猕猴桃溃疡病菌生防放线菌TGNBSA5的鉴定及其活性组分的初步研究
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摘要
由丁香假单孢杆菌猕猴桃致病变种(Pseudomonas syingae pv. actinidiae)所引起的猕猴桃溃疡病（Kiwifruit bacterial canker disease）是一种严重威胁猕猴桃生产的毁灭性病害。目前，生产中对该病的防治多采用化学方法，但化学农药存在严重的污染，因此研究如何利用生物防治，开发对环境无污染的生物源农药成为目前研究的热点问题。经研究发现，从牛蒡茎部分离纯化得到的内生放线菌株TGNBSA5对猕猴桃溃疡病菌有较好抑菌活性，为开发利用该内生放线菌，本论文对该菌株的分类地位、产生活性物质的发酵条件以及活性代谢组分进行了研究。
菌株TGNBSA5的鉴定采用菌落形态观察、生理生化指标测定及16S rDNA序列分析的方法。结果发现TGNBSA5菌株的菌落呈现乌灰色，基丝乌紫，菌苔表面平坦；电镜观察孢子呈卵圆形或柱形，表面光滑，大小约0.5μm×0.7μm，16S rDNA序列分析结果及系统发育树表明，其与孢杆链霉菌(Streptomyces sporovirgulis)序列同源性达到99%，但不产生黑色素。菌株TGNBSA5定名为烬灰类群链霉菌属孢杆链霉菌的牛蒡变种(S.sporovirgulis var. arctium)。
对TGNBSA5菌株种子培养液的优化研究结果表明，最优种子液的组成为：淀粉2.4%、牛肉膏0.3%、葡萄糖0.1%、酵母浸粉0.5%，蛋白胨0.3%，碳酸钙0.4%；最佳培养条件为28℃、36h、摇床转速150r/min、培养基pH值为7.0。
对TGNBSA5菌株发酵条件的优化研究结果表明，最优发酵培养基组成为：淀粉2%、蔗糖2%、蛋白胨1%、酵母粉2%，碳酸钙0.4%；最佳发酵条件为28℃、5d、摇床转速180r/min、发酵培养基pH值为7.0。
对TGNBSA5菌株发酵液有效成分进行萃取和色谱分离，以猕猴桃溃疡病菌为靶标进行活性追踪，用乙酸乙酯萃取，对乙酸乙酯相进行活性检测，得到活性浸膏约1g；再利用硅胶柱层析和制备薄层分离得到了一个活性组分（W），最后利用分析型HPLC检测、凝胶柱层析纯化并获得抑菌活性组分W4，经紫外、NMR鉴定活性组分之一为苯甲醇。
Kiwifruit bacterial canker disease, caused by a pathogenic variant Pseudomonas syingaepv. actinidiae (Abbreviation Psa) is a devastating disease of a serious threat to kiwiproduction. At present, the main prevention and treatment method of the disease is the use ofchemicals, whereas serious pollution exists. Consequently, the study of applying bio-controlmethod and developing pollution free biogenic pesticides are becoming heat issues. Previousresearch found that, an endophytic actinomycete strain TGNBSA5, isolated from Arctiumlappa stem, has strong inhibitory activity against Pseudomonas syringae pv. actinidiae. Inorder to further exploit the biocontrol application of the actinomycete, the taxonomic status ofstrain TGNBSA5, and its fermentation condition of producing bioactive substances againstPsa plus the elucidation of bioactive compound were studied in this research.
Physiological and biochemical characterization, morphological, and16S rDNA sequenceanalysis were conducted to identify the strain TGNBSA5. The results showed that theactinomycete performed black grey colony, raisin substrate mycelia, flat lawn surface. Thespores were long ovoid in shape with smooth surface and0.5μm×0.7μm in size.16S rDNAsequence and its Phyloenetic N-J tree analysis showed that the strain was very close to thegene sequence of Streptomyces sporovirgulis, which was99%homology. Nevertheless, strainTGNBSA5did not produce melanin. Therefore, strain TGNBSA5was identified andnominated as Streptomyces sporovirgulis var. arctium.
The optimization of TGNBSA5seed culture trial showed that the optimized ingredientswere: starch2.4%, beef extract0.3%, glucose0.1%, yeast extract powder0.5%, petptone0.3%, and CaCO30.4%. In addition, the optimized culture condition was28,36h, rotationspeed150r/min, and culture pH7.0.
The optimization of TGNBSA5fermentation culture trial showed that the optimizedingredients were: starch2%, sucrose2%, peptone1%, yeast extract powder2%and CaCO30.4%. In addition, the optimized fermentation condition was28,5d, rotation speed180r/min, and culture pH7.0.
Bioassay-guided method was conducted using the bacterial pathogen Psa as target.Bioactive compounds from ethyl acetate were isolated and purified by chromatographictechnology, and HPLC detection. NMR and UV spectra analysis indicated that one of thebioactive compounds was benzyl alcohol.
菌株TGNBSA5的鉴定采用菌落形态观察、生理生化指标测定及16S rDNA序列分析的方法。结果发现TGNBSA5菌株的菌落呈现乌灰色，基丝乌紫，菌苔表面平坦；电镜观察孢子呈卵圆形或柱形，表面光滑，大小约0.5μm×0.7μm，16S rDNA序列分析结果及系统发育树表明，其与孢杆链霉菌(Streptomyces sporovirgulis)序列同源性达到99%，但不产生黑色素。菌株TGNBSA5定名为烬灰类群链霉菌属孢杆链霉菌的牛蒡变种(S.sporovirgulis var. arctium)。
对TGNBSA5菌株种子培养液的优化研究结果表明，最优种子液的组成为：淀粉2.4%、牛肉膏0.3%、葡萄糖0.1%、酵母浸粉0.5%，蛋白胨0.3%，碳酸钙0.4%；最佳培养条件为28℃、36h、摇床转速150r/min、培养基pH值为7.0。
对TGNBSA5菌株发酵条件的优化研究结果表明，最优发酵培养基组成为：淀粉2%、蔗糖2%、蛋白胨1%、酵母粉2%，碳酸钙0.4%；最佳发酵条件为28℃、5d、摇床转速180r/min、发酵培养基pH值为7.0。
对TGNBSA5菌株发酵液有效成分进行萃取和色谱分离，以猕猴桃溃疡病菌为靶标进行活性追踪，用乙酸乙酯萃取，对乙酸乙酯相进行活性检测，得到活性浸膏约1g；再利用硅胶柱层析和制备薄层分离得到了一个活性组分（W），最后利用分析型HPLC检测、凝胶柱层析纯化并获得抑菌活性组分W4，经紫外、NMR鉴定活性组分之一为苯甲醇。
Kiwifruit bacterial canker disease, caused by a pathogenic variant Pseudomonas syingaepv. actinidiae (Abbreviation Psa) is a devastating disease of a serious threat to kiwiproduction. At present, the main prevention and treatment method of the disease is the use ofchemicals, whereas serious pollution exists. Consequently, the study of applying bio-controlmethod and developing pollution free biogenic pesticides are becoming heat issues. Previousresearch found that, an endophytic actinomycete strain TGNBSA5, isolated from Arctiumlappa stem, has strong inhibitory activity against Pseudomonas syringae pv. actinidiae. Inorder to further exploit the biocontrol application of the actinomycete, the taxonomic status ofstrain TGNBSA5, and its fermentation condition of producing bioactive substances againstPsa plus the elucidation of bioactive compound were studied in this research.
Physiological and biochemical characterization, morphological, and16S rDNA sequenceanalysis were conducted to identify the strain TGNBSA5. The results showed that theactinomycete performed black grey colony, raisin substrate mycelia, flat lawn surface. Thespores were long ovoid in shape with smooth surface and0.5μm×0.7μm in size.16S rDNAsequence and its Phyloenetic N-J tree analysis showed that the strain was very close to thegene sequence of Streptomyces sporovirgulis, which was99%homology. Nevertheless, strainTGNBSA5did not produce melanin. Therefore, strain TGNBSA5was identified andnominated as Streptomyces sporovirgulis var. arctium.
The optimization of TGNBSA5seed culture trial showed that the optimized ingredientswere: starch2.4%, beef extract0.3%, glucose0.1%, yeast extract powder0.5%, petptone0.3%, and CaCO30.4%. In addition, the optimized culture condition was28,36h, rotationspeed150r/min, and culture pH7.0.
The optimization of TGNBSA5fermentation culture trial showed that the optimizedingredients were: starch2%, sucrose2%, peptone1%, yeast extract powder2%and CaCO30.4%. In addition, the optimized fermentation condition was28,5d, rotation speed180r/min, and culture pH7.0.
Bioassay-guided method was conducted using the bacterial pathogen Psa as target.Bioactive compounds from ethyl acetate were isolated and purified by chromatographictechnology, and HPLC detection. NMR and UV spectra analysis indicated that one of thebioactive compounds was benzyl alcohol.
引文
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