Screening,Medium Optimization And Genome Sequencing Of PGRP From The Rhizosphere Of Lycium Barbarum L.

Lycium barbarum L.is an important commercial crop.In recent years,with the disinterment of medicinal value and health value,the cultivated area of Lycium barbarum L.in China is constantly expanding,which Lycium barbarum L.in Ningxia is one of the most famous varieties.Driven by economic interests,the limits of cultivated land,and the constraints of planting conditions,the phenomenon of consecutive monoculture of Lycium barbarum L.has been inevitable,which lead to a variety of consecutive monoculture problem,seriously affecting the yield and quality of Lycium barbarum L.Root rot is one of the most common soil-borne disease.PGPR（Plant Growth-Promoting Rhizobacteria）can control soil-borne diseases,promote the growth and development of plant roots,improve the rhizosphere soil environment.In this study,we screened and identified antagonistic bacterium,proteinase producing bacterium,organophosphorus solubilization bacterium,inorganic phosphorus solubilization bacterium,IAA producing bacterium from rhizosphere soil of Lycium barbarum L..We optimized the culture medium for antagonistic bacteria which have better antagonistic effect.We verified biocontrol effect of antagonistic bacterium by means of field testing.And we performed the whole gene sequencing for antagonistic bacterium which have better antagonistic effect in the field.We isolated a Fusarium solani from the diseased tissue of Lycium barbarum L..And we screened 51 antagonistic strains,121 protease producing strains,105 organophosphorus solubilization strains,8 inorganic phosphorus solubilization strains,7 IAA producing strains from rhizosphere soil of Lycium barbarum L.After repeated tests and determine that the antagonistic strains have steadily ability of producing protease.According to morphological characteristics,physiological and biochemical characteristics and 16S r RNA gene sequence analysis,GQJK2,GQJK4 and GQJK6 are Bacillus subtilis,GQJK17 is Bacillus atrophaeus,GQJK49 is Bacillus velezensis.We optimized the culture medium with bean sprouts as the basic culture medium for GQJK2,GQJK4,GQJK17 and GQJK49.The suitable carbon source,nitrogen source and inorganic salt were determined by single factor experiments.In the single factor experiment,carbon sources we used were sucrose,glucose,lactose,corn flour,soluble starch.Organic nitrogen sources we used were beef extract,peptone,yeast powder,soybean meal.Inorganic nitrogen sources we used were（NH₄）₂SO₄,NH₄NO₃,NH₄Cl,urea.Inorganic salts we used were MgSO₄,CaCO₃,K₂HPO₄,KH₂PO₄.Then we got the optimum medium for each strain by orthogonal test.The optimal medium for GQJK2 was:3%soluble starch,1.5%soybean meal,0.3%NH₄NO₃ and 0.3%of KH₂PO₄.The optimal medium for GQJK4 was 3%sucrose,1.5%soybean meal,0.3%NH₄NO₃,0.3%K₂HPO₄.The optimal medium for GQJK17 was3%glucose,1.5%soybean meal,0.3%NH₄NO₃,0.3%MgSO₄.The optimal medium for GQJK49 was:3%glucose,1.5%soybean meal,0.3%（NH₄）₂SO₄,0.3%K₂HPO₄.The number of colonies was significantly improved by culturing the strain with the optimized medium.GQJK2 colonies increased from 3.83×10⁸cfu/m L to 2.58×10⁹cfu/mL,GQJK4 colonies increased from 3.25×10⁸cfu/mL to 2.76×10⁹cfu/mL,GQJK17 colonies increased from3.21×10⁸cfu/m L to 7.98×10⁹cfu/m L,GQJK49 colonies increased from 2.08×10⁸cfu/m L to1.58×10⁹cfu/m L.The biocontrol effect of 12 antagonistic strains against root rot of Lycium barbarum L.was tested in field testing.Bacillus subtilis GQJK2,Bacillus atrophaeus GQJK17,Bacillus velezensis GQJK49 have significant antagonistic effects.The experimental results showed that compared with the control group,the incidence of root rot was reduced by 55,48.33 and43.33 percentage points,respectively,and the yields of Lycium barbarum L.were increased by 550%,484%and 434%,respectively.The whole genome sequence of Bacillus subtilis GQJK2 was performed.The genome size of GQJK2 was 4,072,961bp,the GC content was 43.76%,the number of coding genes was 4,190,including 4,069 coding protein genes,30 rRNA,86 tRNA,5 sRNA.Repeated sequence,transposon analysis and CRISPR analysis were carried out,and one possible CRISPR was found in the genome of GQJK2.By comparing with COG database,the gene was divided into 21 categories.By comparing with the GO database,the gene was divided into 38 categories,including 7 categories related to cell components,10 categories related to molecular function,21 categories related to biological processes.By comparing with the KEGG database,the gene was divided into 40 types,including 4 types of related to cellular processes,3 types of related to environmental information processing,4 types of related to genetic information processing,8 types of associated with human disease,and 12 types of metabolism,and 9 types of biological systems.We also made analyses for specific function,including carbohydrate active enzyme（CAZy）annotation analysis,transport protein annotation analysis,secretory protein prediction,secretion of the system protein prediction,secondary metabolic gene cluster analysis.In the secondary metabolites gene cluster analysis,two of the 10 secondary metabolites genes clusters of GQJK2 were highly similar to those of the previously reported gene cluster.The whole genome sequence of Bacillus atrophaeus GQJK17 was performed.The genome size of GQJK17 was 4,325,818bp,the GC content was 43.26%,the number of coding genes was 4,487,including 4,376 coding protein genes,18 rRNA,84 tRNA,9 sRNA.By comparing with COG database,the gene was divided into 21 categories.By comparing with the GO database,the gene was divided into 41 categories,including 9 categories related to cell components,10 categories related to molecular function,22 categories related to biological processes.By comparing with the KEGG database,the gene was divided into 40types,including 4 types of related to cellular processes,3 types of related to environmental information processing,4 types of related to genetic information processing,8 types of associated with human disease,and 12 types of metabolism,and 9 types of biological systems.We also made analyses for specific function,including carbohydrate active enzyme（CAZy）annotation analysis,transport protein annotation analysis,secretory protein prediction,secretion of the system protein prediction,secondary metabolites gene cluster analysis.In the secondary metabolites gene cluster analysis,Among the 14 secondary metabolites gene clusters of GQJK17,one gene cluster showed high degree of similarity between the predecessors reported.The whole genome sequence of Bacillus velezensis GQJK49 was performed.The genome size of GQJK49 was 3,929,760bp,the GC content was 46.498%,the number of coding genes was 4,049,including 3,936 coding protein genes,27 rRNA,86 tRNA.By comparing with COG database,the gene was divided into 22 categories.By comparing with the GO database,the gene was divided into 44 categories,including 14 categories related to cell components,13 categories related to molecular function,17 categories related to biological processes.We also made analyses for specific function,including carbohydrate active enzyme（CAZy）annotation analysis,and secondary metabolites gene cluster analysis.In the secondary metabolites gene cluster analysis,12 secondary metabolites gene clusters were predicted in GQJK49.Six clusters had a similarity of 100%to the previously reported gene cluster.