【作者】 何红；

【导师】 胡方平； 谢联辉；

【作者基本信息】 福建农林大学 ， 植物病理学， 2003， 博士

【摘要】 本研究对辣椒内生细菌及其拮抗菌进行了分离筛选，着重对其中2株分别来自辣椒叶片和茎杆的BS-2和BS-1菌株的鉴定、内生定殖、防病促生作用及其机制，B-2菌株的抗菌物质和发酵条件等进行了研究。 辣椒内生细菌分离结果表明，在所选用的分离条件下，辣椒组织体内含有2.83×10~3～1.35×10~4 cfu／g(fw)的细菌；不同品种或同一品种不同组织器官内，体内细菌的数量有所不同，叶片内细菌的含量最高，果中最少，叶、根、茎和果中细菌含量分别为3.4×10~3～4.5×10~4 cfu／g(fw)、0.75×10~3～1.3×10~3 cfu／g(fw)、0.1×10~3～3.25×10~3 cfu／g(fw)和0.3×10~3～1.8×10~3 cfu／g(fw)。室内拮抗作用测定表明，所获得的108株细菌中，有31株(占28.7％)对香蕉、黄瓜枯萎病等10多种植物病原真菌具有拮抗作用，其中以分别来自辣椒叶片和茎杆中的BS-2和BS-1菌株的拮抗作用最强、最稳定。经形态和生化特征等测定，上述具有拮抗作用的31株细菌均为芽孢杆菌(Bacillus spp．)；其中BS-2和BS-1菌株经Biolog鉴定为枯草芽孢杆菌(B．subtilis)，再经16S rRNA序列分析比较，2菌株进一步鉴定为枯草芽孢杆菌内生亚种(B．subtilis subsp．endophyticus)。 以抗利福平(300μg／ml)和拮抗病原真菌双抗性为标记，测定BS-2和BS-1菌株的内生定殖寄主范围及其在辣椒和白菜体内的定殖动态表明，浸种、灌根和涂抹叶片等方法接种，两菌株均可以进入辣椒、茄子、白菜、甜瓜、西瓜、丝瓜等植物体内定殖，除此之外，BS-2菌株还可以在蕃茄、黄瓜、豇豆、水稻和小麦等植物体内定殖；BS-2菌株的内生寄主范围比BS-1菌株广、内生定殖作用强。室内测定2菌株在辣椒体内和室外田间测定BS-2在白菜体内定殖动态表明，浸种、灌根及涂抹叶片等方法接种后，菌株均可主动进入辣椒和白菜体内定殖，并迅速向接种点上部、未接种的组织器官转移传导，并可在辣椒和白菜体内长期定殖。 室内盆栽和大田小区试验测定菌株对植物生长作用影响表明，BS-2和BS-1菌株对辣椒、白菜等作物有明显的促进生长作用。室内盆栽测定，菌液浸种辣椒，出苗后15d鲜重分别增加146.72％和58.58％；大田小区测定，菌液浸种辣椒苗鲜重分别增加72.02％和63.04％；菌液浸种白菜20d后鲜重分别增加91.20％～138.04％和25.43％～55.43％；BS-2菌株比BS-1菌株的促生作用福建农林大学博士学位论文强。BS一2菌株浸种处理辣椒和白菜后，植株内源激素测定表明，菌株浸种处理后，植株体内的生长素(IAA)、玉米素(ZR4)和赤霉素(GA3)等激素的含量比清水对照高，而脱落酸(ABA)含量比对照减少。菌株进入植株体内后，调节其内源激素的变化可能是菌株促生作用的主要机制之一。 室内防病测定表明，BS一2和BS一1菌株对辣椒炭疽病、白菜炭疽病和香蕉炭疽病均有良好的预防效果。BS一2菌株对辣椒苗、辣椒果、白菜和香蕉炭疽病的防治效果分别为:51.49%一93.34%、50.00/0一100%、45.120/0和71.54%;BS一1菌株对上述各病害的防治效果分别为:66.13%一79.23%、60.0%一100%、68.45%和43.28%。BS一2菌株对植物炭疽病的防病效果比BS一1菌株高。防病机制研究表明，菌株分泌抗菌物质抑制病菌菌丝生长、分生抱子产生和萌发，主动进入植株内生定殖、优先占领病菌入侵位点，促进植株生长、增强寄主生长势;诱导寄主植物过氧化物酶(POD)、超氧化物歧化酶(s OD)、过氧化氢酶(CAf)、苯丙氨酸解氨酶(PAL)等参与的植物防御性反应，降低寄主植物体内的丙二醛(MAD)的产生等，可能是菌株防病的主要机制。 BS一2菌株抗菌物质研究表明，其培养液经硫酸钱盐析，100℃处理30min，可获得热稳定、抗紫外线、能强烈抑制植物病原真菌和细菌、并对辣椒炭疽病具有69.79%(接种后gd)防病效果的抗菌物质;经SDS.PAGE、高效液相色谱(HPLC)、州叭上Dl一TOF质谱等检测，该物质为分子量为落2884.39Da的多肤类物质。 BS一2菌株发酵条件研究表明，以黄豆粉培养基培养、培养基初始pH值6.7(灭菌后)、温度为28℃、培养时间48h及良好的通气条件，为该菌株的最佳发酵条件。更多还原

【Abstract】 The results of isolation by the surface sterilized with 0.1% mercury bichloride showed that 2.83×103~1.35×104cfu/g(fw) endophytic bacteria were obtained from the plants of capsicum; 3.4x103~4.5xl04cfu/g(fw) from leaves, 0.75×10-3-1.3×103 cfu/g(fw) from roots, 0.1×103~3.25×103 cfu/g(fw) from stems and 0.3xl03~1.8xl03 cfu/g(fw) from fruits. A total of 108 endophytic bacteria isolates, 31, or 28.7%, had in vitro inhibitory activity to phytopathogens of fungi, such as Fusarium oxysporum f.sp.cubense, Fusarium oxysporum f.sp.cucumberinum, and Colletotrichum sp.,etc. The strongest inhibitory activities were produced by the BS-1 and BS-2 strains which isolated from the leaf and stem respectively. Based on the basic properties including cell shape, Gram’s stain, sporulation, production of catalase and oxidase, all of the 31 antagonistic isolates were identified as Bacillus spp., BS-1 and BS-2 were further identified as B. subtilis by the Biolog analysis, and based on the colonized in the plants and signature sequences of 16S rRNA, the two strains may be belonging to the B.subtilis subsp. endophyticus.The mutanted by antagonism against the rifampicin, and inoculated by dipping seeds, daubing leaves, and watering roots, the two strains were proved to be able to colonize in the many kinds of plants, such as eggplant, cabbage, muskmelon, watermelon, loofah goard, besides that the BS-2 was able to colonize in more plants which were tomato, cucumber, cowpea, rice and wheat etc. The results also showed that the two strains were able to live and move in the plants of capsicum and the BS-2 in the cabbage plant for long time.The growth of capsicum and cabbage was significantly promoted by BS-2 and BS-1 strains with their cells suspension dipping seeds. Compare with the water control, the fresh weight of the 15d old capsicum seedling was increased 146.72% and 58.58% respectively tested in the pots; and 72.02% and 63.04% respectively tested in fields; and that of the 20d old cabbage was increased 91.20%~138.04% and 25.43%~55.43% respectively tested in fields. Inducing the change of plant hormone concentration may be one of the key mechanisms in promoting growth activities of the two strains.The anthracnoses of capsicum seedling and fruits, cabbage and banana, which were caused by Colletotrichum gloeosporioides, C. higgisianum and C. musae respectively, were found effective controlled by the BS-2 and BS-1 strains. The control effects of the BS-2 strain on the above anthracnoses were 81.49%~93.34%, 80.0%~100%> 45.12% and 71.54% respectively; and that of the BS-1 were66.13%~79.23%. 60.0%~100%?68.45% and 43.28% respectively. The results also showed that the secreting antibiotics which had strong inhibitory activity against the pathogens mycelial growth and conidia formation and germination, colonizing in the plants and promoting their growth, and inducing the plants defense responses were proposed to be the key mechanism in the disease control activity of the two strains.The BS-2 antifungal compound obtained through ammonium sulfate precipitation was primarily identified as a polypeptide (MW <=2884.39D) by SDS-PAGE and MALDI-TOF. This antifungal polypeptide of the nature of thermostable and UV-tolerant showed strong inhibitory activity against various plant pathogenic fungi and bacteria, such as Colletotrichum sp. and Ralstonia solanaceance, and had 69.79% control effect (9d after inoculating the pathogen) against the anthracnose on the capsicum postharvest fruits.Optimum fermentation conditions for BS-2 might culture for 48h at 28℃ with the medium made of soybean powder(pH6.7), and increase the oxygen supply as much as possible.更多还原