Isolation And Identification Of Tomato Rhizosphere Bacteria And Screening Of Antagonistic Bacteria Against Soil-borne Diseases

Most microbial species inhabiting natural environments have not been isolated yet.One of the main issues is that different organisms require a different set of nutrients in varying concentrations and formation.Therefore,exploring the cultivation conditions of different nutrient environments will contribution to develop culture-dependent microbial resources from the natural environment.In this study,the rhizosphere soil samples of 6 healthy tomato plants collected from tomato fields with serious soilborne diseased in China were used as experimental materials.At firstly,we compared the bacterial communities of the media types based on nutrient level to isolated bacteri and analyzed the microbial community differences among the tested culture media.Furthermore,we focused on screening antagonistic potential of isolates recovered from the rhizosphere soil of tomato,which could be used to control common soil borne diseases.Finally,the novel species were identified in polyphasic approaches.The main results are as follows:1.294 strains of bacteria were isolated and cultured from healthy tomato rhizosphere soil in different media,including 3 phyla(Firmicutes,Actinobacteria and Proteobacteria),21 families,26 genera.To evaluate the culturability of microorganisms on the effects of different media,a greater diversity of isolates was obtained on DTS and PYG media than R2A,SE and WA media across all environmental sources.The Shannon and Simpon’s diversity indexes indicated that DBT and DXF had most diverse culturable bacteria,but the diversity of QSY is lowest.2.In this study,we found that 14.47%of test strains had antagonism to Fusarium graminearum through the plate-promoting experiment,4.93%to Phytopathora nicotianae,10.2%to Clavibacter michiganensis subsp.michiganensis,7.56%to Ralstonia solanaecearum and 7.89%to Pseudomonas syringae pv.tomato.3.Based on16S high-throughput analysis of the DXF,HY and WXZ regions,Proteobacteria was found to be the most abundant phyla in each region,contained 12 genera.At least 229 genera were detected in 16S rRNA gene amplicon sequencing but only 26 genera were cultivated by using different media.4.Two potential novel species were isolated from SE medium and WA medium,assigned to the genus Rhizobium,Paenibacillus.1)The strain DBTS2T was characterized using the polyphasic taxonomic approach:Blast and Phylogenetic analysis using 16S rRNA gene sequences showed this strain belonged to the genus Rhizobium and was most closely related to Rhizobium subbaraonis JC85T(99.1%)and Rhizobium daejeonense CCBAU 10050T(97%).In phenotypic characterization,cells of strain DBTS2T were Gram-negative,short rod,aerobic and non-motile.This novel strain was found to grow at 20-45℃(optimum 25-37℃),pH 5-9(optimum 8)and in the presence of 4%NaCl.It was positive for catalase and oxidase.The predominant cellular fatty acids were Summed Feature 8(52.7%)and C19:0 cyclo ω8c(23.3%).The polar lipids of strain DBTS2T consisted of diphosphatidylglycerol(DPG),phosphatidylglycerol(PG),phosphatidylethanolamine(PE),unidentifed aminophospholipid(APL),unidentifed aminolipid(AL),four unidentifed phospholipids(PL),unidentifed lipid(L),phosphatidylcholine,unknown glycolipid and unknown aminophosphoglycolipids.Q-10 was the major quinone.The DNA-DNA hybridization similarity values between the strain DBTS2T and R.subbaraonis JC85T,R.daejeonense CCBAU 10050T and Rhizobium azooxidifex DSM100211T were 46.4%,20.7%and 25.5%,respectively.The ANI value was 91.96%between strain DBTS2T and R.subbaraonis JC85T and 75.18%between strain DBTS2T and R.daejeonense CCBAU 10050T.The DNA G+C content of the genomic DNA was 63.1 mol%.The type strain is DBTS2T(=CICC 24887T=ACCC61707T=JCM 34245T),for which the name Rhizobium rhizolycopersici sp.nov.is proposed.2)The strain DXFW5T was characterized using the polyphasic taxonomic approach:Phylogenetic analysis using 16S rRNA gene sequences showed this strain was most closely related to Paenibacillus timonensis DSM 16943T(98.77%),Paenibacillus barengoltzii DSM 22255T(98.31%)and Paenibacillus macerans DSM 24T(98.07%),respectively.In phenotypic characterization,cells of strain was Gram-positive,rod-shaped,motile,endospore-forming.Strain DXFW5T grew at 20℃-50℃(optimum 25℃-37℃),pH 5-8(optimum 7)and in the presence of 3%NaCl.It was positive for catalase and oxidase.The major fatty acids were anteiso-C15:0(55.1%),iso-C16:0(13.2%)and C16:0(10%);minor fatty acids are anteiso-C17:0(6.1%),iso-C15:0(5.4%),anteiso-C13:0(0.5%)and iso-C17:0(1.9%).The polar lipid of strain DXFW5T consisted of diphosphatidylglycerol(DPG),phosphatidylglycerol(PG),phosphatidylethanolamine(PE),unidentified aminophospholipid(APL),unidentified aminolipid(AL),unidentified phospholipid(PL),unidentified lipid(L).The MK-7 was the major quinone.The DNA G+C content of the genomic DNA was 52.9 mol%.The digital DNA-DNA hybridization value between strain DXFW5T and P.timonensis DSM 16943T,P.barengoltzii DSM 22255T and P.macerans DSM 24T were 33.1%,24.9%and 21.2%,respectively.The ANI value between strain DXFW5T and P.timonensis DSM 16943T,P.barengoltzii DSM 22255T and P.macerans DSM 24T were 86.93%,81.77%and 75.98%,respectively.The type strain is DXFW5T(=ACCC 61751T=JCM 34488T),for which the name Paenibacillus rhizolycopersici sp.nov.is proposed.