苦参内生真菌抑菌物质纯化鉴定及作用机制研究
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摘要
本论文采用16Sr-DNA测序法、响应面分析法、捷克八层析法、1H-NMR、13C-NMR和LC-MS等研究方法,从苦参种子中分离筛选出具有抑菌活性的5种内生真菌,并对BS001菌株的分类地位和发酵条件进行了系统研究。纯化鉴定了BS001菌株的抑菌活性物质,并对其抑菌作用机制进行了深入研究。BS001菌株鉴定为土曲霉(Aspergillu terreus);明确了BS001菌株的最佳液体发酵条件；分离获得一种新型抑菌活性物质；阐明了该抑菌物质对黄瓜枯萎病菌的抑菌机制及对土壤微生物的影响。本研究成果为苦参内生真菌及其抑菌活性物质的有效利用奠定了坚实基础。
本论文测定了分离获得的5种苦参内生真菌对25种植物病原真菌和14种常见细菌的拮抗作用,其中BS001菌株对油菜菌核病菌、苹果斑点落叶病菌、番茄炭疽病菌、黄瓜枯萎病菌、小麦纹枯病菌、辣椒疫霉病菌有抑菌效果,其中对黄瓜枯萎病菌抑菌效果明显,抑菌直径达25mm；对铜绿假单胞菌、苏云金杆菌、白葡萄球菌、蜡样芽孢杆菌、产气肠细菌、普通变形杆菌、姜瘟病菌、地衣芽孢杆菌及白菜软腐病菌有抑菌效果,其中对产气肠细菌有抑菌效果明显,抑菌直径达14mm；
用Placket Burman分析法设计试验,选择影响的主效应因子,用统计学的原理、对试验数据进行极差分析,最后用RSM (Response surface methodology)分析法并借助Design expert 8.0软件分析数据,确定了BS001菌株的最佳发酵条件。确定接种量为107cfu/mL;250 mL锥形瓶装液量为100 mL；转速为150 r/min;培养温度为25℃；代谢因子KH2P04的加入量为1 mmol/mL;培养基初始pH为5；马铃薯加入量为335 g/L；最佳碳源为葡萄糖的其加入量为35g/L；最佳氮源NH4N03的加入量为1.80g/L为最佳发酵条件。
在LC-10AT VP高效液相色谱输液泵(日本岛津公司),SPD-10A VP紫外检测器(日本岛津公司),分析色谱柱：DiamonsilTM-C18 (5μm,250mm×4.6 mm I.D.),流动相：甲醇：2%磷酸=2：8,流速：0.5mL/min,检测波长：220nm,进样量：10μL的分析条件下,对BS001菌株发酵液成分进行分析。确定了内生真菌BS001的次生代谢产物中存在与药用植物苦参相同的成分——氧化苦参碱和苦参碱,测定二者的保留时间分别为：9.282 min和13.298 min。
利用大孔树脂HPD-100吸附,乙酸乙酯萃取,10-20%乙醇梯度洗脱,得到了BS001菌株发酵液中对黄瓜枯萎菌具有抑制作用的抑菌物质结构,确定其为：(2E,2E')-((Z)-2,3-dihydroxy-5,8-dioxocyclooct-6-ene-1,4-diyl)dibut2-enoate,为一新化合物。初步命名为千山霉素。
初步明确了新抗菌活性成分对黄瓜枯萎菌的作用机理。确定抑菌物质对黄瓜枯萎病菌丝生长起抑制作用,同时抑制孢子萌发。并且可以使细胞膜结构受损,但不能产生几丁质酶水解几丁质或者破坏几丁质的合成。千山霉素对蛋白质合成没有显著影响,但对合成羧甲基纤维素酶有抑制作用。
调查了千山霉素对室内黄瓜小苗的生物防治效果。从室内试验结果看,千山霉素160μg/mL及320μg/mL溶液15d调查的防效分别为80.10%和81.82%,与对照液30%噁霉灵水剂40μg/mL溶液的防效84.83%效果相当。在F=0.01水平上,千山霉素160μg/mL溶液15d调查的防效效果显著,考虑经济成本,认为千山霉素160μg/mL为最佳试验浓度。
千山霉素对土壤中的细菌,放线菌及真菌都有一定影响,能够增加细菌数量,同时可以减少真菌的数量。可见千山霉素能够增加有益菌群,减少有害菌群,从而达到预防黄瓜枯萎病的目的。
This research screened 5 strains endophytic fungi from Sophora flavescens that have antagonistic activity, and studied the classification and the optimum condition of the active substance BS001 by using 16Sr-DNA method, response surface methodology, eight kinds of solvent system,1H-NMR,13C-NMR and LC-MS. The antagonistic activity of the BS001 was purified and identified in this paper, also the mechanism. After identifying, the strain of BS001 was confirmed as Aspergillu terreus. This study confirmed the conditions of liquid fermentation of BS001 strain and isolated a new antibacterial active component from its fermentation liquor. And the antagonistic mechanism and the effect on soil microorganism of the active component were dealt with. These results provided a solid foundation for the application of the endophytic fungi from Sophora flavescens and its antagonistic active components.
The antifungal activity of 5 strain endophytic fungi to 25 agricultural pathogenic fungi and 14 bacterial were studied. The strain of BS001 could inhibited the growth of Sclerotinia sclerotiorum, Alternaria mali Roberts, Colletotrichum coccodes Hughes., Fusarium oxysporum f.sp. cucumerinum, Rhizoctonia cerealis, Phytophthora capsici, Pseudomonas aeruginosa, Bacillus thuringiensis, Herba Pogostemonis, Bacillus cereus. Frankland, Enterobacter aerogenes, Proteusbacillus vulgaris, Ralstoia solanacearum Smith, Bacillus subtilis (Ehrenberg) Cohn and Erwinia carotovora subsp, in which the inhibition to Fusarium oxysporum f.sp. cucumerinum and Enterobacter aerogenes were the biggest with the antibacterial diameters of 25 mm and 14 mm, respectively.
The optimum condition for BS001 fermentation was obtained by using the Placket Burman analysis, and then selected the main effect factor, analyzed the data through Duncan Analysis, RSM (Response surface methodology) and Design expert 8.0 Soft. The best condition for fermenting BS001 was 100 mL medium volume by adding 107 cfu/mL inoculums concentration,1 mmol/mL KH2PO4,5 pH value,335 g/L potato,35 g/L glucose, 1.80 g/L NH4NO3, with the revolution of 150 rpm and the culture temperature of 25℃.
The determination of chemical contents of BS001 through HPLC with the DiamonsilTM-C18 chromatographic column, the mobile phase was methanol:2% phosphate =2:8, the flow rate was 0.5mL/min, the detection wavelength was 220 nm and the linear range was 10μL. The oxymatrine and matrine, main components of Sophora flavescens, were presented in secondary metabolite of BS001 with the retention time of 9.282 min and 13.298 min, respectively.
The structure of antifungal substance, had inhibition to F. oxysporum f.sp. cucumerinum, in BS001 fermentation was identified by using macrospore absorbed resin and 10-20% ethanol gradient elution. The component was a new compound, (2E,2E')- ((Z)-2,3-dihydroxy-5,8-dioxocyclooct-6-ene-1,4-diyl)dibut2-enoate, named Qianshanmycin, primarily.
The mechanism of active substance to fusarium wilt was defined. In this study, the active substance inhibited the mycelia growth and the spore germination, destroyed the cell membrane structure. But this substance could not produce chitosanase to hydrolyzing the chitin or preventing the synthesis of the chitin. Qianshanmycin had no significant effect on the protein synthesis, while inhibited the synthesis of the carboxymethylcellulose enzyme.
The efficacy of biological control of Qianshanmycin on cucumber seedlings was investigated. The results showed that at the concentration of 160μg/mL and 320μg/m, the control effect on fusarium wilt of cucumber were 80.1% and 81.82% after treating 15 d, which has the same result with 30% hymexazo at 40μg/mL. At F=0.01 level, the concentration of 160μg/mL of Qianshanmycin had better effect, and that was considered the best test concentration.
The active substance increased the number of bacteria and actinomycetes, while decreased fungi number. From this result we concluded that Qianshanmycin could increase the number of beneficial flora, while decrease the deleterious number to prevent the fusarium wilt.
本论文测定了分离获得的5种苦参内生真菌对25种植物病原真菌和14种常见细菌的拮抗作用,其中BS001菌株对油菜菌核病菌、苹果斑点落叶病菌、番茄炭疽病菌、黄瓜枯萎病菌、小麦纹枯病菌、辣椒疫霉病菌有抑菌效果,其中对黄瓜枯萎病菌抑菌效果明显,抑菌直径达25mm；对铜绿假单胞菌、苏云金杆菌、白葡萄球菌、蜡样芽孢杆菌、产气肠细菌、普通变形杆菌、姜瘟病菌、地衣芽孢杆菌及白菜软腐病菌有抑菌效果,其中对产气肠细菌有抑菌效果明显,抑菌直径达14mm；
用Placket Burman分析法设计试验,选择影响的主效应因子,用统计学的原理、对试验数据进行极差分析,最后用RSM (Response surface methodology)分析法并借助Design expert 8.0软件分析数据,确定了BS001菌株的最佳发酵条件。确定接种量为107cfu/mL;250 mL锥形瓶装液量为100 mL；转速为150 r/min;培养温度为25℃；代谢因子KH2P04的加入量为1 mmol/mL;培养基初始pH为5；马铃薯加入量为335 g/L；最佳碳源为葡萄糖的其加入量为35g/L；最佳氮源NH4N03的加入量为1.80g/L为最佳发酵条件。
在LC-10AT VP高效液相色谱输液泵(日本岛津公司),SPD-10A VP紫外检测器(日本岛津公司),分析色谱柱：DiamonsilTM-C18 (5μm,250mm×4.6 mm I.D.),流动相：甲醇：2%磷酸=2：8,流速：0.5mL/min,检测波长：220nm,进样量：10μL的分析条件下,对BS001菌株发酵液成分进行分析。确定了内生真菌BS001的次生代谢产物中存在与药用植物苦参相同的成分——氧化苦参碱和苦参碱,测定二者的保留时间分别为：9.282 min和13.298 min。
利用大孔树脂HPD-100吸附,乙酸乙酯萃取,10-20%乙醇梯度洗脱,得到了BS001菌株发酵液中对黄瓜枯萎菌具有抑制作用的抑菌物质结构,确定其为：(2E,2E')-((Z)-2,3-dihydroxy-5,8-dioxocyclooct-6-ene-1,4-diyl)dibut2-enoate,为一新化合物。初步命名为千山霉素。
初步明确了新抗菌活性成分对黄瓜枯萎菌的作用机理。确定抑菌物质对黄瓜枯萎病菌丝生长起抑制作用,同时抑制孢子萌发。并且可以使细胞膜结构受损,但不能产生几丁质酶水解几丁质或者破坏几丁质的合成。千山霉素对蛋白质合成没有显著影响,但对合成羧甲基纤维素酶有抑制作用。
调查了千山霉素对室内黄瓜小苗的生物防治效果。从室内试验结果看,千山霉素160μg/mL及320μg/mL溶液15d调查的防效分别为80.10%和81.82%,与对照液30%噁霉灵水剂40μg/mL溶液的防效84.83%效果相当。在F=0.01水平上,千山霉素160μg/mL溶液15d调查的防效效果显著,考虑经济成本,认为千山霉素160μg/mL为最佳试验浓度。
千山霉素对土壤中的细菌,放线菌及真菌都有一定影响,能够增加细菌数量,同时可以减少真菌的数量。可见千山霉素能够增加有益菌群,减少有害菌群,从而达到预防黄瓜枯萎病的目的。
This research screened 5 strains endophytic fungi from Sophora flavescens that have antagonistic activity, and studied the classification and the optimum condition of the active substance BS001 by using 16Sr-DNA method, response surface methodology, eight kinds of solvent system,1H-NMR,13C-NMR and LC-MS. The antagonistic activity of the BS001 was purified and identified in this paper, also the mechanism. After identifying, the strain of BS001 was confirmed as Aspergillu terreus. This study confirmed the conditions of liquid fermentation of BS001 strain and isolated a new antibacterial active component from its fermentation liquor. And the antagonistic mechanism and the effect on soil microorganism of the active component were dealt with. These results provided a solid foundation for the application of the endophytic fungi from Sophora flavescens and its antagonistic active components.
The antifungal activity of 5 strain endophytic fungi to 25 agricultural pathogenic fungi and 14 bacterial were studied. The strain of BS001 could inhibited the growth of Sclerotinia sclerotiorum, Alternaria mali Roberts, Colletotrichum coccodes Hughes., Fusarium oxysporum f.sp. cucumerinum, Rhizoctonia cerealis, Phytophthora capsici, Pseudomonas aeruginosa, Bacillus thuringiensis, Herba Pogostemonis, Bacillus cereus. Frankland, Enterobacter aerogenes, Proteusbacillus vulgaris, Ralstoia solanacearum Smith, Bacillus subtilis (Ehrenberg) Cohn and Erwinia carotovora subsp, in which the inhibition to Fusarium oxysporum f.sp. cucumerinum and Enterobacter aerogenes were the biggest with the antibacterial diameters of 25 mm and 14 mm, respectively.
The optimum condition for BS001 fermentation was obtained by using the Placket Burman analysis, and then selected the main effect factor, analyzed the data through Duncan Analysis, RSM (Response surface methodology) and Design expert 8.0 Soft. The best condition for fermenting BS001 was 100 mL medium volume by adding 107 cfu/mL inoculums concentration,1 mmol/mL KH2PO4,5 pH value,335 g/L potato,35 g/L glucose, 1.80 g/L NH4NO3, with the revolution of 150 rpm and the culture temperature of 25℃.
The determination of chemical contents of BS001 through HPLC with the DiamonsilTM-C18 chromatographic column, the mobile phase was methanol:2% phosphate =2:8, the flow rate was 0.5mL/min, the detection wavelength was 220 nm and the linear range was 10μL. The oxymatrine and matrine, main components of Sophora flavescens, were presented in secondary metabolite of BS001 with the retention time of 9.282 min and 13.298 min, respectively.
The structure of antifungal substance, had inhibition to F. oxysporum f.sp. cucumerinum, in BS001 fermentation was identified by using macrospore absorbed resin and 10-20% ethanol gradient elution. The component was a new compound, (2E,2E')- ((Z)-2,3-dihydroxy-5,8-dioxocyclooct-6-ene-1,4-diyl)dibut2-enoate, named Qianshanmycin, primarily.
The mechanism of active substance to fusarium wilt was defined. In this study, the active substance inhibited the mycelia growth and the spore germination, destroyed the cell membrane structure. But this substance could not produce chitosanase to hydrolyzing the chitin or preventing the synthesis of the chitin. Qianshanmycin had no significant effect on the protein synthesis, while inhibited the synthesis of the carboxymethylcellulose enzyme.
The efficacy of biological control of Qianshanmycin on cucumber seedlings was investigated. The results showed that at the concentration of 160μg/mL and 320μg/m, the control effect on fusarium wilt of cucumber were 80.1% and 81.82% after treating 15 d, which has the same result with 30% hymexazo at 40μg/mL. At F=0.01 level, the concentration of 160μg/mL of Qianshanmycin had better effect, and that was considered the best test concentration.
The active substance increased the number of bacteria and actinomycetes, while decreased fungi number. From this result we concluded that Qianshanmycin could increase the number of beneficial flora, while decrease the deleterious number to prevent the fusarium wilt.
引文
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