核桃内生真菌G8次生代谢产物及其生物活性的研究
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摘要
本文从核桃根中筛选出一株真菌G8,对G8进行了内生真菌确认,证明G8是核桃内生真菌。明确了核桃内生真菌分离方法的可行性。并根据G8的菌株形态及显微结构特征,初步鉴定为葡萄壳小圆孢菌。
以8种病原真菌和3种植物种子为活性实验材料,对内生真菌G8的发酵产物进行活性测定。结果表明:内生真菌发酵液对8个病原真菌都有一定的抑制作用,其中对小麦赤霉和玉米大斑病原真菌的抑制率大于50%,说明发酵液中含有抑制病原真菌的活性物质。对3种植物种子进行化感试验说明:G8菌株发酵液对3种植物生长有抑制作用,其中,发酵液对芽的抑制率大小为:油菜>绿豆>萝卜;发酵液对根的抑制率大小为:绿豆>萝卜>油菜,发酵液对测试植物绿豆的抑制活性总体较强。以此把内生真菌G8作为实验菌株。
对G8进行大量发酵,发酵液各萃取相进行抑菌活性,以28种病原真菌和5种细菌作为供试菌株进行活性试验,结果表明:(1)发酵液的提取物对28种植物病原真菌均有不同程度的抑制作用,各萃取相粗提物在40mg.mL-1时,乙酸乙酯萃取物的抑制效果最好,其次为正丁醇萃取物,萃余物最弱,但正丁醇萃取物对水稻纹枯、杨树溃疡、小麦雪腐病原菌的抑制率大于其它两相萃取物。(2)不同浓度的乙酸乙酯萃取物对11种病原真菌进行活性实验,结果表明:对苹果轮纹、苹果腐烂、小麦根腐、稻瘟4种病原菌的抑制率为100%,不同浓度的萃取物对苜蓿炭疽、小麦赤霉、番茄早疫、苹果炭疽和玉米小斑5种病原菌的抑制率表现出极显著差异,对玉米大斑和辣椒疫霉2种病原菌的抑制率无显著差异。(3)乙酸乙酯萃取物对5种细菌的抑菌试验表明,对蜡状芽孢杆菌和马铃薯芽孢杆菌2种细菌表现为抗药性,但不同浓度处理间差异不显著。以(2)的试验结果确定了菌株次生代谢物分离的部位,活性跟踪的菌种和浓度。
G8发酵液乙酸乙脂萃取得31.2g样品,在活性跟踪的指导下,采用硅胶柱层析,sephadex LH-20柱层析和重结晶等手段分离得到两种化合物。通过理化性质分析和NMR分析,最终确定了其中1个化合物为胆甾醇,另外一个尚未进一步鉴定。
An fungus G8 was screened from the root of walnut, the identification experiment whether the fungus is endophyte have been done, the result showed that the fungus was endophyte, and the method separating endophyte from walnut is feasible. The work which identified the endophyte have been carried according to the shape and the character of micro-structure, and confirmed it as Coniothyrium. vitivora Mitura initially.
The 8 stains of pathogen and 3 kinds of plant was used as the materials to test the activity of the endophytic fermentation , result showed that the fermentation of the endophyte G8 had some inhabitation to the 8 strains, and the rate of inhabitation was over 50% to the Helminthosporium turcicum Pass and Fusarium graminearum, it indicate that the fermentation have some compounds to reduce the pathogen; the fermentation had effect on the 3 kinds plant, the inhabited rate of the bud was rape>mung>radis, and the inhabited rate of root was mung>radis>rape, the inhabited rate of mung was highest of all. so the endophyte G8 was screened as the material to study.
G8 was fermented largely. The extract of its fermentation was tested to inhabit 28strains pathogen and also the ethyl acetate extract of it to 5 kinds of bacteria. The results showed that (1) For the extract of acetyl acetate, butanol and residue. The different extracts of fermentation all have different range activity to 28 kinds of pathogenic fungi. At concentration 40mg.mL-1, the inhabited effect of the phases is ethyl acetate, butanol and residue from strong to weak, but the inhabitation of butanol extract is better than the two other phases to Thanatephorus cucumeris, Dothiorella gregaria and Monographella nivialis. (2)Different concentrations of ethyl acetate extract have been measured to 11 kinds of pathogenic fungus. The results show the inhabiting rate of the extract is 100% to Physalospora piricola, Valsa mali miyabe et, Cochliobolus sativus and Magna porthe. The inhabited rate of Colletotrichum hum trifolii, Fusarium graminearum, Alternaria solani, Glomerella cingnlata.schr.et.and Bipolaris maydis to different concentration have been obvious difference. (3)The extract of ethyl acetate had studied on inhabited effect of 5 species bacteria. Bacillus megaterium and Bacillus mesenterius is sensitive to drugs, but not dominant to different concentration. The section of the secondary metabolites was sure through the test , also the density of inhabitation and the pathogen needed to test from the result (2).
The fermentation of the endophyte was carried, the extract of ethyl acetate was 31.2g. two compounds was separated following its activity, passing the silica gel column chromatography, passing Sephadex LH-20 column chromatography and re-crystallizing. The structure of the compound was elucidated through physical and chemical properties and spectrum data. One of them is cholesterol, and the other wasn’t elucidated.
以8种病原真菌和3种植物种子为活性实验材料,对内生真菌G8的发酵产物进行活性测定。结果表明:内生真菌发酵液对8个病原真菌都有一定的抑制作用,其中对小麦赤霉和玉米大斑病原真菌的抑制率大于50%,说明发酵液中含有抑制病原真菌的活性物质。对3种植物种子进行化感试验说明:G8菌株发酵液对3种植物生长有抑制作用,其中,发酵液对芽的抑制率大小为:油菜>绿豆>萝卜;发酵液对根的抑制率大小为:绿豆>萝卜>油菜,发酵液对测试植物绿豆的抑制活性总体较强。以此把内生真菌G8作为实验菌株。
对G8进行大量发酵,发酵液各萃取相进行抑菌活性,以28种病原真菌和5种细菌作为供试菌株进行活性试验,结果表明:(1)发酵液的提取物对28种植物病原真菌均有不同程度的抑制作用,各萃取相粗提物在40mg.mL-1时,乙酸乙酯萃取物的抑制效果最好,其次为正丁醇萃取物,萃余物最弱,但正丁醇萃取物对水稻纹枯、杨树溃疡、小麦雪腐病原菌的抑制率大于其它两相萃取物。(2)不同浓度的乙酸乙酯萃取物对11种病原真菌进行活性实验,结果表明:对苹果轮纹、苹果腐烂、小麦根腐、稻瘟4种病原菌的抑制率为100%,不同浓度的萃取物对苜蓿炭疽、小麦赤霉、番茄早疫、苹果炭疽和玉米小斑5种病原菌的抑制率表现出极显著差异,对玉米大斑和辣椒疫霉2种病原菌的抑制率无显著差异。(3)乙酸乙酯萃取物对5种细菌的抑菌试验表明,对蜡状芽孢杆菌和马铃薯芽孢杆菌2种细菌表现为抗药性,但不同浓度处理间差异不显著。以(2)的试验结果确定了菌株次生代谢物分离的部位,活性跟踪的菌种和浓度。
G8发酵液乙酸乙脂萃取得31.2g样品,在活性跟踪的指导下,采用硅胶柱层析,sephadex LH-20柱层析和重结晶等手段分离得到两种化合物。通过理化性质分析和NMR分析,最终确定了其中1个化合物为胆甾醇,另外一个尚未进一步鉴定。
An fungus G8 was screened from the root of walnut, the identification experiment whether the fungus is endophyte have been done, the result showed that the fungus was endophyte, and the method separating endophyte from walnut is feasible. The work which identified the endophyte have been carried according to the shape and the character of micro-structure, and confirmed it as Coniothyrium. vitivora Mitura initially.
The 8 stains of pathogen and 3 kinds of plant was used as the materials to test the activity of the endophytic fermentation , result showed that the fermentation of the endophyte G8 had some inhabitation to the 8 strains, and the rate of inhabitation was over 50% to the Helminthosporium turcicum Pass and Fusarium graminearum, it indicate that the fermentation have some compounds to reduce the pathogen; the fermentation had effect on the 3 kinds plant, the inhabited rate of the bud was rape>mung>radis, and the inhabited rate of root was mung>radis>rape, the inhabited rate of mung was highest of all. so the endophyte G8 was screened as the material to study.
G8 was fermented largely. The extract of its fermentation was tested to inhabit 28strains pathogen and also the ethyl acetate extract of it to 5 kinds of bacteria. The results showed that (1) For the extract of acetyl acetate, butanol and residue. The different extracts of fermentation all have different range activity to 28 kinds of pathogenic fungi. At concentration 40mg.mL-1, the inhabited effect of the phases is ethyl acetate, butanol and residue from strong to weak, but the inhabitation of butanol extract is better than the two other phases to Thanatephorus cucumeris, Dothiorella gregaria and Monographella nivialis. (2)Different concentrations of ethyl acetate extract have been measured to 11 kinds of pathogenic fungus. The results show the inhabiting rate of the extract is 100% to Physalospora piricola, Valsa mali miyabe et, Cochliobolus sativus and Magna porthe. The inhabited rate of Colletotrichum hum trifolii, Fusarium graminearum, Alternaria solani, Glomerella cingnlata.schr.et.and Bipolaris maydis to different concentration have been obvious difference. (3)The extract of ethyl acetate had studied on inhabited effect of 5 species bacteria. Bacillus megaterium and Bacillus mesenterius is sensitive to drugs, but not dominant to different concentration. The section of the secondary metabolites was sure through the test , also the density of inhabitation and the pathogen needed to test from the result (2).
The fermentation of the endophyte was carried, the extract of ethyl acetate was 31.2g. two compounds was separated following its activity, passing the silica gel column chromatography, passing Sephadex LH-20 column chromatography and re-crystallizing. The structure of the compound was elucidated through physical and chemical properties and spectrum data. One of them is cholesterol, and the other wasn’t elucidated.
引文
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