Development of environmentally friendly and multifunctional microbial pesticides by using Bacillus subtilis

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(1) Development of chemical pesticide-resistance strains of B. subtilisTwo chemical pesticides, Flutolanil and Flsulfamide were selected. B. subtilis RB 14-C was found to be resistant to 100 mg/l Flutolanil. A strain resistant to 10 mg/l Flsulfamide was selected from B. subtilis NB22 by spontaneous mutation. Those chemical pesticide-resistant strains showed almost the same suppressive spectrum to plant pathogens as the parent strains. Co-utilization of flutolanil-resistant strain and flutolanil were applied to damping-off of tomato in a pot test and the co-utilization of them decreased the amount of flutolanil used to one-fourth of that when flutolanil was alone used.The seedlings of a Chinese cabbage were grown in a commercial seedling culture medium and a newly developed medium containing the cells of flsulfamide-resistant mutant where both media were infested with spores of fungi of club disease root. 100 % of the occurrence of the disease was observed in a commercial medium, but si … More gnificant reduction of disease occurrence was observed in a newly developed medium. The field where the club disease root of broccoli was severely expressed was used and the combination of the cells of flsulfamide-resistant mutant and the newly developed medium was effective to reduce the disease occurrence.(2) Suppression of damping-off and root rot diseases by B. subtlis RB 14-CCombination of pouring of RB14-C culture into soil or seed coating with RB14-C with Flutolanil treatment showed high suppressive effect on the occurrence of damping-off of tomato. Dipping of roots of cucumber in the RB14-C culture broth was effective to suppress the disease of root rot caused by Phmopsis sp.(3) Cloning of surfactin-resistance gene in B. subtilisTransposon mutagenesis was performed in B, subtilis 168, and a surfactin-susceptible mutant strain, 801, was isolated. Analysis of the transposon-inserted region revealed that yerP is the determinant of surfactin self-resistance. YerP has homology with the RND family, which is a proton-motive-force-dependent efflux pump only identified in Gram-negative strains. The yerP-deficient strain, 802, which was constructed from 168, showed properties associated with drug resistance to acriflavin and ethidium bromide. When strain 802 was converted to a surfactin producer by introducing a functional sfp which is mutated in 168, this yerP deficient strain produced surfactin, although surfactin production was significantly reduced. yerP is the first RND-like gene found in Gram-positive strains, and is supposed to be involved in the efflux of surfactin.(4) Cloning of iturin operonThe iturin A synthetase operon of RB14 was identified and cloned, and its sequence was determined. The iturin A operon spans a region of more than 38 kb, and is composed of four ORFs, ituD, ituA, ituB, and ituC. The gene ituD encodes a putative malonyl-CoA transacylase. The second gene, ituA, encodes a 449 kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third, ituB, and fourth, ituC, encode 609 kDa and 297 kDa peptide synthetases that harbor four and two amino acid modules, respectively. Comparison of the amino acid sequences of the iturin A operon and mycosubtilin operon revealed that ItuD, ituA, and ituB have high homologies to each counterpart gene, fenF (89 %), mycA (79 %), and mycB (79 %), respectively.(5) Expression of chitinase gene in B. subtilisThe chitinase gene of Kurthia zopfii was transformed into B. subtilis and the recombinant B, subtilis inhibited the mycelial growth of R. solnai. Less