防控小麦禾谷孢囊线虫病芽胞杆菌筛选及生防机制研究

发布时间：2018-06-26 14:52

本文选题：小麦禾谷孢囊线虫病 + 生物防治　；参考：《中国农业大学》2015年博士论文

【摘要】：禾谷孢囊线虫又名燕麦孢囊线虫(Heterodera avenae Wollen-weber1924, Cereal Cyst Nematodes,简称CCN),为世界性禾谷类作物病原线虫,现已广泛分布于我国16个省市(自治区、直辖市)小麦产区。近几年土壤虫量逐年增加,病害逐年加重,造成小麦严重减产,对小麦生产构成威胁,现在已经成为小麦生产上的主要防治对象。该病害发生在小麦根部,导致防治难度加大。由于化学剧毒农药的限制使用和抗原缺乏,目前还未找到对该病害有效的防治方法,对该病害芽胞杆菌的生物防治也未见报道。本项研究主要从生物防治角度出发,通过筛选对小麦禾谷孢囊线虫病具有防控效果的有益芽胞杆菌,评价其室内、盆栽和田间病害防治效果,并对菌株进行了鉴定,探索了生防芽胞杆菌小麦禾谷孢囊线虫病的生防机制,结果表明：本研究从小麦根内和根际土壤中共分离得到293株芽胞杆菌,通过室内快速筛选获得8株对小麦禾谷孢囊线虫J2具有稳定杀线活性的芽胞杆菌,分别是菌株BMT19、B202、BMT20、BMT8、 YLT24、YCG3、YCG4和BMT11。通过温室盆栽复筛和3年田间防效验证,筛选出菌株B202具有稳定防治小麦禾谷孢囊线虫病效果,田间最高防效达到63.6%。菌株B202可以促进小麦植株和根系生长,增加了小麦植株根系对土壤中全氮、速效P和速效K的吸收。通过Biolog检测碳源利用和16S rDNA基因序列分析,鉴定菌株B202为短小芽胞杆菌(Bacillus pumilus),命名为为短小芽胞杆菌(Bacillus pumilus) B202。菌株B202可以形成生物膜,能够产生蛋白酶、嗜铁素等多种生防相关物质。通过双抗标记法检测菌株B202在小麦根际定殖定殖情况,结果表明菌株B202可以在小麦根际土壤定殖65天以上,定殖量维持在107CFU/g；菌剂B202应用到田间小麦根部后,对根际土壤线虫群落组成成分无明显改变作用,对种群优势度有调节作用,调节优势属双胃属Panagrolaimus为常见属,稀有属短体属Pratylenchus上升为次优势属。施用菌剂B202后增加了根际土壤中线虫总丰度,提高了食细菌类群线虫、食真菌类群线虫结构比例,对植物寄生类群线虫和杂食/捕食类群线虫有镇压作用。施用菌剂B202后,线虫多样性指数中香农一威纳指数(H’)无明显变化,营养类群指数(TD)下降,优势度指数(Ig)呈升高趋势,植物寄生线虫指数(PPI)呈下降趋势；采用DGGE-PCR技术对田间应用菌株B202后小麦根际土壤中细菌真菌多样性进行了检测,结果表明施用菌株B202后提高了土壤细菌类群中绿弯菌门(Chloroflexi)、芽单胞菌门(Gemmatimonadetes)、拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)和酸杆菌门(Acidobacteria)的菌群数量,增加了土壤真菌优势菌群中新丽鞭毛菌门(Mastigomycota)和子囊菌门(Ascomycotina)的菌群数量,降低了接合菌门(Zygomycota)的菌群数量；通过柱层析和HPLC制备技术分离纯化获得主要存在于菌株B202发酵上清液杀线活性物质1-16和3-11,结合HPLC-MS和NMR测定,鉴定1-16为：邻苯二甲酸二丁酯,分子式为：C16H2204,鉴定3-11为：4,4'-二苯甲烷二氨基甲酸甲酯,分子式为：C17H18N2O4。两种化合物为首次在短小芽胞杆菌中发现。
[Abstract]:Cereal cyst nematode, also known as Heterodera avenae Wollen-weber1924 (Cereal Cyst Nematodes, CCN), is a worldwide cereal crop nematode, and is widely distributed in wheat producing areas in 16 provinces and municipalities (autonomous regions and municipalities) in China. In recent years, the amount of soil insects increased year by year, and the disease was aggravated year by year, causing serious reduction of wheat. Production, which poses a threat to the production of wheat, has now become the main control object in wheat production. This disease occurs at the root of wheat, which leads to more difficulty in prevention and control. Due to the limited use of chemical toxic pesticides and the lack of antigen, the effective prevention and control method for the disease has not been found, and the biological control of the bacillus is also not found. This study mainly from the biological control point of view, through screening the beneficial bacillus that has the control effect on the wheat cereal cyst nematode disease, evaluating the effect of the prevention and control of the indoor, potted and field diseases, and the identification of the strains, and exploring the biocontrol mechanism of the Bacillus spore bacillus cereus cyst nematode disease.
In this study, 293 strains of Bacillus were isolated from Magene and rhizosphere soil, and 8 strains of Bacillus with stable nematode activity to wheat cereal cyst nematode J2 were screened through indoor rapid screening. The strain BMT19, B202, BMT20, BMT8, YLT24, YCG3, YCG4 and BMT11. were screened by greenhouse potted rescreening and 3 year field effectiveness verification. The strain B202 has a stable effect on the control of wheat cereal cyst nematode disease. The highest efficiency of 63.6%. strain B202 in the field can promote the growth of wheat plant and root, and increase the absorption of total nitrogen in the soil, the available P and the available K by the root of the wheat plant. The strain B202 is identified by the use of Biolog detection carbon source and the sequence analysis of 16S rDNA gene. Bacillus cereus (Bacillus pumilus) is named Bacillus pumilus (B202.).
Strain B202 can form a biofilm to produce a variety of biocontrol related substances, such as protease, ferric, and so on. The colonization of strain B202 in wheat rhizosphere was detected by double resistance labeling. The results showed that the strain B202 could be colonized for more than 65 days in the rhizosphere soil of wheat and the colonization of the strain was maintained at 107CFU/g. After the inoculant B202 was applied to the root of wheat in the field, the strain B202 was applied to the root of wheat in the field. There was no obvious change in the composition of the rhizosphere soil nematode community, which had a regulating effect on the population dominance, and the regulatory dominance of the genus bis Panagrolaimus was the common genus, and the rare genus Pratylenchus increased to the secondary dominant genus. The application of microbial inoculant B202 increased the total abundance of line worms in the rhizosphere soil and improved the bacteria eating nematodes and the edible fungi. The structure ratio of the group nematode has a repression effect on the plant parasitic nematode and the omnivorous / predatory nematode. After the use of B202, the Shannon one Wiener index (H ") of the nematode diversity index has no obvious change, the nutrient group index (TD) decreases, the dominance index (Ig) shows a rising trend, the plant parasitic nematode index (PPI) shows a downward trend, and uses DG. The bacterial and fungal diversity in the rhizosphere soil of wheat after field application of strain B202 was detected by GE-PCR technology. The results showed that after the application of strain B202, the phylum green gate (Chloroflexi), butoomonas (Gemmatimonadetes), Pseudomonas (Bacteroidetes), deformable bacteria gate (Proteobacteria) and acid bacilli (Acidobac) in soil bacteria groups were improved. The number of teria bacteria increased the number of Mastigomycota and Ascomycotina in the dominant fungi group of the soil fungi, and reduced the number of bacteria groups of the conjugative bacteria gate (Zygomycota). The isolation and purification by column chromatography and HPLC preparation were mainly found in the nematoactive substance 1- of the strain B202 fermentation supernatant. 16 and 3-11, in combination with HPLC-MS and NMR, 1-16 were identified as: dibutyl phthalate, the molecular formula was C16H2204, and 3-11 was identified as 4,4'- two methanate two carbamate, and the molecular formula was found for the first time in Bacillus cereus, C17H18N2O4. two compounds.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S476.1

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