Prevention Effects On Major Cotton Soilborne Diseases And Itu Gene Cloning Of Bacillus Subtilis S37,S44

Object:To make clear the prevention effect of S37 and S44 to major soilborne disease of cotton and the control mechanism.Methods:Pot trail, plot and field experiment to appraise the prevention effects of S37 and S44 to control cotton Rhizoctonia solami-induced damping-off disease and cotton Verticillium wilt. The two bacteria were identified by morphology and molecular biology. The antagonistic functional protein from S37 and S44 were separated,,purificatied and physiological and biochemical characteristics were studied.Through specific primers of Bacillus subtilis functional gene reported recently, The relevant functional gene was searched by polymerase chain reaction.Results:1.The control efficacy of S44 was better than FUDUOJIA, its control efficacy was57%.The control efficacy of S37 was 47.8%. The field trials of antagonistic strain S37 indicated that the control efficacy to cotton Verticillium wilt was 51.3% in regiment 143 and 55.1% in regiment 147, increased yield 7.43% and 17.62%, respectively. The field trials of antagonistic strain S44 indicated that the control efficacy to cotton Verticillium wilt was 35.2% in regiment 143 and 39.3% in regiment 147, increased yield 5.56% and 15.51%, respectively.2.Physiological and biochemical characteristics of S44 crude protein were studied. The results show that S44 crude protein was thermostable for it had strong inhibitory activities after treated at 120℃for 30min.Proteinase K had no effect on its activity. The inhibitory titer of protein exposed uder ultraviolet radiation was similar to visiable light. The antagonistic effect of protein treated with various kinds of organic solvent was displayed that it was stable in methanol,ethnol, acetone, ethyl acetate and so on. Several pathogens were strongly inhibited by the protein, such as sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, fusarium oxysporum f.sp.vasinfectum, fusarium oxysporum f.sp.niverum, verticillium dahliae kelb, alternaria solani etc.3.In polymerase chain reaction,genomic DNA of strain S44 as a template,the five specific PCR primers was designed by literature.We detected the presence of iturin gene (itu44) in strain S44 by iturin gene specific primers.Conclusion:1.Morphological and 16S rDNA sequence analysis were made to identify the two antagonistic bacteria.Results indicated that both S37 and S44 were Bacillus subtilis.2.The ituD fragment (itu44) was used to blast-search related sequences from GenBank database. The itu44 had high sequence homology with iturin gene from the Bacillus subtilis (84%), which confirmed the presence of iturin gene similar to above iturin in strain S44. By alignment and selection of highly conserved regions of the homologous sequences, one pair of primers was designed to amplify the open reading frame (ORF) of the iturin from strain S44 by PCR. The results revealed that the itu44 ORF was consisted of 942 nucleotides encoding a protein of 314 amino acid residues.