权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

利用枯草芽孢杆菌开发环保多功能微生物农药

基本信息

批准号：
11450312
负责人：
SHODA Makoto
金额：
$ 8.51万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

(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

(1)B类芽孢杆菌化学农药抗性菌株的选育筛选出氟酰胺和氟磺酰胺两种化学农药。B。发现枯草杆菌RB 14-C对100 mg/l氟酰胺具有抗性。从B中筛选出对10 mg/l氟磺酰胺具有抗性的菌株。枯草杆菌NB 22的突变。这些化学农药抗性菌株对植物病原菌的抑菌谱与亲本菌株基本一致。将抗氟酰胺菌与氟酰胺菌共用，在盆栽条件下进行番茄立枯病的防治试验，氟酰胺菌与氟酰胺菌共用后，氟酰胺菌的用量仅为氟酰胺菌单独使用时的1/4。抗性突变体，其中两种培养基都被棒病根的真菌孢子侵染。在商业培养基中观察到100%的病害发生，但在商业培养基中观察到100%的病害发生。 ...更多信息在新开发的培养基中观察到疾病发生的显著减少。利用青花菜根肿病严重的田间，将抗氟磺酰胺突变体细胞与新开发的培养基组合，可有效地减少病害的发生。(2)B对水稻立枯病和根腐病的抑制作用RB 14-C培养物灌土或包衣与氟酰胺处理的组合对番茄立枯病的发生有较好的抑制作用。用RB 14-C培养液浸根对黄瓜根腐病有较好的抑制作用。(3)B抗表面活性素基因的克隆在B，subtilis 168中进行转座子诱变，并分离出表面活性素敏感的突变株801。转座子插入区域的分析表明，yerP是表面活性素自身抗性的决定因素。YerP与RND家族具有同源性，RND家族是仅在革兰氏阴性菌株中鉴定的质子动力依赖性外排泵。由168构建的yerP缺陷型菌株802显示出与对吖啶黄和溴化乙锭的耐药性相关的性质。当通过引入在168中突变的功能性sfp将菌株802转化为表面活性素生产者时，该yerP缺陷型菌株产生表面活性素，尽管表面活性素产量显著降低。yerP是在革兰氏阳性菌株中发现的第一个RND样基因，并且被认为参与表面活性素的外排。(4)鉴定并克隆了RB 14的伊枯草菌素A合成酶操纵子，并测定了其序列。iturin A操纵子跨越超过38 kb的区域，并且由四个ORF组成，ituD、ituA、ituB和ituC。基因ituD编码假定的丙二酰辅酶A转酰酶。第二个基因ituA编码一个449 kDa的蛋白质，该蛋白质具有与脂肪酸合成酶、氨基酸转移酶和肽合成酶同源的三个功能模块。第三，ituB，和第四，ituC，编码609 kDa和297 kDa的肽合成酶，港口四个和两个氨基酸模块，分别。iturin A操纵子和mycobtilin操纵子的氨基酸序列的比较显示ItuD、ituA和ituB分别与每个对应基因fenF（89%）、mycA（79%）和mycB（79%）具有高同源性。(5)几丁质酶基因在B.枯草杆菌将库尔氏菌几丁质酶基因转化到B中。枯草芽孢杆菌和重组B，subtilis均能抑制红曲霉菌丝生长。索尔奈少