【作者】 薛鹏琦；

【导师】 高学文；

【作者基本信息】 南京农业大学 ， 植物病理学， 2010， 硕士

【摘要】 芽孢杆菌属细菌(Bacillus spp.)是一种革兰氏阳性,产芽孢杆状细菌,好氧或兼性厌氧,广泛分布在自然环境中。其中,芽孢杆菌产生的内生芽孢对高温、干旱、低温和紫外等不良环境有较高的抗逆性。芽孢杆菌作为一类重要的植物根围促生细菌(plant growth promoting rhizobacteria, PGPR),可以产生各种抑菌物质和诱导植物产生系统获得抗性(induced systemic resistance)防治植物病害。其中,脂肽化合物是芽孢杆菌产生的最主要抑菌化合物,包括伊枯草菌素(Iturin)、表面活性素(Surfactin)和芬枯草菌素(Fengycin)三大类,在植物病害防治过程中发挥着重要的作用。青藏高原为世界上海拔最高的高原,平均海拔4000米以上,由于其独特的极端环境,如低温、缺氧和强辐射等,在该地区可能孕育着特殊的芽孢杆菌。本研究首先对采自西藏地区的35个土样进行芽孢杆菌的分离,共分离得到1388个菌株。以水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae)和油菜菌核病菌(Sclerotinia sclerotiorum)为指示菌,对分离的1375个菌株进行抑菌效果测定,最后得到311株抑菌效果较好的菌株(抑菌圈直径≥5 mm),然后,对311个抑菌效果好分离菌株进行基因组rep-PCR (repetitive extragenic palindromic PCR)指纹图谱筛选,选取指纹图谱不同的菌株,共得到150个菌株。对经过指纹图谱筛选获得的150个菌株进行生理生化鉴定,然后按照Gordon等人描述的经典分类体系将150个菌株分为7个类群,从每个类群中选取代表性的菌株共31个菌株进行脂肪酸甲酯(fatty acid methyl ester, FAME)鉴定。接着对这31菌株进行分子鉴定,首先进行16S rDNA和gyrB基因序列分析鉴定,然后进行基因组rep-PCR指纹图谱鉴定,包括BOX-PCR和ERIC-PCR指纹图谱分析鉴定。综合表型鉴定和一系列分子鉴定的结果,将分离的有生防潜力的31个芽孢杆菌菌株鉴定为短小芽孢杆菌(B. pumilus)11株,蜡样芽孢杆菌(B. cereus)7株,苏云金芽孢杆菌(B. thuringiensis)3株,解淀粉芽孢杆菌(B. amyloliquefaciens)3株,萎缩芽孢杆菌(B. atrophaeus)3株,B. axarquiensis2株,枯草芽孢杆菌(B. subtilis)1株,地衣芽孢杆菌(B. licheniformis)1株。从31个菌株中选择有代表性的11个芽孢杆菌菌株进行脂肽化合物MALDI-TOF-MS分析,发现菌株LLCG23能产生surfactin,bacillomycins D和fengycin,菌株NMTD81能产生脂肽类化合物surfactin, iturin和fengycin,菌株YBWC18,LNXM78能产生surfactin和fengycin,菌株NMTD54和GBSW11均能产生bacillomycins L和fengycin两种脂肽类化合物,菌株LSSC22能产生iturin和fengycin两种脂肽类化合物,菌株GBSW2和菌株GBSW19产生了surfactin,菌株DJFZ40只检测到脂肽化合物bacillomycins D,菌株菌株NMSW23在m/z为200-2000的范围内,没有检测到有抑菌活性的脂肽类化合物。并对分离得到的两株对油菜菌核病菌抑菌效果好的菌株YBWC43 (Bacillus amyloliqufaciens)和RJGP18 (Bacillus atrophaeus)进行脂肽类化合物分析发现菌株YBWC43产生BacillomycinD和fengycin,菌株RJGP16产生脂肽化合物surfactin和fengycin,对这两个菌株在油菜离体叶片上进行生防活性检测,菌株YBWC43和RJGP16对油菜菌核病菌的防治效果分别为100%和50.24%。更多还原

【Abstract】 The genus Bacillus is comprised of Gram-positive, spore-forming, and rod-shaped organisms with an aerobic or facultatively anaerobic metabolism, are ubiquitous in many environments. And their endospores are highly resistant to hostile physical and chemical conditions, such as high temperature, low temperature, arid and UV. The Bacillus spp., which is one of the most important plant growth promoting rhizobacteria(PGPR), can also produce various antibacterial substances or elicit induced systemic resistance of plant. And the lipopeptide compounds are the major antibiotics produced by Bacillus spp., play an very important role in the plant disease controlling, include three categories:iturin, surfactin and fengycin. In this article, the MALDI-TOF-MS was also used to analysis the kinds of lipopeptides produced by the Bacillus isolates.Qinghai-Tibet Plateau is the highest plateau in the world, and the average altitude is 4000 meters above sea level. This isolated region has an extremely hostile environment, such as low temperature, hypoxia and strong ultraviolet exposure, and it is likely that may produce abundant Bacillus spp. specific to this region. In this work,1388 isolates were obtained from thirty-five soil samples from rhizospheres of plants were collected from Nyingchi, Lhasa, Shannan and Xigaze in Tibet. Xanthomonas oryzae pv. oryzae and Sclerotinia sclerotinorium were chosen as the indicator bacteria and fungi, respectively, for antagonistic activity screening of bacteria, and a total of 311 isolates were found to be antagonistic (diameter of the inhibition zone≥5mm) to at least one of the two pathogens. 150 isolates were obtained after the genomic fingerprints screening based on results of the rep-PCR (repetitive extragenic palindromic PCR)The 150 Bacillus isolates were identified according to the classical classification system described by Gordon et al. (1973), and the isolates were clustered into seven branches. Then 31 Bacillus isolates were chosen for phylogenetic analysis. The phylogenetic trees were constructed using fatty acid methyl ester (FAME),16S rDNA partial sequences, gyrB gene partial sequences, and repetitive extragenic palindromic PCR (rep-PCR). The 31 isolates were identified to be most closely related to Bacillus pumilus (11 isolates), Bacillus cereus (7 isolates), Bacillus thuringiensis (3 isolates), Bacillus amyloliquefaciens (3 isolates), Bacillus atrophaeus (3 isolates), Bacillus axarquiensis (2 isolates), Bacillus subtilis (1 isolate) and Bacillus licheniformis (1 isolate).Then 11 representative Bacillus isolates were chosen for lipopeptides compounds analysis by the MALDI-TOF-MS detection. The results showed that strain LLCG23 can produce surfactin, bacillomycins D and fengycin; strain NMTD81 can produce surfactin, iturin and fengycin; strains YBWC18 and LNXM78 can produce surfactin and fengycin; strains NMTD54 and GBSW11 can produce bacillomycins L and fengycin; strain LSSC22 can produce iturin and fengycin; strains GBSW2 and GBSW19 can produce surfactin; strain DJFZ40 can produce bacillomycins D only; strain NMSW23 can not produce surfactin, iturin family(Mycosubtilins, iturin, bacillomycins D and bacillomycins L) or fengycin. Two strains YBWC43 and RJGP16 that have high antagonistic activity towards Sclerotinia sclerotinorium were used for lipopeptides compounds analysis. The result showed that strain YBWC43 produced bacillomycins D and fengycin, strain RJGP16 produced surfactin and fengycin. And biocontrol efficacy of strains YBWC43 and RJGP16 to rape selerotiniose on the detached leaves were 100% and 50.24%, respectively.更多还原