Preliminary Study On The Mechanism Of Bacillus Velezensis ZJ1 On Alternaria Solani And Botrytis Cinerea And Its Control Effect

In this study,an endophytic bacterium ZJ1 isolated from the stems of Buddleia solani with good antimicrobial activity against Alternaria solani and Botrytis cinerea was classified,its biological characteristics and the antibacterial activity was determined,the constituents of its volatile substances were identified,and the antibacterial mechanism was investigated;the conditions for the preparation of the ZJ1 strain oil suspension were screened,and the quality of the preparation was tested,as well as the prevention and growth-promoting effects.The results of the research lay a theoretical foundation for the application of plant endophytic bacteria ZJ1 in biological control and the later marketization.The specific research content is as follows:1.Through morphological identification,supplemented by 16 S r DNA and gyr A specific genes for phylogenetic research,the Buddleia endophytic bacteria ZJ1 was identified as Bacillus Velezensis.2.The lethal temperature and lethal pH of the ZJ1 strain were measured.The results showed that the lethal temperature of the ZJ1 strain was 96℃ for 20 min,and the lethal pH was less than or equal to 4 or greater than or equal to 14;The formation of biofilm in the base was observed,and the influence of different metal ions on the formation of biofilm was measured.The results showed that 0.3% of Zn²⁺,Mn²⁺,Cu²⁺ and Fe²⁺ inhibited the production of biofilm by the ZJ1 strain.It can promote the production of ZJ1strain’s biofilm,and Mg²⁺ can significantly promote the production of ZJ1 strain’s biofilm;the 96-well plate quantitative method was used to determine the ability of ZJ1 strain’s biofilm formation,and the results showed that the result was found to contain 0.3% Mg SO4 The most biofilm was produced on the second day of culture in the culture medium,and ZJ1 strain is a strong biofilm forming strain.3.The indoor antibacterial activity of the ZJ1 strain and its volatile substances were measured by the plate confrontation method and the plate pairing method.The results showed that the inhibition rates of the ZJ1 strain against A.solani and B.cinerea were 59.15% and 71.62%,respectively.The inhibitory rates of ZJ1 strain volatiles on A.solani and B.cinerea were 40.58% and 50.90%,respectively.The effect of ZJ1 strain fermentation broth on tomato early blight and gray mold disease was determined by indoor pot culture,and the results showed that ZJ1 strain The potted control effects of fermentation broth and different dilution multiples on these two diseases were all above 70%.4.Through the progressive GC-MS detection of the volatile components of the ZJ1 strain,it was found that the main components of the volatiles were 2-Tridecanone,2-Undecone,1-Tridecene,2-Nonanone,2-Ethyl^-1-hexanol,2-Heptanone,6-methyl-,2-Nonanol,1-Eicosanoland other 8 substances.among which isooctyl alcohol,2-Ethyl^-1-hexanol,2-Nonanone,2-Nonanol,2-Undecone alcohol are for A.solani and B.cinerea has good antibacterial activity;the growth rate method is used to determine the virulence of 2-Ethyl^-1-hexanol and 2-Nonanol to A.solani and B.cinerea,and the different ratios of2-Nonanol and 2-Ethyl^-1-hexanol are screened and enhanced.The determination of the coefficient shows that 2-Nonanol is more toxic to A.solani and B.cinerea,with EC50 of 1.82 μL·m L^-1 and 0.54 μL·m L^-1,respectively.Isooctyl alcohol is effective against these two pathogens.The EC50 is 7.12 μL·m L^-1 and 1.64μL·m L^-1,respectively.The mixture of 2-Nonanol and 2-Ethyl^-1-hexanol at 1:1 and 4:1 has a synergistic effect on A.solani and B.cinerea;The fumigation and control effects of volatile substances and ZJ1 strain against two kinds of diseases were determined by in vitro control effect test.It was found that when the concentration of 2-Nonanol and 2-Ethyl^-1-hexanol 1:1 was 0.0625 μL·m L^-1,the effect of A.solani The control effect of B.cinerea is the best.When the concentration of 2-Ethyl^-1-hexanol is 0.25 μL·m L^-1,the control effect of B.cinerea is the best,and the control effect can reach 95.24%.The different concentration of ZJ1 bacterial suspension has the control effect of two kinds of pathogenic fungi.Between 56.67% and81.00%.5.Detect the antibacterial active substances produced by the ZJ1 strain,and use A.solani as the indicator bacteria to observe the influence of the ZJ1 strain and its volatiles on the hyphae of the pathogenic fungus by scanning electron microscopy.The results showed that the ZJ1 strain can secrete extracellular proteases and cellulase,but cannot produce chitinase,β^-1,3-glucanase and IAA,and does not have the ability to dissolve phosphorus.Observed by scanning electron microscope,it was found that the ZJ1 strain and its volatiles would make A.solani hyphae thin,twisted,deformed,bifurcated,broken,wrinkled and cracked on the surface of the hyphae,and volatile substances can also cause the hyphae content Flow out causes the hyphae to shriveled.6.After the compatibility test of ZJ1 strain with different solvents and auxiliaries,the best formula of ZJ1 strain oil suspension was screened out: emulsifier 7%（v/v）Tween 80,antibiotic 50 μg·m L^-1Amphotericin,antifreeze 2% Ca Cl2,wetting agent 1% SDS,stabilizer 1% Ca CO3,dispersant 0.5% calcium lignosulfonate,protective agent 5% dextrin,the balance is made up with solvent peanut oil,and in accordance with the country The quality inspection standard tests its various indicators,and the results show that the indicators of 575 million cfu·m L^-1 Bacillus Velez ZJ1 oil suspension are in line with and higher than the national standard.7.By treating greenhouse potted tomatoes with different dilutions of Bacillus Velezensis ZJ1 oil suspension,the control and growth-promoting effects were tested.The results showed that 575 million cfu·m L^-1 Bacillus Velezensis ZJ1 oil suspension The 200-fold dilution has the best control effect on A.solani and B.cinerea,and will significantly increase the growth rate of stem thickness,but at the same time significantly reduce the growth rate of plant height.