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

(1)枯草芽孢杆菌化学抗药性菌株的选育筛选出氟虫胺和氟磺胺两种化学农药。枯草芽孢杆菌Rb14-C对10 0 mg/L氟虫腈具有抗性。以枯草杆菌NB22为出发菌株，通过自发诱变，获得了一株耐10 mg/L磺胺的菌株。这些化学农药抗性菌株对植物病原菌的抑制谱与亲本菌株几乎相同。盆栽试验表明，在番茄立枯病的盆栽试验中，抗氟虫胺菌株和氟虫胺共用可使氟虫胺的使用量减少到单独使用时的1/4。100%的疾病发生在商业介质中观察到，但si…在一种新开发的培养基中观察到了更显著的疾病发生减少。在青花菜球根病严重表达的田间试验中，将抗氟磺胺突变体的细胞与新研制的培养基组合使用，可有效地减少番茄的立枯病和根腐病的发生。(2)细小芽孢杆菌Rb14-C对番茄立枯病和根腐病的抑制效果表现出较好的抑制效果。将黄瓜根部浸泡于RB14-C发酵液中，能有效地抑制根腐病的发生。(3)利用枯草杆菌转座子诱变技术，对枯草杆菌耐表面素基因进行了诱变，获得了一株对表面素敏感的突变株801。对转座子插入区域的分析表明，YERP是表面蛋白自身耐药的决定因素。YERP与RND家族有同源性，RND家族是一种依赖质子动力的外排泵，仅在革兰氏阴性菌株中发现。YERP缺陷菌株802由168株构建而成，显示出与阿克里黄素和溴化乙锭耐药有关的特性。当菌株802通过引入168位突变的功能性SFP而转化为表面蛋白产生菌时，该YERP缺陷菌株产生表面素，尽管表面素的产量显著减少。YERP是在革兰氏阳性菌中发现的第一个RND样基因，可能与表面蛋白的外流有关。(4)ITURIN操纵子的克隆鉴定和克隆了RB14的ITU合成酶操纵子，并测定了其序列。Iturin A操纵子大小超过38kb，由ituD、ituA、ituB和ituC四个ORF组成。基因ituD编码一个可能的丙二酰辅酶A转酰酶。第二个基因Itua编码一个449 kDa的蛋白质，它有三个功能模块，分别与脂肪酸合成酶、氨基酸转移酶和多肽合成酶同源。第三个是ituB，第四个是ITUC，分别编码609 kDa和297 kDa的多肽合成酶，分别含有四个和两个氨基酸模块。ItuD、ItuA和ituB分别与对应基因fenF(89%)、myca(79%)和mycB(79%)有较高的同源性。(5)几丁质酶基因在枯草杆菌中的表达。较少

(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