生防细菌菌株AbⅢ745-6的筛选、鉴定及其产生的抑菌活性物质的初步研究
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摘要
本试验旨在从植物根际土壤中分离并筛选出对水稻稻瘟病菌等造成地上部病害的病原菌有良好颉颃效果的生防细菌，探求用于植物病害生物防治的新资源，并对其产生的抑菌活性物质进行初步的研究和分析。
通过对分离得到的117株细菌的发酵液进行抑菌活性测定，筛选出对稻瘟病菌和小麦全蚀病菌有一定抑菌效果的原始菌株11个。其中对稻瘟病菌抑制率达50％以上的菌株有8个，占总颉颃菌株数的72.73％；对水稻稻瘟病菌和小麦全蚀病菌的抑制效果均达50％以上的菌株有AbⅢ745、AbⅢ763和AbⅢ716，尤以AbⅢ745的颉颃活性最强。依据颉颃活性测定结果，确定AbⅢ745的单孢分离后代AbⅢ745-6为最优菌株，其对稻瘟病菌的抑制率可达96.88%。经初步鉴定，AbⅢ745-6属于假单胞菌科假单胞菌属荧光假单胞杆菌。
菌株AbⅢ745-6的抑菌谱测定结果表明：该菌株抑菌谱较广，其发酵液（108-9 cfu/ml）对多种病原菌有很高的颉颃活性，对稻瘟病菌、番茄枯萎病菌、番茄早疫病菌、黄瓜枯萎病菌及小麦全蚀病菌的抑制效果均达90%以上；对番茄灰霉病菌、烟草赤星病菌、梨黑星病菌及小麦根腐病菌的抑制率在60%以上；对番茄叶霉病菌、玉米大斑病菌、小麦赤霉病菌和茄青枯病菌也有一定的颉颃作用。
通过温室盆栽试验，对颉颃菌株的最佳应用途径及使用方法做了初步探索，发现利用发酵液混以载体进行种子包衣处理防治稻瘟病效果最佳（病情指数降低22.54，病叶抑制率可达75.36%），且适于大面积推广；土壤处理也有良好效果；叶面喷雾效果相对较差，防治效果仅为45.68%，病情指数下降14.38。
以番茄、水稻、烟草和黄瓜为供试植物，就菌株AbⅢ745-6对植物的促生效应进行了初步研究。试验结果显示：AbⅢ745-6发酵液（108-9 cfu/ml）在不同浓度下对植物生长可表现促进或抑制两个方面的效应，总体趋势是较低浓度下促进幼苗生长，而在较高浓度下则有抑制幼苗生长的倾向。发酵液稀释1100X时促生效果最好，尤以对番茄和水稻幼苗的根长及株高影响较大。由此可见，AbⅢ745-6菌株兼具防治植物病害和促进植物生长的潜能。
从菌株AbⅢ745-6的发酵液中提取具有强抑菌活性的物质，经纯化后对活性最强组分中含量较高的单一化合物进行化学分析和结构鉴定，初步命名为苯胺基甲基乙酸，这是在荧光假单胞杆菌中发现的一种新抑菌活性物质。
This research aimed at isolating and screening of antagonistic bacteria with great ability to suppress above-ground-plant-disease-causing agents such as Pyricularia orazye, searching for new resources for plant disease biological control, studying and analyzing the inhibitory substances produced by them preliminarily.
Eleven antagonistic bacterial strains showing inhibiting effect on Pyricularia oryzae and Gaeumannomyces graminis were screened out of 117 bacteria strains isolated from rhizosphere soil on the basis of their suppressiveness in the in-vitro bioassay. A result of over 50% suppression of Pyricularia oryzae was obtained by eight of those suppressive original strains with the treatment of their fermentation filtrate, which amounted to 72.83% of the total antagonistic ones. Among those eight strains, there were three strains(namely AbⅢ745,AbⅢ763 and AbⅢ716) showing an inhibiting rate of over 50% against both indicator pathogens, AbⅢ745 particularly. According to the result of the suppression assay, strain AbⅢ745-6，the single-cell isolated offspring of AbⅢ745,was selected as the idealest one. It could inhibit the mycelium growth of Pyricularia oryzae by 96.88% and was preliminarily identified as Pseudomonas Fluorescens.
The determination of suppression spectrum by strain AbⅢ745-6 showed that this bacterial strain possessed a wide inhibiting spectrum. It inhibited many pathogens with high antagonistic effect, with over 90% inhibition against Pyricularia oryzae, Fusarium oxysporum f.lycopersici（sacc）.snyder et Hanse, Altermaria solani, Fusarium oxysporum（scho）.f.sp.cucumbrum Owen and Gaeumannomyces graminis; over 60% against Botrytis cirerea, Alternaria alternata, Venturia nashicola and Bipolaris sorokiniana. Also, it showed certain suppression of other indicator pathogens involved in this assay.
Tests were taken in this study to figure out the best way for application of strain AbⅢ745-6. Practice showed that seed-treatment with bacterial fermentation filtrate was the most effective and convenient way, with an inhibiting rate of 75.36% and a decrease of 22.54 in disease index against rice blast disease. Disease-controlling effect was fair with soil-treatment but not so good with leaf-spraying treatment with which the inhibiting rate was only 45.68% and decrease in disease index 14.38.
Preliminary study on growth-promoting effects of strain AbⅢ745-6 were conducted, with tomato, rice, tobacco and cucumber plants as tested plants. The results indicated that the
bacterial fermentation（108-9cfu/ml） had different or even contrary effects on plant growth as different concentration treatments. There was a tendency of growth-promoting effects when treated with low concentrations while grow-inhibiting effects when the concentration was high enough. As a whole, the growth-promoting effect was the best when treated with fermentation diluted by 1100 times and it performed better on tomato and rice seedlings with their root length and plant height. One could easily tell that strain AbⅢ745-6 possesses both plant disease control and plant growth-promoting potential based on above bioassay results.
Substances with strong inhibiting activity were extracted from the filtrate of strain AbⅢ745-6. After further purified, a single compound predominant in the fraction showing the most desirable suppressive activity was selected for closer study. It was primarily named Phenaminomethylacetic based on chemical analysis and structural identification, which turned out to be a new kind of inhibitory substance ever found in Fluorescent Pseudomonas spp..
通过对分离得到的117株细菌的发酵液进行抑菌活性测定，筛选出对稻瘟病菌和小麦全蚀病菌有一定抑菌效果的原始菌株11个。其中对稻瘟病菌抑制率达50％以上的菌株有8个，占总颉颃菌株数的72.73％；对水稻稻瘟病菌和小麦全蚀病菌的抑制效果均达50％以上的菌株有AbⅢ745、AbⅢ763和AbⅢ716，尤以AbⅢ745的颉颃活性最强。依据颉颃活性测定结果，确定AbⅢ745的单孢分离后代AbⅢ745-6为最优菌株，其对稻瘟病菌的抑制率可达96.88%。经初步鉴定，AbⅢ745-6属于假单胞菌科假单胞菌属荧光假单胞杆菌。
菌株AbⅢ745-6的抑菌谱测定结果表明：该菌株抑菌谱较广，其发酵液（108-9 cfu/ml）对多种病原菌有很高的颉颃活性，对稻瘟病菌、番茄枯萎病菌、番茄早疫病菌、黄瓜枯萎病菌及小麦全蚀病菌的抑制效果均达90%以上；对番茄灰霉病菌、烟草赤星病菌、梨黑星病菌及小麦根腐病菌的抑制率在60%以上；对番茄叶霉病菌、玉米大斑病菌、小麦赤霉病菌和茄青枯病菌也有一定的颉颃作用。
通过温室盆栽试验，对颉颃菌株的最佳应用途径及使用方法做了初步探索，发现利用发酵液混以载体进行种子包衣处理防治稻瘟病效果最佳（病情指数降低22.54，病叶抑制率可达75.36%），且适于大面积推广；土壤处理也有良好效果；叶面喷雾效果相对较差，防治效果仅为45.68%，病情指数下降14.38。
以番茄、水稻、烟草和黄瓜为供试植物，就菌株AbⅢ745-6对植物的促生效应进行了初步研究。试验结果显示：AbⅢ745-6发酵液（108-9 cfu/ml）在不同浓度下对植物生长可表现促进或抑制两个方面的效应，总体趋势是较低浓度下促进幼苗生长，而在较高浓度下则有抑制幼苗生长的倾向。发酵液稀释1100X时促生效果最好，尤以对番茄和水稻幼苗的根长及株高影响较大。由此可见，AbⅢ745-6菌株兼具防治植物病害和促进植物生长的潜能。
从菌株AbⅢ745-6的发酵液中提取具有强抑菌活性的物质，经纯化后对活性最强组分中含量较高的单一化合物进行化学分析和结构鉴定，初步命名为苯胺基甲基乙酸，这是在荧光假单胞杆菌中发现的一种新抑菌活性物质。
This research aimed at isolating and screening of antagonistic bacteria with great ability to suppress above-ground-plant-disease-causing agents such as Pyricularia orazye, searching for new resources for plant disease biological control, studying and analyzing the inhibitory substances produced by them preliminarily.
Eleven antagonistic bacterial strains showing inhibiting effect on Pyricularia oryzae and Gaeumannomyces graminis were screened out of 117 bacteria strains isolated from rhizosphere soil on the basis of their suppressiveness in the in-vitro bioassay. A result of over 50% suppression of Pyricularia oryzae was obtained by eight of those suppressive original strains with the treatment of their fermentation filtrate, which amounted to 72.83% of the total antagonistic ones. Among those eight strains, there were three strains(namely AbⅢ745,AbⅢ763 and AbⅢ716) showing an inhibiting rate of over 50% against both indicator pathogens, AbⅢ745 particularly. According to the result of the suppression assay, strain AbⅢ745-6，the single-cell isolated offspring of AbⅢ745,was selected as the idealest one. It could inhibit the mycelium growth of Pyricularia oryzae by 96.88% and was preliminarily identified as Pseudomonas Fluorescens.
The determination of suppression spectrum by strain AbⅢ745-6 showed that this bacterial strain possessed a wide inhibiting spectrum. It inhibited many pathogens with high antagonistic effect, with over 90% inhibition against Pyricularia oryzae, Fusarium oxysporum f.lycopersici（sacc）.snyder et Hanse, Altermaria solani, Fusarium oxysporum（scho）.f.sp.cucumbrum Owen and Gaeumannomyces graminis; over 60% against Botrytis cirerea, Alternaria alternata, Venturia nashicola and Bipolaris sorokiniana. Also, it showed certain suppression of other indicator pathogens involved in this assay.
Tests were taken in this study to figure out the best way for application of strain AbⅢ745-6. Practice showed that seed-treatment with bacterial fermentation filtrate was the most effective and convenient way, with an inhibiting rate of 75.36% and a decrease of 22.54 in disease index against rice blast disease. Disease-controlling effect was fair with soil-treatment but not so good with leaf-spraying treatment with which the inhibiting rate was only 45.68% and decrease in disease index 14.38.
Preliminary study on growth-promoting effects of strain AbⅢ745-6 were conducted, with tomato, rice, tobacco and cucumber plants as tested plants. The results indicated that the
bacterial fermentation（108-9cfu/ml） had different or even contrary effects on plant growth as different concentration treatments. There was a tendency of growth-promoting effects when treated with low concentrations while grow-inhibiting effects when the concentration was high enough. As a whole, the growth-promoting effect was the best when treated with fermentation diluted by 1100 times and it performed better on tomato and rice seedlings with their root length and plant height. One could easily tell that strain AbⅢ745-6 possesses both plant disease control and plant growth-promoting potential based on above bioassay results.
Substances with strong inhibiting activity were extracted from the filtrate of strain AbⅢ745-6. After further purified, a single compound predominant in the fraction showing the most desirable suppressive activity was selected for closer study. It was primarily named Phenaminomethylacetic based on chemical analysis and structural identification, which turned out to be a new kind of inhibitory substance ever found in Fluorescent Pseudomonas spp..
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