新疆苦豆子内生细菌多样性及其抗菌作用研究
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摘要
植物内生细菌不仅因其抗病促生作用成为生防菌剂的重要来源,同时也是人、植物和动物疾病治疗新药开发的源泉。本论文对新疆苦豆子不同组织部位的内生细菌进行了分离、纯化与保存,从苦豆子内生细菌中筛选了拮抗棉花枯萎病菌、棉花黄萎病菌的菌株,研究了拮抗棉花枯萎病菌和棉花黄萎病菌菌株的多样性及系统发育地位,对拮抗性菌株进行了盆栽控病实验及其拮抗机制研究,对抗MRSA菌株进行了筛选及其活性成分分析,得到以下主要结论:
1.从新疆苦豆子不同组织部位中共分离获得内生细菌202株,不同组织部位分离获得的内生细菌菌株数存在明显的差异,总体上来看,内生细菌在苦豆子植株中的分布规律为根瘤>叶>种子>根>茎>花。
2. 202株苦豆子内生细菌对棉花枯萎病菌、棉花黄萎病菌均表现出一定的拮抗作用,其中125株对棉花枯萎病菌、棉花黄萎病菌的相对抑菌率均超过50%,48株内生细菌胞外分泌物对棉花枯萎病菌、棉花黄萎病菌有抑制作用,胞外分泌物的拮抗性与平板对峙培养中的拮抗性不存在对应关系。
3.拮抗棉花枯、黄萎病菌的48株苦豆子内生细菌表型多样性丰富,在Waston距离为0.23时可聚为7个表观类群和12个独立成群的菌株。ERIC-PCR指纹图谱分析表明,在Watson距离为0.31时48株菌被划分为6个ERIC群和两个独立成群的菌株。两种分析方法结果基本一致,从两种分析结果中挑选代表性的菌株测定16S rDNA进行系统发育分析,结果表明这些菌株分别属于Bacillus atrophaeus、Bacillus subtilis、Bacillus cereus、Bacillus megaterium、Bacillus pumilus、Serratia marcescens。
4.盆栽控病实验结果表明,5株拮抗性苦豆子内生细菌对棉花枯萎病都有较好的防效,KDRE51和KDEB30比目前应用的棉花枯萎病生防药剂宁楠霉素的防治效果更好;对棉花黄萎病也有较好的防效,KDRE51和KDRE01比目前应用的棉花黄萎病生防药剂宁楠霉素的防治效果更好。
5. KDRE51可以通过产生活性化学物质、产生羧酸型铁载体、对棉花产生诱导系统抗性等多种方式对棉花枯萎病菌、棉花黄萎病菌进行拮抗,其中产生活性化学物质是最主要的拮抗方式。
6. KDRE51对MRSA具有较强的拮抗活性,它所产生的活性化合物ZSC-6对MRSA的抑菌圈直径达到22 mm,通过多种波谱技术最终将活性化合物ZSC-6鉴定为1-(5-氯-5-甲氧基正戊烷基)吡咯烷-2-酮。
Because of antimicrobial activity and plant growth-promoting effect, endophytic bacteria become an important source of biocontrol agents and new drug for people, plants and animal disease treatment. This paper isolated and purified endophytic bacteria lived in different tissues of Sophora alopecuroides at Xinjiang province, screened antagonistic endophytic bacteria against the cotton pathogens such as Verticillium dahliae and Fusarium oxysporum, studied the diversity and phylogenetic position of the antagonistic strains, researched antagonistic pot experiment and mechanism of bial-control disease, screened antagonistic strain against the MRSA and its active components. The main conclusions are following.
1. 202 endophytic bactria strains were isolated from different organization of Sophora alopecuroides at Xinjiang province. The species of endophytic bacteria in difference organization of Sophora alopecuroides were obviously difference, which distributed rule was decreasing progressively in nodule, leaf, seed, root, stem and flower.
2. All of 202 endophytic bacteria had antagonist activity against to Verticillium alboatrum and Fusarium oxysporum and inhibition rate of 125 strains against to Verticillium alboatrum and Fusarium oxysporum was more than 50%. Extracellular secretion of 48 antagonistic endophytic bacteria had the inhibitory action against to Verticillium alboatrum and Fusarium oxysporum.
3. Phynotype of 48 antagonistic endophytic bacteria was divided into 7 cluster apparently and 12 independence strains at 0.23 of Waston distance. The ERIC-PCR fingerprint analysis of 48 antagonistic endophytic bacteria was indicated into 6 ERIC groups and two independence strain at 0.31 of Watson distance. The result of two kind of analysis method was consistent. Ten type strains belong to Bacillus atrophaeus, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus pumilus, Serratia marcescens.
4 Five antagonistic endophytic bacteria had the control effects of against to Verticillium alboatrum and Fusarium oxysporum in pot tests. The control effects of KDRE51 and KDEB30 against to Verticillium alboatrum and KDRE51 and KDRE01 against to Fusarium oxysporum was rather better than ningnanmycin.
5 KDRE51 had antagonistic action against to Verticillium alboatrum and Fusarium oxysporum by producing the active chemical substance, iron siderophore and the induce system resistant. Producing the active chemical substance was main oppresses mechanism.
6 KDRE51 has strongly antagonistic activity against to MRSA. The inhibitary circle diameter of compound ZSC-6 proceded by KDRE51 achieved 22 mm to the MRSA. Compound ZSC-6 was determined as 1-(5-chlorine-5-methoxyn-pentane base)the tetrahydropyrrole-2-alkone by comparison of the GC-MS total ionic chromatographic, infrared absorption spectrum, 1H- NMR and 13C-NMR spectral data.
1.从新疆苦豆子不同组织部位中共分离获得内生细菌202株,不同组织部位分离获得的内生细菌菌株数存在明显的差异,总体上来看,内生细菌在苦豆子植株中的分布规律为根瘤>叶>种子>根>茎>花。
2. 202株苦豆子内生细菌对棉花枯萎病菌、棉花黄萎病菌均表现出一定的拮抗作用,其中125株对棉花枯萎病菌、棉花黄萎病菌的相对抑菌率均超过50%,48株内生细菌胞外分泌物对棉花枯萎病菌、棉花黄萎病菌有抑制作用,胞外分泌物的拮抗性与平板对峙培养中的拮抗性不存在对应关系。
3.拮抗棉花枯、黄萎病菌的48株苦豆子内生细菌表型多样性丰富,在Waston距离为0.23时可聚为7个表观类群和12个独立成群的菌株。ERIC-PCR指纹图谱分析表明,在Watson距离为0.31时48株菌被划分为6个ERIC群和两个独立成群的菌株。两种分析方法结果基本一致,从两种分析结果中挑选代表性的菌株测定16S rDNA进行系统发育分析,结果表明这些菌株分别属于Bacillus atrophaeus、Bacillus subtilis、Bacillus cereus、Bacillus megaterium、Bacillus pumilus、Serratia marcescens。
4.盆栽控病实验结果表明,5株拮抗性苦豆子内生细菌对棉花枯萎病都有较好的防效,KDRE51和KDEB30比目前应用的棉花枯萎病生防药剂宁楠霉素的防治效果更好;对棉花黄萎病也有较好的防效,KDRE51和KDRE01比目前应用的棉花黄萎病生防药剂宁楠霉素的防治效果更好。
5. KDRE51可以通过产生活性化学物质、产生羧酸型铁载体、对棉花产生诱导系统抗性等多种方式对棉花枯萎病菌、棉花黄萎病菌进行拮抗,其中产生活性化学物质是最主要的拮抗方式。
6. KDRE51对MRSA具有较强的拮抗活性,它所产生的活性化合物ZSC-6对MRSA的抑菌圈直径达到22 mm,通过多种波谱技术最终将活性化合物ZSC-6鉴定为1-(5-氯-5-甲氧基正戊烷基)吡咯烷-2-酮。
Because of antimicrobial activity and plant growth-promoting effect, endophytic bacteria become an important source of biocontrol agents and new drug for people, plants and animal disease treatment. This paper isolated and purified endophytic bacteria lived in different tissues of Sophora alopecuroides at Xinjiang province, screened antagonistic endophytic bacteria against the cotton pathogens such as Verticillium dahliae and Fusarium oxysporum, studied the diversity and phylogenetic position of the antagonistic strains, researched antagonistic pot experiment and mechanism of bial-control disease, screened antagonistic strain against the MRSA and its active components. The main conclusions are following.
1. 202 endophytic bactria strains were isolated from different organization of Sophora alopecuroides at Xinjiang province. The species of endophytic bacteria in difference organization of Sophora alopecuroides were obviously difference, which distributed rule was decreasing progressively in nodule, leaf, seed, root, stem and flower.
2. All of 202 endophytic bacteria had antagonist activity against to Verticillium alboatrum and Fusarium oxysporum and inhibition rate of 125 strains against to Verticillium alboatrum and Fusarium oxysporum was more than 50%. Extracellular secretion of 48 antagonistic endophytic bacteria had the inhibitory action against to Verticillium alboatrum and Fusarium oxysporum.
3. Phynotype of 48 antagonistic endophytic bacteria was divided into 7 cluster apparently and 12 independence strains at 0.23 of Waston distance. The ERIC-PCR fingerprint analysis of 48 antagonistic endophytic bacteria was indicated into 6 ERIC groups and two independence strain at 0.31 of Watson distance. The result of two kind of analysis method was consistent. Ten type strains belong to Bacillus atrophaeus, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus pumilus, Serratia marcescens.
4 Five antagonistic endophytic bacteria had the control effects of against to Verticillium alboatrum and Fusarium oxysporum in pot tests. The control effects of KDRE51 and KDEB30 against to Verticillium alboatrum and KDRE51 and KDRE01 against to Fusarium oxysporum was rather better than ningnanmycin.
5 KDRE51 had antagonistic action against to Verticillium alboatrum and Fusarium oxysporum by producing the active chemical substance, iron siderophore and the induce system resistant. Producing the active chemical substance was main oppresses mechanism.
6 KDRE51 has strongly antagonistic activity against to MRSA. The inhibitary circle diameter of compound ZSC-6 proceded by KDRE51 achieved 22 mm to the MRSA. Compound ZSC-6 was determined as 1-(5-chlorine-5-methoxyn-pentane base)the tetrahydropyrrole-2-alkone by comparison of the GC-MS total ionic chromatographic, infrared absorption spectrum, 1H- NMR and 13C-NMR spectral data.
引文
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