Screening Of Endophytic Bacteria From Aluminum Resistant Alfalfa Seeds And Trial Production Of Bacterial Agents

Alfalfa,the "king of grass",is a perennial leguminous plant with the most extensive planting area and rich protein in the world.It plays an important ecological and economic role in the agricultural ecosystem.Aluminum toxicity is the main factor limiting the yield and persistence of alfalfa in acid soil.In this study,endophytic bacteria of alfalfa seeds were studied.Through 16 S and ITS high-throughput sequencing technology and network analysis,the relationship between microbial community abundance of alfalfa seeds and plant growth was explored.Microbiota were prepared by isolation and culture,and the effects of microbiota s on the growth of alfalfa under aluminum stress were tested by tieback.The main research results are as follows:1.Analysis of endophytic microbial community and structure in seeds of different alfalfa varieties showed that the endophytic bacterial and fungal resources of alfalfa seed were abundant,and different varieties significantly affected the diversity of endophytic fungi but not bacteria.At the generic level,the dominant bacterial genus were Pseudomonas,Bradyhizobium,Pantoea and Paenibacillus.The dominant fungal genus were Aspergillus,Aitemaria,Stemphylium,Talaromyces and Cercospora.The dominant microbiota genus of different varieties were basically similar,but their relative abundance was significantly different.2.The correlation between growth characteristics of alfalfa and seed microorganisms under aluminum stress showed that the seedling height,aboveground biomass,root biomass,root length,root surface area,mean diameter and root volume were significantly positively or negatively correlated with the relative abundance of endophytic bacteria and fungi under aluminum stress(p < 0.05).3.The endophytic bacteria of alfalfa seeds were screened,and five strains of endophytic bacteria and four strains of fungi were identified,which were as follows:Bacteria Erwinia,Enterobacter,Stenotrophomonas,Bacillus b10＿12,Bacillus b14,Fungi Cladosporium,Talaromyces,Aspergillus fn4,Aspergillus fc6.Compared with non-inoculation,inoculation of selected bacteria significantly decreased aboveground biomass and height(p<0.05),while inoculation of selected fungi Cladosporium significantly increased aboveground biomass and height of seedings(p<0.05)under aluminum stress.4.In the trial production of fungal Cladosporium,the fermentation condition screening experiment based on carbon and nitrogen source replacement showed that the fermentation effect was ranked as soluble starch > sucrose > lactose > PDB(basic fermentation liquid,potato glucose,control)≈ maltose > yeast extract ≈ peptone.The results showed that the optimal fermentation effect was obtained by replacing the glucose in PDB with soluble starch.The fermentation inoculant verification experiment showed that compared with PDB,the fermentation inoculant replaced by soluble starch and sucrose significantly increased the height of alfalfa seedlings(p<0.05),but the fermentation inoculant replaced by lactose had no significant effect on the seedlings(p > 0.05).Although there was no significant difference in efficacy between the soluble starch and sucrose replaced fermentants(p > 0.05),the mean effect of the soluble starch replaced fermentant was higher than that of the sucrose replaced fermentant.Therefore,the fermentation agent which replaces the glucose in PDB with soluble starch has the best effect.