The Iturin A Producted By Bacillus Atrophaeus Strain B44 And Its Effect Of Control And Growth Promotion To Cotton Rhizoctoniosis

Bacillus strain B44 was isolated from the rhizosphere of cotton in the Xinjiang Uyghur Autonomous Region. Laboratory tests indicated that strain B44 had strong antagonism against several soil-borne fungal pathogens. In field tests, strain B44 exhibited significant biocontrol effects to cotton rhizoctoniosis, cotton Verticillium wilt and processing tomato Pythium root rot. The purposes of this research were(i) to identify the classification of strain B44 and(ii) to study the biocontrol mechanisms of B44 against cotton rhizoctoniosis. Strain B44 was confirmed the classification status based on the sequences of the gyr A gene and the whole-genome sequences. The different lipopeptide homologues produced by strain B44 were detected and identified using specific primers of lipopeptide biosynthesis genes, reverse phase high-performance liquid chromatography(RP-HPLC), and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry(MALDI-TOF-MS). The itu C gene, which is related to iturin A synthesis, was cloned from strain B44. A knock-out mutant of the itu C gene was created through homologous recombination. The results clearly indicated that the itu C gene had an important role in the antagonistic activity and biocontrol effect of strain B44, furthermore, iturin A was key antifungi lipopeptide in antagonism of B44 toward cotton rhizoctoniosis caused by R. solani. Single factor and orthogonal experiments were conducted to compare the effects of carbon source, nitrogen source, and mineral salts on the growth(i.e. biomass) of strain B44 and its production of iturin A. The biocontrol mechanisms of strain B44 against cotton rhizoctoniosis were investigated using both pot and field tests. The main results of this dissertation are listed below:1. Bacillus atrophaeus and B. subtilis are phenotypically similar and can be easily confused. The 16 S r RNA gene is used as a framework for conventional bacterial classification, but it often shows limited variation for closely related Bacillus species. In a previous study, B44 was initially assigned to B. subtilis, and it was named B. subtilis S44. However, in this study, the results of two analyses of homology(phylogenetic analysis based on gyr A gene sequences and whole-genome sequence compared with the NCBI gene bank) indicated that the strain belongs to B. atrophaeus. Therefore, it was renamed B. atrophaeus B44.2. Mycosubtilin, fengycin, plipastatin, surfactin and iturin A were detected from strain B44 using specific primers for the detection of lipopeptide biosynthesis genes. The lipopeptide compounds were analyzed using MALDI-TOF-MS analysis. The mass peaks of the B44 samples were detected at m/z 1028.5, 1058.8, 1106.4, 1121.4, 1463.7, 1477.7 and 1505.8. These data, which correspond well to those determined by other authors, indicate the presence of C13-iturin A, C18-mycosubtilin, C15-surfactin, C16-fengycin A and C16-fengycin B. The peak at m/z 1106.4 represents an unknown lipopeptide.3. The itu C gene was amplified from B44 and cloned into the plasmid p UC19. The recombination plasmid was constructed by inserting the tet gene into the p UC19 plasmid containing the ituc gene. Strain B44 containing the recombination plasmid was selected on tet-containing medium. The iturin A production and disease efficacy of the itu C mutant strain were compared with those of the wild-type strain. The results indicated that the itu C mutant strain decreased iturin A production by 75.9% compared with the wild-type strain. The lipopeptide products of B44 and the itu C mutant strain were identified using MALDI-TOF-MS. The mutant did not produce iturin A. Furthermore, the relative abundance of fengycin and surfactin were significantly lower in the mutant than in the wild-type strain. The average disease efficacy of the itu C mutant strain(18.5%) was 60.5% less than that of the wild-type strain(46.7%). In conclusion, the itu C gene has a major impact on the synthesis of iturin A and iturin A is the key antimicrobial substance affecting the antagonism of B44 toward cotton rhizoctoniosis caused by R. solani.4. The effect of C source, N source, and mineral salts on the production of iturin A by strain B44 was tested using RP-HPLC analysis and well-diffusion assays. Beef-extract peptone medium without the addition of C, N, or mineral salts was used as the control treatment. The results showed that iturin A production significantly increased when strain B44 grow on medium containing α-lactose, fructose, or corn flour. Iturin A production decreased when strain B44 grow on medium containing glucose, maltose, galactose, and sucrose. Among these, iturin A production was lowest in the sucrose treatment. The soluble starch and d-xylose treatments had no significant effect on iturin A production. Among the N source treatments, iturin A production significantly increased when strain B44 grow on KNO3. The Na NO3 and pea meal treatments increased iturin A production; however the increases were not statistically significant. Iturin A production decreased in the Ca(NO3)2, NH4NO3, yeast extract, NH4 Cl,(NH4)2SO4, and urea treatments. Among these, iturin A production was lowest in the urea treatment. Among the mineral salt treatments, iturin A production and the antimicrobial activity of B44 both increased significantly in the Mn SO4, Ca CO3, and Cu SO4 treatments and decreased significantly in the Fe SO4, K2HPO4, KCl, KH2PO4, and Zn SO4 treatments. The medium containing Mg SO4 had no significant effect on iturin A production and antimicrobial activity. An orthogonal experiment was conducted to determine the medium composition that maximized the production of iturin A by strain B44. A significant positive correlation was found between the antagonistic activity and iturin A production of strain B44. The maximum iturin A production(204.3 mg/L) was observed when strain B44 was cultured on beef-extract peptone optimum medium containing 10 g/L corn flour, 5 g/L pea meal, 10 g/L fructose, 10 g/L KNO3, 0.05 g/L Mn SO4, and 1.0 g/L Mg SO4.5. Pot and field plot tests were conducted to determine the ability of strain B44 to control rhizoctoniosis. The results indicated that the biocontrol effect of strain B44 was most closely related with its iturin A production. The production of gibberellins and siderophores by strain B44 were also measured. The greater plant growth promotion was most likely attributable to gibberellin concentration. The disease efficacy of B44 grown on the optimum culture medium(identified by orthogonal tests) was 72.8%, which was 10.2% higher than the disease efficacy in KNO3 treatment in the pot plot test. The disease efficacy of B44 grown on the optimum culture medium(identified by orthogonal tests) was 62.3%, which was 9.1% higher than the disease efficacy in KNO3 treatment in the field plot tests. Cotton seeds were treated with culture medium that had been used for the culture of B44. The activities of SOD, POD, and CAT in the cotton seedlings were measured. The results indicated that disease efficacies were higher when the enzyme activities were high. This finding indicated that the disease efficacy of strain B44 was related to the promotion of seedling growth as well as the induction of disease resistance.