棉花黄萎病拮抗芽孢菌的筛选及其活性物质研究

发布时间：2018-05-11 04:21

本文选题：棉花黄萎病 + 大丽轮枝菌　；参考：《新疆师范大学》2017年硕士论文

【摘要】：棉花黄萎病是由大丽轮枝菌(Verticillium dahliae)引起的土传性维管束病害,是我国棉花生产危害最严重的真菌病害。寻找对其有效的防治方法对于我国棉花高产稳产具有重大意义。本文从新疆连作棉田棉花根际土壤中分离筛选出大丽轮枝菌拮抗芽孢细菌,探究拮抗菌的抑菌特性和拮抗机理,为新疆棉花黄萎病的生物防治提供菌株资源和理论指导。试验共分离到146株芽孢细菌,29株能拮抗大丽轮枝菌,9株拮抗性极强;经鉴定,这9株菌中J-02和J-29属短小芽孢杆菌(Bacillus pumilus),J-15和其他菌株与枯草芽孢杆菌(B.subtilis)相近。J-02热稳定性较好,pH耐受范围在5-9,培养至衰亡期的抑菌效果最好;J-02能抑制大丽轮枝菌芽管伸长形成菌丝;在盆栽试验中对棉花黄萎病也表现出明显防治作用。J-15在盆栽试验中显示出对棉花黄萎病的防治效果。硫酸铵盐析粗提J-15抑菌物质结果显示,80%硫酸铵饱和度提取效果最好。J-15对温度敏感,100℃可致菌体死亡,但其抑菌物质经100℃处理1 h后活性仅降低18.3%;J-15经pH7-9处理后抑菌性与对照组无显著差异,而其抑菌物质能耐受pH 2-10,且pH 2-4能完全沉淀抑菌物质,却不影响抑菌活性;J-15培养至稳定期抑菌性最强,而摇菌12-24 h,OD600=1.7以上为抑菌物质最佳提取时间;此外,J-15抑菌粗提物对胃蛋白酶、蛋白酶K、紫外照射和丙酮都不敏感。利用pH 2酸沉淀后丙酮抽提J-15抑菌物质,进行SDS-PAGE电泳检测和光谱扫描,并利用DEAE-Sepharose Fast Follow阴离子交换层析纯化后质谱鉴定。结果显示,J-15粗提抑菌物质经丙酮沉淀去除非活性蛋白后,电泳未检测到特异蛋白;在210 nm和270 nm处有吸收峰,280 nm处光吸收值较低;采用0.1 mmol/L Tris-HCl缓冲溶液(pH=6.5),0-1 mol NaCl线性洗脱得到较纯抑菌物质,在280 nm不具吸光性;质谱鉴定J-15抑菌物质分子量可能为828 Da。J-15抑菌物质对大丽轮枝菌孢子萌发、菌丝生长和防御酶活力影响的研究结果表明,J-15抑菌物质低浓度抑制孢子萌发,高浓度可致萌发力丧失;平板试验发现抑菌物质可致菌丝短粗畸形,产孢能力降低;与对照组相比,J-15抑菌物质可抑制大丽轮枝菌SOD、POD和CAT活力,且导致MDA含量较对照高。表明J-15抑菌物质的作用机制主要表现在,抑制孢子萌发,菌丝伸长,并造成大丽轮枝菌的抗氧化系统和膜系统的损伤。
[Abstract]:Cotton Verticillium wilt is a soil borne vascular disease caused by Verticillium dahliae, which is the most serious fungal disease in China's cotton production. It is of great significance to find the effective control method for the high and stable yield of cotton in our country. This paper isolated and screened the Dali wheel from the cotton rhizosphere soil of Xinjiang continuous cropping cotton field. Mycobacteria antagonized the spore bacteria, explored the antimicrobial properties and antagonistic mechanisms of the antagonistic bacteria, provided strain resources and theoretical guidance for the biological control of Verticillium Wilt in Xinjiang. 146 strains of spores were isolated, 29 strains could antagonize Dali and 9 strains were extremely resistant; the 9 strains of J-02 and J-29 were identified as Bacillus pum Ilus), J-15 and other strains and Bacillus subtilis (B.subtilis) similar to.J-02 thermal stability, pH tolerance range of 5-9, culture to the decay period of the best bacteriostasis effect; J-02 can inhibit the elongation of the bud tube of rhizo Dali to form mycelium; in pot experiment, the cotton Verticillium wilt also showed obvious control effect.J-15 in pot experiment. The results of the prevention and control of Cotton Verticillium wilt. Ammonium sulfate salting out J-15 bacteriostasis results showed that the best extraction effect of 80% ammonium sulfate was.J-15 sensitive to temperature, and 100 C could cause the death of the bacteria, but the activity of the bacteriostat was only 18.3% after 1 h treatment at 100, and there was no significant difference between the J-15 and the control group after pH7-9. Bacteria can tolerate pH 2-10, and pH 2-4 can completely precipitate bacteriostatic substances, but do not affect bacteriostasis activity; J-15 culture to stable period is the most bacteriostasis, while the bacteria 12-24 h, OD600=1.7 above the best extraction time of bacteriostat; in addition, J-15 antibacterial crude extracts are not sensitive to pepsin, egg white enzyme K, ultraviolet radiation and acetone. Use pH 2 acid precipitation J-15 bacteriostat was extracted by acetone, detected by SDS-PAGE electrophoresis and spectrum scanning, and identified by DEAE-Sepharose Fast Follow anion exchange chromatography and purified by mass spectrometry. The results showed that the specific protein was not detected by electrophoresis, and there was a peak of absorption, 280 nm at 210 nm and 270 nm. The light absorption value was low; 0.1 mmol/L Tris-HCl buffer solution (pH=6.5), 0-1 mol NaCl linear elution obtained more pure bacteriostat and no light absorption in 280 nm; mass spectrometry identified that the molecular weight of J-15 bacteriostat may be the effect of 828 Da.J-15 bacteriostat on spore germination, mycelial growth and defense enzyme activity of Dali The low concentration of J-15 inhibited the germination of spores and the loss of germinating force at high concentration; the plate test found that bacteriostatic substances could induce mycelial mycelium deformity and decrease the sporulation ability. Compared with the control group, J-15 inhibitory substances could inhibit the activity of SOD, POD and CAT, and lead to the higher MDA content. The mechanism of J-15 bacteriostasis was shown to be the mechanism of action of J-15 bacteriostat. It is mainly manifested in Inhibiting Germination of spores, elongation of mycelia and damage of antioxidant system and membrane system of Verticillium dahliae.

【学位授予单位】：新疆师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.62;S476.1

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