Study On The Prevention And Mechanism Of Compound Biocontrol Microbial On Onion Root Rot Disease

Onion stem basal rot is also called onion root rot.It is a soil-borne disease caused by Fusarium.It occurs at all stages of onion growth and during storage,which seriously affects onion production.In order to choose a green and effective method to prevent and control onion diseases,this study took Fusarium proliferatum as the control object,and isolated and screened out Bacillus amyloliquefaciens and Trichoderma asperellum which have strong antagonism to F.proliferatum.By studying the optimal fermentation conditions of the two strains,using their metabolites for in vitro tests,pot experiments,etc.,the effects of the two strains and their composite microbial on the prevention and control of F.proliferatum were further determined,and the effects of the two strains on the onion rhizosphere preliminary explorations were made on the colonization of onions,the growth promotion and resistance induction of onions,which provided a theoretical basis for the application of composite microbial in the field.The main findings are as follows:1.The pathogenic F.proliferatum was isolated from the diseased onion bulbs collected from the Jiayuguan onion planting base in Gansu province.The bacteria XG and the fungus M2 were screened out from the soil.In addition,the two strains are compatible,and the inhibition rates of F.proliferatum are 65% and 84.6%,respectively;The inhibition rate of the spore germination of the mixed fermentation filtrate of the two strains was 96.15%;Using flat plate qualitative tests,it was found that the strains XG and M2 both contained protease,cellulase,chitinase,siderophore,glucanase,IAA and other secondary metabolites,and had the performance of dissolving phosphorus and lowering p H;Through morphology and molecular biology,the two antagonistic microbial were identified as Bacillus amyloliquefaciens and Trichoderma asperellum.2.Based on the biomass of strains XG and M2 and the inhibitory rate against F.proliferatum,selected the most suitable fermentation medium for strains XG and M2,and used a combination of response surface analysis and single factor test to optimize the amount of strain fermentation medium added and fermentation conditions.The results show: The most suitable fermentation medium for strains XG and M2 are YMC medium,which is 2%yeast powder,2% sucrose,and 2% corn gluten meal;The best fermentation conditions for strain XG are the initial p H of the medium 6.5,the fermentation temperature of 32°C,the rotation speed of 180 r/min,the inoculation amount of 5%,and the fermentation time of 72h;The optimal fermentation conditions for strain M2 were the initial p H of the medium 7.0,the fermentation temperature of 26°C,the rotation speed of 180 r/min,the inoculum amount 3%,and the fermentation time of 7 days.After optimizing the culture medium and fermentation conditions,in vitro test,it was found that spraying the filtrate of the composite microbial XG+M2 first and then inoculating the F.proliferatum had the best control effect on onion root rot,reaching 72.5%.3.The strain XG with GFP marker was constructed by genetic engineering technology,and the strain XG-p GFP was obtained,and strain M2 with RFP marker,get strain M2-RFP.The wild-type strain XG and transformation XG-p GFP were detected by the growth curve method and the biomass method respectively.The growth rate of the transformation XG-p GFP is basically the same;There is no significant difference in sporulation and antifungal effect between wild-type fungus M2 and transformant M2-RFP;After the marker strains XG-p GFP and M2-RFP were inoculated in the rhizosphere of onion plants,it was found that both strains could colonize the rhizosphere of onion.4.The effects of strains XG,M2 and composite microbial XG+M2 on the growth of onion seeds were studied through seed germination experiments.It was found that the fermentation filtrate diluted 100 times by composite microbial XG+M2 had the strongest promotion effect on onion seeds.The stem length,fresh weight and dry weight increased by103.73%,166.14%,57.21%,67.22% respectively;The pot experiment showed that the composite microbial XG+M2 has a good control effect on onion root rot and can promote the growth of onion plants;Enzyme activity determination showed that onion plant root activity,chlorophyll content and enzyme activity of POD,PPO,and PAL were significantly improved after treatment with composite strains;Fluorescence quantitative method was used to detect protein genes related to onion plants.After onion plants were treated with composite microbial XG+M2,the expression levels of Ac PR1,Ac PAL1,and Ac EIN3 protein genes were all significantly up-regulated,except that the expression of Ac LOX1 protein gene was significantly down-regulated.