植物病原菌拮抗微生物的筛选、鉴定及拮抗机理研究
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摘要
在农业生产过程中，植物病原真菌和细菌很易感染植物，每年因此而造成数千万元的经济损失，如何控制疾病、维持植物的正常生长一直是科研工作者研究的热点。化学农药可以有效的控制真菌和细菌病害，在植病防治上发挥了巨大的作用。但是随着人们对化学农药更多的认识，比如它引起土壤、水体和大气的污染，农副产品中农药残留增加，直接危害了人类的健康及生存，以及病原菌的抗药性等问题，致使许多农药的使用受到了限制，由此加速了人们去寻求新的安全有效的植物病害防治途径。利用自然环境中的拮抗微生物防止植物病原的侵袭，也就是我们说的生物防治，这样不仅可以避免象化学农药引起的抗药性问题而且对环境和人类健康都是安全的。
本研究用稀释分离法对成都、简阳、雅安等地区的农田、自然保护区的13个土壤样品进行放线菌和细菌的分离，共分离到放线菌119株，细菌67株。以玉米弯孢菌为供试菌，采用传统五点对峙法和改良的点对峙培养筛选法对80株放线菌进行筛选，结果显示，前者在同步培养5天时间筛选出具有拮抗活性放线菌14株，后者在同步培养5天时间筛选出具有拮抗活性放线菌21株，由此确定改良点对峙培养筛选法适宜病原真菌拮抗性放线菌的筛选。用改良的点对峙培养筛选法从119株放线菌筛选出对病原真菌有拮抗作用的放线菌共39株，用传统的点对峙法从67株细菌中筛选出对病原真菌都有拮抗作用的细菌5株，即BS04、ST1、ST8、C3和C4。其中对山葵墨入菌有拮抗活性的放线菌36株，玉米弯孢菌的拮抗放线菌29株，水稻纹枯病菌的拮抗放线菌27株，玉米小斑病菌的拮抗放线菌28株，玉米大斑病菌的
拮抗放线菌28株，小麦赤霉菌的拮抗放线菌25株，玉米圆斑病菌的拮抗放
线菌27株，枯斑拟盘多饱菌的拮抗放线菌27株，交链抱的拮抗放线菌25株。
用改良的琼脂扩散法从119株放线菌和67株细菌筛选对病原细菌有拮抗作
用的放线菌1株，编号为Pll，且只对姜瘟青枯假单胞菌有作用，筛选到拮
抗细菌4株，其中BS04、STI、STS对柑桔溃疡病菌和姜瘟青枯假单胞菌均
有拮抗作用，P18只对柑桔溃疡病菌有拮抗作用。
由于拮抗菌 BS04对病原真菌和细菌都具有很好的拮抗作用，具有作为生
防制剂的潜力，故对其进行了深入的研究。传统的细菌系统鉴定方法鉴定拮抗
菌BS04结果发现，BSO4与多粘芽抱杆菌和多粘类芽抱杆菌都很相似，无法精
确确定其分类地位。采用165 rDNA序列分析方法从分子角度确定拮抗菌BSO4
的分类地位，结果表明细菌BS04的165 rDNA的序列与类芽抱杆菌属细菌有很
高的同源性(92%一99%)，与多粘类芽抱杆菌同源性为99%，最终鉴定其为多粘
类芽抱杆菌。
通过正交设计试验，优化了多粘类芽抱杆菌BS04发酵产拮抗物质的试验条
件。确定了BS04最佳发酵条件，培养基组成(g/L)为葡萄糖10，糊精7.5，
氯化钠5，玉米浆15，大豆粉30，碳酸钙3，自然pH，发酵接种量为30ml发
酵液接种1.5ml种子液(10日Cfu/ml含菌量)，28oC培养2天(140r/min)。
以青枯假单胞菌为供试菌，采用改良的琼脂孔扩散法来进行生物跟踪，对
多粘类芽抱杆菌BSO4拮抗成分进行了分离纯化。在对发酵液处理上发现，
20一100%的(NH4)250;不能使拮抗成分沉淀，从而不能达到初步分离的目的:正己
烷、乙醚、乙酸乙醋三种有机溶剂均不能把拮抗成分从发酵液中萃取出来，说
明采用萃取法获得拮抗成分粗提液的方案也欠可行。用不同浓度的乙醇处理也
不能促使拮抗成分产生沉淀，但是通过上清液减压浓缩可获得拮抗成分浓缩液，
适合拮抗成分粗提液的制备。将发酵液经65%乙醇沉淀后，上清液减压浓缩经
过Sp一Sepharose Fast Flow柱层析和Souree SRPe反相柱层析后得到棕黄色的
纯化样品。将样品溶解在水中，测定其对青枯假单胞菌的拮抗作用，结果显示
BS04拮抗成分能耐受广泛的pH，并且热稳定性好，活性不受蛋白酶K和胰蛋白
酶影响，但是纯化的拮抗成分对病原真菌没有拮抗作用。拮抗成分的薄层层析
荀三酮显色阳性和红外光谱结果暗示此活性成分可能为肤类，质谱结果显示此
了
活性成分为4个分子量相近的混合物，其分子量分别为1143.7Da、1125.6Da、
1104.7Da和1085.7Da，但是是否这4个组分均有活性，还是只是其中一个或几
个有活性，以及活性组分的结构和抗菌谱还有待进一步研究。另外，拮抗组分
的200一40Onm扫描发现，其在209nm有一最高吸收峰，但是这并不代表每个组
分的最大吸收峰，这一指标对拮抗组分大批量分离纯化提供了监测手段。
通过对BSO4产酶能力的研究发现，BS04虽然没有几丁质酶活性，但是具
有纤维素酶和蛋白酶活性，这就说明其产生的纤维素酶和蛋白酶在抗真菌上发
挥了一定的作用，当然，这并不排除除降解纤维素和蛋白质以外的其他酶或未
知拮抗成分也在拮抗过程中发挥着作用。
通过显微观察，BS04代谢产物的拮抗成分可能是破坏了青枯假单胞菌的细
胞膜，致使胞内物外溢，菌体最后发生死亡。BSO4作用于真菌，使真菌发生菌
丝断裂解体，菌丝变形，内容物外溢。
关键词:植物病原菌;拮抗菌;筛选;鉴定;
多粘类芽袍杆菌;作用机理
In production agriculture, Fungal and bacterial plant pathogens infect plants and crops easily and often seriously, causing millions of dollars worth of economic damage each year. So, how to control these diseases and maintain the normal growth of plants is always the highlight. Application of chemical pesticides is the effective measure to control the fungal and bacterial infection and plays an important role in preventive defence. With the further knowledge to chemical pesticides, however, many concerns, such as the pollution of soil, water and atmosphere, the increase of the leftover of germicides in agricultural produce which imperils human health and life directly, the unbalance of ecological environment resulting from the pesticides, and resistance of phytopathogenic fungi and bacteria to germicides, have caused the withdrawal of many of them. So, new and safe alternative control measures are much sought after. Biological control by natural antagonistic organisms is a potential nonchemical tool for crop protection against phytopathogenic fungi and bacteria and offers a promising alternative to synthetic pesticides, in part because it is perceived as safe to the environment and human health and can avoid the appearance of drug-resistance.In our study, 13 soil samples were gathered from fields in Chengdu,
Jianyang and Yaan, from which 119 actinomyces strains and 67 bacterial strains were isolated by tube dilution assay. With Curuvularia Lunata (Walk) Boed as the tested stain, 21 antibiotic actinomyces stains were screened out by modified hyphal extension inhibition assay and 14 antibiotic actinomyces stains by traditional hyphal extension inhibition assay from 80 actinomyces stains, which indicated that the modified method was suitable to screen the antibiotic actinomyces. From these isolates, 39 antibiotic actinomyces strains and 5 bacterial strains (BS04, ST1 ST8, C3 and C4) against phytopathogenic fungi were screened by modified hyphal extension inhibition assay and traditional hyphal extension inhibition assay, respectively. Among 39 antibiotic actinomyces strains, there were 36, 29, 27, 28, 28, 25, 27, 27 and 25 strains against Phoma wasabiae Yokogi Curuvularia Lunata (Walk) Boed Thantephorus cucumris , Cochlibolus Heterostrophus , Exserohilum Turcicum Fusarium graminearum Cochliobolus carbonum Pestalotiopsis foedans and Altalaria altanata, respectively. With modified agar diffusion assay, 1 actinomyces strain, named P11, was screened out to be antibiotic against to phytopathogenic bacteria, and it was antibiotic against to Pseudomonas solanacearum only. Bacteria BS04, ST1 and ST8 were antibiotic against Xanthomonas axonopodis pv. Citri (Hasse) Dye and Pseudomonas solanacearum, while bacterium P18 was antibiotic only against to Xanthomonas axonopodis pv. Citri (Hasse) Dye.Because of good antibiosis against phytopathogenic fungi and bacteria, bacterium BS04 had the potential as biocontrol agent, and so further study on it was carried out. In order to determine the taxonomic placement, the identification of BS04 was performed with traditional bacterial systemic identification method. The result showed that the physiological and biochemical character of BS04 is similar to that of Bacillus polymyxa and that of Paenibacillus polymyxa so that it was
difficult to ascertain its systematic position. Therefore, the molecular identification of BS04 was performed. Through the comparative 16s rDNA sequences analysis, the 16s rDNA sequence of BS04 had high homology with typical Paenibacillus bacteria from the RDP library (from 92% to 99%). And the constructed phylogenetic tree by using maximum-likelihood method with Bootstrap Trial 1000 proved that BS04 was subjected to Paenibacillus polymyxa.Through orthogonal test, the optimal fermentation conditions of producing antibiotic products of Paenibacillus polymyxa BS04 were given out. The fermentation cultures contained (g/liter): dextrose 10g, dextrin7.5g, sodium chloride5g, corn syrupl5g, soy meal30g, calcium carbonate 3g and pH5. 5-6.5. 3% seed media(V/V) with 108cfu/mL
本研究用稀释分离法对成都、简阳、雅安等地区的农田、自然保护区的13个土壤样品进行放线菌和细菌的分离，共分离到放线菌119株，细菌67株。以玉米弯孢菌为供试菌，采用传统五点对峙法和改良的点对峙培养筛选法对80株放线菌进行筛选，结果显示，前者在同步培养5天时间筛选出具有拮抗活性放线菌14株，后者在同步培养5天时间筛选出具有拮抗活性放线菌21株，由此确定改良点对峙培养筛选法适宜病原真菌拮抗性放线菌的筛选。用改良的点对峙培养筛选法从119株放线菌筛选出对病原真菌有拮抗作用的放线菌共39株，用传统的点对峙法从67株细菌中筛选出对病原真菌都有拮抗作用的细菌5株，即BS04、ST1、ST8、C3和C4。其中对山葵墨入菌有拮抗活性的放线菌36株，玉米弯孢菌的拮抗放线菌29株，水稻纹枯病菌的拮抗放线菌27株，玉米小斑病菌的拮抗放线菌28株，玉米大斑病菌的
拮抗放线菌28株，小麦赤霉菌的拮抗放线菌25株，玉米圆斑病菌的拮抗放
线菌27株，枯斑拟盘多饱菌的拮抗放线菌27株，交链抱的拮抗放线菌25株。
用改良的琼脂扩散法从119株放线菌和67株细菌筛选对病原细菌有拮抗作
用的放线菌1株，编号为Pll，且只对姜瘟青枯假单胞菌有作用，筛选到拮
抗细菌4株，其中BS04、STI、STS对柑桔溃疡病菌和姜瘟青枯假单胞菌均
有拮抗作用，P18只对柑桔溃疡病菌有拮抗作用。
由于拮抗菌 BS04对病原真菌和细菌都具有很好的拮抗作用，具有作为生
防制剂的潜力，故对其进行了深入的研究。传统的细菌系统鉴定方法鉴定拮抗
菌BS04结果发现，BSO4与多粘芽抱杆菌和多粘类芽抱杆菌都很相似，无法精
确确定其分类地位。采用165 rDNA序列分析方法从分子角度确定拮抗菌BSO4
的分类地位，结果表明细菌BS04的165 rDNA的序列与类芽抱杆菌属细菌有很
高的同源性(92%一99%)，与多粘类芽抱杆菌同源性为99%，最终鉴定其为多粘
类芽抱杆菌。
通过正交设计试验，优化了多粘类芽抱杆菌BS04发酵产拮抗物质的试验条
件。确定了BS04最佳发酵条件，培养基组成(g/L)为葡萄糖10，糊精7.5，
氯化钠5，玉米浆15，大豆粉30，碳酸钙3，自然pH，发酵接种量为30ml发
酵液接种1.5ml种子液(10日Cfu/ml含菌量)，28oC培养2天(140r/min)。
以青枯假单胞菌为供试菌，采用改良的琼脂孔扩散法来进行生物跟踪，对
多粘类芽抱杆菌BSO4拮抗成分进行了分离纯化。在对发酵液处理上发现，
20一100%的(NH4)250;不能使拮抗成分沉淀，从而不能达到初步分离的目的:正己
烷、乙醚、乙酸乙醋三种有机溶剂均不能把拮抗成分从发酵液中萃取出来，说
明采用萃取法获得拮抗成分粗提液的方案也欠可行。用不同浓度的乙醇处理也
不能促使拮抗成分产生沉淀，但是通过上清液减压浓缩可获得拮抗成分浓缩液，
适合拮抗成分粗提液的制备。将发酵液经65%乙醇沉淀后，上清液减压浓缩经
过Sp一Sepharose Fast Flow柱层析和Souree SRPe反相柱层析后得到棕黄色的
纯化样品。将样品溶解在水中，测定其对青枯假单胞菌的拮抗作用，结果显示
BS04拮抗成分能耐受广泛的pH，并且热稳定性好，活性不受蛋白酶K和胰蛋白
酶影响，但是纯化的拮抗成分对病原真菌没有拮抗作用。拮抗成分的薄层层析
荀三酮显色阳性和红外光谱结果暗示此活性成分可能为肤类，质谱结果显示此
了
活性成分为4个分子量相近的混合物，其分子量分别为1143.7Da、1125.6Da、
1104.7Da和1085.7Da，但是是否这4个组分均有活性，还是只是其中一个或几
个有活性，以及活性组分的结构和抗菌谱还有待进一步研究。另外，拮抗组分
的200一40Onm扫描发现，其在209nm有一最高吸收峰，但是这并不代表每个组
分的最大吸收峰，这一指标对拮抗组分大批量分离纯化提供了监测手段。
通过对BSO4产酶能力的研究发现，BS04虽然没有几丁质酶活性，但是具
有纤维素酶和蛋白酶活性，这就说明其产生的纤维素酶和蛋白酶在抗真菌上发
挥了一定的作用，当然，这并不排除除降解纤维素和蛋白质以外的其他酶或未
知拮抗成分也在拮抗过程中发挥着作用。
通过显微观察，BS04代谢产物的拮抗成分可能是破坏了青枯假单胞菌的细
胞膜，致使胞内物外溢，菌体最后发生死亡。BSO4作用于真菌，使真菌发生菌
丝断裂解体，菌丝变形，内容物外溢。
关键词:植物病原菌;拮抗菌;筛选;鉴定;
多粘类芽袍杆菌;作用机理
In production agriculture, Fungal and bacterial plant pathogens infect plants and crops easily and often seriously, causing millions of dollars worth of economic damage each year. So, how to control these diseases and maintain the normal growth of plants is always the highlight. Application of chemical pesticides is the effective measure to control the fungal and bacterial infection and plays an important role in preventive defence. With the further knowledge to chemical pesticides, however, many concerns, such as the pollution of soil, water and atmosphere, the increase of the leftover of germicides in agricultural produce which imperils human health and life directly, the unbalance of ecological environment resulting from the pesticides, and resistance of phytopathogenic fungi and bacteria to germicides, have caused the withdrawal of many of them. So, new and safe alternative control measures are much sought after. Biological control by natural antagonistic organisms is a potential nonchemical tool for crop protection against phytopathogenic fungi and bacteria and offers a promising alternative to synthetic pesticides, in part because it is perceived as safe to the environment and human health and can avoid the appearance of drug-resistance.In our study, 13 soil samples were gathered from fields in Chengdu,
Jianyang and Yaan, from which 119 actinomyces strains and 67 bacterial strains were isolated by tube dilution assay. With Curuvularia Lunata (Walk) Boed as the tested stain, 21 antibiotic actinomyces stains were screened out by modified hyphal extension inhibition assay and 14 antibiotic actinomyces stains by traditional hyphal extension inhibition assay from 80 actinomyces stains, which indicated that the modified method was suitable to screen the antibiotic actinomyces. From these isolates, 39 antibiotic actinomyces strains and 5 bacterial strains (BS04, ST1 ST8, C3 and C4) against phytopathogenic fungi were screened by modified hyphal extension inhibition assay and traditional hyphal extension inhibition assay, respectively. Among 39 antibiotic actinomyces strains, there were 36, 29, 27, 28, 28, 25, 27, 27 and 25 strains against Phoma wasabiae Yokogi Curuvularia Lunata (Walk) Boed Thantephorus cucumris , Cochlibolus Heterostrophus , Exserohilum Turcicum Fusarium graminearum Cochliobolus carbonum Pestalotiopsis foedans and Altalaria altanata, respectively. With modified agar diffusion assay, 1 actinomyces strain, named P11, was screened out to be antibiotic against to phytopathogenic bacteria, and it was antibiotic against to Pseudomonas solanacearum only. Bacteria BS04, ST1 and ST8 were antibiotic against Xanthomonas axonopodis pv. Citri (Hasse) Dye and Pseudomonas solanacearum, while bacterium P18 was antibiotic only against to Xanthomonas axonopodis pv. Citri (Hasse) Dye.Because of good antibiosis against phytopathogenic fungi and bacteria, bacterium BS04 had the potential as biocontrol agent, and so further study on it was carried out. In order to determine the taxonomic placement, the identification of BS04 was performed with traditional bacterial systemic identification method. The result showed that the physiological and biochemical character of BS04 is similar to that of Bacillus polymyxa and that of Paenibacillus polymyxa so that it was
difficult to ascertain its systematic position. Therefore, the molecular identification of BS04 was performed. Through the comparative 16s rDNA sequences analysis, the 16s rDNA sequence of BS04 had high homology with typical Paenibacillus bacteria from the RDP library (from 92% to 99%). And the constructed phylogenetic tree by using maximum-likelihood method with Bootstrap Trial 1000 proved that BS04 was subjected to Paenibacillus polymyxa.Through orthogonal test, the optimal fermentation conditions of producing antibiotic products of Paenibacillus polymyxa BS04 were given out. The fermentation cultures contained (g/liter): dextrose 10g, dextrin7.5g, sodium chloride5g, corn syrupl5g, soy meal30g, calcium carbonate 3g and pH5. 5-6.5. 3% seed media(V/V) with 108cfu/mL
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