Screening And Biocontrol Effects Of Biocontrol Antagonistic Bacterial Against Botrytis Cinerea

Tomato gray mold is a devastating soil-borne disease caused by Botrytis cinerea,which poses a great threat to tomato production.The use of biocontrol bacteria to control tomato gray mold is considered as an effective green control method.The objective of this research project is to isolate an antagonistic bacterium that has a good control effect on tomato gray mold,analyze its fermentation products,colonization in the tomato rhizosphere,and conduct preliminary explorations on tomato growth promotion and resistance induction.It provides a theoretical basis for the prevention and treatment of grey mould of tomato.The main findings are as follows:1.Strain XF,a bacterium with strong antagonistic effect against B.cinerea,was isolated from the soil of the virgin forest of Xinglong Mountain,Gansu Province,and the inhibition rate was 66.35%.By observing the morphological characteristics of the strain and analyzing the 16 S rDNA sequence,it was determined that strain XF was Bacillus cereus.2.Taking B.cinerea as an indicator,the biomass and bacteriostatic rate were used as indicators,and a combination of single factor test and orthogonal optimization test was used to optimize the fermentation medium and fermentation conditions of strain XF.The results showed that the optimal medium for fermentation of strain XF was: glucose 1.00%,peptone1.00%,yeast powder 0.50%,NaCl 1.00%,MgSO4 ·7H2O 0.10%,tryptophan 100 mg/L;Optimal fermentation conditions: initial pH 6.0,the temperature was 30?,the fermentation period was 60 hours,the speed of the shaker was 200 r / min,and the inoculation amount was5%.After optimization,when the fermentation broth of strain XF was diluted 10 times,the inhibition rate against tomato gray mold was 98.19%,and the control effect on tomato leaves and pots could reach 66.58% and 83.97%.3.Analysis of antibacterial substances of strain XF: First,it was determined that the fermentation product of strain XF contains volatile substances and high temperature resistant substances by using the double-plate buckle method and high temperature boiling method;secondly,the plate validation test showed that the secondary metabolites of strain XF contained Protease,glucanase,chitinase,and iron carrier;then Salkowski colorimetry was used to determine the auxin(IAA)content in the fermentation broth of strain XF;LC-MS comparative results analysis found that the substances with increased content in the fermentation broth mainly include caryophyllin,ursolic acid,chlorogenic acid,cinnamic acid,chloramphenicol,3,4-dimethoxybenzoic acid,and various amino acids.However,specific metabolites with resistance need to be further analyzed.4.Colonization of the strain XF in the rhizosphere of tomato: First,the strain XF was labeled with GFP green fluorescent protein to obtain a labeled strain XF-pGFP.By examining the growth rate,it was found that the introduction of plasmid pGFP4412 had little effect onthe normal life activity of strain XF.Then the spraying method was used to inoculate the labeled strain XF-pGFP in the tomato plant rhizosphere,it was found by laser confocal microscopy that it could colonize in tomato roots,the number of marker strains XF-pGFP observed in the roots gradually decreased over time.5.The growth-promoting effect of tomato strain XF and the induction of systemic resistance were studied by flask test and pot test.First,the fermentation filtrate(V: V = 1: 100)of strain XF was added to the MS semi-solid medium to show that the fermentation filtrate can promote the germination of the seeds.Compared with the control group,the seedlings' shoot length,root length,number of whiskers,wet weight,and dry weight within 7 days of germination were increased.Secondly,by spraying tomato seedlings with the fermentation broth of strain XF,it was shown that the dilution of 100 fermentation broths had a significant promotion effect on the biomass of tomato plants treated for 20 days.The total chlorophyll content,resistance-related POD and PAL enzyme activities in the leaves were all improved.Finally,the analysis of PR1,PR2,PR8,and PAL protein gene expression using fluorescence quantitative analysis showed that PR1,PR2,PR8,and PAL were in the relative expression multiples of tomato plant roots,stems and leaves increased.