Screening,Identification And Evaluation On Biocontrol Potential Of Antagonistic Bacterium Against Tobacco Black Shank

Using the plate dilution method,three strains of bacteria XCS007,YJC-4 and YBM-4 against black shank were screened from rhizosphere soil suffered by tobacco black shank.Dual-culture and Oxford cup were used to test the antifungi activity against tobacco black shank.The optimum growth conditions of these three strains were investigated.Molecular morphology,physiological and biochemical characteristics were used to clarify the taxa of these three stains.Furthermore,chitins and?-1,3-glucanase activities were investigated.To investigated disease prevention and disease control effect the fermentation products of strain XCS007 and crude extract together with mixture of XCS007+YBM-4+YJC-4?1:1:1 ratio?were applied in pot and field experiment.The results were as followed.1.Three bacteria strains named XCS007,YBM-4 and YJC-4 with significant antifungi activities were screened from 324 bacteria isolates,the antifungi activities against tobacco black shank were91.80%,85.34%and 72.99%,respectively.In addition to a significant inhibition zone,the edge of black shank were found to be dissolved.Which indicated that these three strains have significant antifungi activities.Using the Oxford cup method,the antibacterial effect of XCS007 antagonistic crude extract was the most obvious,the YBM-4 and YJC-4 crude extracts had relatively lower antibacterial effect.2.The optimum growth temperature of XCS007 antagonistic bacteria was 30?,and the most suitable pH was 7.5,and the yield of bacteria was high at 48h.The optimum growth condition for YBM-4 is 32.5^oC,pH=7.5 and incubate for 60 hours.The maximum amount of YJC-4 antagonistic bacteria was 35 ^oC and 60 hours.The bacterium cannot growth at pH 4.5.3 XCS007 was gram negative bacteria and had obvious motility,which showed gelatin liquefaction and can produce oxidase,nitrite and N₂.It could utilize of citrate and glucose decomposition.It could not utilize V-P,methyl red,fructose and starch.When the sodium concentration was 3%and the maximum growth was 2.11×10⁸ cfu/mL.According to the morphological,physiological,biochemical characteristics and the phylogenic analysis 16S rDNA,XCS007 was identified as Pseudomonas aeruginosa.YBM-4 was gram positive bacteria,V-P test and methyl red test were positive.It could use citrate,hydrolyzed starch and glucose,besides it can produced nitrite and N₂.Oxidase and gelatin liquefaction were negative,and not decomposed chitosan.With the increase of sodium salt concentration,the growth and reproduction of antagonistic bacteria were obviously inhibited.YBM-4had the closest relationship with Bacillus subtilis?KT985478.1?,and the sequence similarity was 99%.YJC-4 was gram positive bacteria,the V-P oxidase and methyl red test were positive.It could use citrate,glucose and fructose,and produced large amounts of nitrite and N₂.The strain cannot utilize gelatin and starch.The maximum growth rate of antagonistic bacteria was about 2.21×10⁹ cfu/mL with 2%NaCl?v/w?in the culture medium.The sequence homology of strain YJC-4 with Bacillus safensis?JF411315.1?was 99%,which suggested that YJC-4 was a member of Bacillus safensis.4 The chitinase and?-1,3-glucanase XCS007 antagonistic bacteria produced a showed the tendency to decrease after the first increase.When the antagonistic cultured for 5 d,the highest activity?1.23 U/mL?of chitinase wasobtained,while the antagonistic cultured for 6 d,the highest activity of?-1,3-glucanase was 1.96 U/mL.The highest chitinase activity of YJC-4 antagonistic bacteria was 0.67U/mL for 6 d,while the activity of?-1,3-glucanas was 0.88 U/mL for 4 d.Interestingly,the highest activity of YBM-4 for?-1,3-glucanase and chitinase were close to zero.5 In the pot experiment,after spraying antagonistic bacteria for tenth days,control effect of seven treatments on tobacco black shank was significant.The control effect of other six treatments,such as XCS007 and its crude extracts,YBM-4,YJC-4 and XCS007+YBM-4+YJC-4 mixture?1:1:1 ratio?,were 77.27%,75.78%,73.03%,78.94%and 80.41%at the fourteenth days,respectively.The AUDPC were 75.87,80.65,88.59,72.01,66.98,64.81,and 111.62.The results showed that the mixture of three antibacterial antagonistic could effectively enhance the control effect on tobacco black shank,and control or delay the infection of tobacco black shank.In the field experiment,after spraying for tenth and twentieth days respectively,the best control effect of 58%metalaxyl mancozeb was found at thirtieth days in the three survey,followed by XCS007+YBM-4+YJC-4,which showed the antagonistic mixture has potential value of application and promotion.