Screening And Biologyical Effects Of B8 Antagonistic Bacteria Against Ralstonia Solanacearum

Due to continuous cropping,single cultivars,improper planting mode and management,the tomato bacterial wilt disease is very serious,which has a great influence on the sustainable development of tomato planting.Biological control is highly widely used because of its friendly environment.In this paper,an antagonistic bacterial strain B8 was screened from the rhizosphere soil of healthy tomato.The fermentation conditions and physiological and biochemical characteristics were studied.The interaction between the strain B8 and the pathogen Ralstonia solanacearum was investigated by the transcriptional analysis The biocontrol effect of the strain B8 was estimated by pot and field test.The results are as follows:1.The antagonistic strain B8 was screened from the rhizosphere soil of healthy tomato plants in the disease field.Through physiological and biochemical and sequence analysis of 16S rRNA gene(Genbank serial number KX961177),B8 strain was preliminarily identified as Bacillus amyloliquefaciens.B8 strain could produce some antibiotics,such as polymyxin B,actinomycosis,bacitracin and tetracycline and showed strongly antagonistic against Ralstonia solanacearum and Fusarium oxysporum.B8 strain can also produce plant growth promotion compounds such as IA A.The optimal culture conditions of strain B8 were as follows:30℃,50ml(250ml bottle)of liquid media,the initial pH 7.0,starch as the carbon source and peptone as nitrogen source.2.The results of soil culture experiment showed that the B8 strain had better colonization ability and suppression to the Ralstonia solanacearum in the soil.In the hydroponic culture test,the B8 strain showed a well promoting effect on growth of tomato plants.3.Results of pot experiment showed that the different use ways of antagonistic strain B8 significantly affected its colonization and biocontrol effects.The colonization number of the antagonistic strain B8 in the treatment of ZB8,TZB8 and TDZB8 was significantly different,and the colonization numbers in TDZB8 treatment was greater than in TZB8 treatment.The colonization number of TZB8 was greater than that of the processed ZB8.The addition of biochar and starch could improve the colonization number of strain B8 in tomato rhizosphere soil.Application of organic fertilizer and amino acid fertilizer also promoted the colonization of strain B8,reaching 4.8 x 106cfu/g dW soil,2.1 x 106cfu/g dW soil respectively.The higher colonization of the strain B8 in application of amino acid liquid fertilizer and organic fertilizer significantly inhibited the number of R.solanacearum.The biocontrol efficiency of tomato bacterial wilt in treatments of applying amino acid liquid fertilizer and organic fertilizer could reach 80%and 77%respectively in pot experiment.In the field experiment,the colonization of the antagonistic strain B8 in the rhizosphere soil could reach 106cfu/g dW soil,indicating that the strain B8 could be well colonized in the rhizosphere of tomato plant and effectively inhibited the growth of Ralstonia solanacearum,and effectively prevented the occurrence of tomato bacterial wilt.4.Detection of antibiotic synthesis genes by PCR showed that antagonistic strain B8 has the encoding genes of antagonistic substance synthesis such as Iturin,Surfactin,Fengycin,Bacillomycin and Biotin.When the pathogen R.solanacearum and B8 strain were cocultured,the growth of pathogen R.solanacearum is inhibited obviously,while the growth of B8 strain showed little effect.The transcriptome analysis results showed that the B8CK vs B8,there were total 1285 up-regulated genes and 2183 down regulated genes.There were 795 significant difference genes,in which there were 248 up-regulated and 547 down-regulation.RS vs RSCK samples,there were total 1275 up-regulated genes and 2323 down regulated genes.There were 218 significant difference genes,in which there were 62 up-regulated genes and 156 down-regulated genes.The expression of most antibiotic synthesis genes in strain B8 and the most of pathogenetic genes in R.solanacearum were inhibited in cocultured condition.The results showed that not only the antagonistic strains could affect the expression of the pathogenic genes,but the pathogen also affected the expression of the antibiotic synthetic genes in the antagonist strain.5.The mutation of strain B8 by UV-NTG obtained mutant strain B8-1 which showed higher antagonistic ability than wild strain B8.The best medium of strain B8-1 is as following,soluble starch 20.00g/L,sucrose 20.00g/L,yeast powder 5.00g/L,peptone 30.00g/L,KH=PO4 5.00g/L,MgS04·7H2O 2.00g/L,pH value 7.0,30℃ incubation time 24h.