251 gene,which is related to the polyadenylation factor?complex.The q PCR results confirmed that the expression of the four genes above was up-regulated after induction by styrene:VDAG?02838,VDAG?09554,VDAG?04557 and VDAG?08251 genes were respectively up-regulated 3.18,78.83,2.71 and 2.92 times compared with the control group.The results of this study initially explored the antifungal mechanism of the T6 strain at the molecular level.Construction of mixed strains and activity verification.The results of affinity experiments confirmed that Enterobacteriaceae sp.V1 with inhibitory activities obtained in previous study,and the T6 strain obtained in this study have good affinity.The V1 and T6 strains were mixed and plated in different proportions to test the inhibitory activity against V.dahliae.The results showed that:when OD₆₀₀=0.8 and the volume ratio of the strains V1 and T6 was 1:3,the inhibition rate of the mixed bacteria was up to 80.5%,which was 5.5%higher than that of the single strain T6(75%),and 19.5%higher than that of single strain V1(61.0%).Other combinations of mixed bacteria did not show higher antibacterial activity than single strain T6.The results of pot experiment confirmed that the mixed bacteria of V1 and T6 could promote the growth of cotton in soil roots.The root length of cotton treated with mixed bacteria reached 20.3 cm,which was 28.5%higher than that of normal cotton(19.5 cm),5.72%higher than that of the cotton treated with single T6 strain,and 0.9%higher than that of the cotton treated with single V1 strain.The stem length of cotton treated with the mixed strains was 22.9 cm,which was 16.9%longer than that of normal growth cotton(19.5 cm),16.2%longer than that of the cotton treated with single T6 strain(19.7 cm),and0.4%longer than that of the cotton treated with single V1 strain(20.1 cm).The growth-promoting capabilities of the mixed bacteria was also measured.The results showed that strain V1 could grow in nitrogen-fixing medium,but strain T6 strain could not.It showed obviously transparent circles when the V1 strain grown in inorganic phosphorus medium,while T6 strain had no transparent circles.Neither the two strains can grow on organophosphorus medium or fixed potassium medium.The indole acetic acid reagent Fe Cl₃was used to detect the fermentation broth of each strain.The results showed no discoloration,indicating that no indole acetic acid was produced in all the tested strains.The results above showed that strain V1 had the ability of fixing nitrogen and dissolving inorganic phosphorus,but had no ability of dissolving organic phosphorus,potassium and growth factor.Strain T6 showed no capabilities of nitrogen fixation,phosphorus and potassium solubilization or growth hormone production.Finally,the control effect of mixed bacteria on other plant pathogens was tested.The mixed bacteria were used to control the main pathogens of apple mold heart disease Cladospora sp.,the main pathogen of tree canker Coniothyrium sp.,and main pathogen of jujube fruit brown spot Cladosporium tenuissimum.The inhibition effects of the mixed bacteria were 66.7%,13.3%and 33.3%,respectively.