黄芪根腐病复合生防放线菌筛选鉴定及防治作用
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摘要
为了研制出有效的黄芪根腐病生物防治产品,本研究利用从疏勒河流域盐碱土壤中分离出对其致病菌有良好防治作用的放线菌进行多菌复配,以期提高生防菌对黄芪根腐病的防效。采用平板对峙法和生长速率法筛选高效生防菌,并对其进行形态特征、生理生化和16S rRNA基因序列鉴定,并采用盆栽试验对黄芪根腐病的防治效果进行测试。结果表明:从供试的244株放线菌中,经初筛有11株放线菌对黄芪根腐病菌的抑菌率均高于50%,复筛后对黄芪根腐病菌的抑菌率为7%～57%。结合筛选效果和菌株间亲和性试验,将菌株DA4-3-12、DA8-4-10和221作为复合菌的研究对象。经鉴定,菌株DA4-3-12和221为Streptomyces alboflavus,DA8-4-10为Streptomyces alanosinicus。单一菌株DA4-3-12、DA8-4-10和221及其不同组合混合菌株(DA4-3-12+DA8-4-10、DA4-3-12+221、DA8-4-10+221、DA4-3-12+DA8-4-10+221)对黄芪根腐病均有防治效果,其防效分别为60%、46%、70%、55%、70%、64%、72%,以混合菌株DA4-3-12+DA8-4-10+221的效果最为理想,混合菌株中各菌株间通过协同作用充分发挥其生防效果,且抑菌谱较广。
To develop effective biocontrol products for controlling the root rot of Astragalus membranaceus,actinomycete strains with excellent prevention and controlling effects to pathogenic bacteria were isolated from the saline-alkali soil of Shule River basin,which were further combined into compound agents to improve the anti-bacterial effect on root rot of A. membranaceus.The plate-to-limulus method and growth rate method were used to screen high-efficiency biocontrol actinomycete strains. Morphological characteristics,physiological and biochemical characteristics,and 16 S rRNA DNA sequence analysis were used to identify strains. After that,a pot experiment was conducted to test the controlling effect on root rot of A. membranaceus. The results showed that among the total 244 tested strains of actinomycetes,11 strains had inhibitory rates of more than 50% against the root rot disease. After rescreening,the inhibitory rate was 7%-57%.Combined with the screening effect and the inter-strain affinity test,the strains DA4-3-12,DA8-4-10 and 221 were used as the research targets for the compound strains. The strains DA4-3-12 and 221 were identified as Streptomyces alboflavus and strain DA8-4-10 as Streptomyces alanosinicus. Single strains DA4-3-12,DA8-4-10 and 221 and their compound strains in different combinations( DA4-3-12 + DA8-4-10,DA4-3-12 + 221,DA8-4-10 + 221,DA4-3-12 + DA8-4-10 +221) all had controlling effects on root rot of A. membranaceus,and their control efficiencies were 60%,46%,70%,55%,70%,64%,and 72% respectively. Among them,the compound DA4-3-12+DA8-4-10+221 had the best controlling effect. The synergistic effects of various strains in the mixed strains were fully expressed,with wide antimicrobial spectrum.
To develop effective biocontrol products for controlling the root rot of Astragalus membranaceus,actinomycete strains with excellent prevention and controlling effects to pathogenic bacteria were isolated from the saline-alkali soil of Shule River basin,which were further combined into compound agents to improve the anti-bacterial effect on root rot of A. membranaceus.The plate-to-limulus method and growth rate method were used to screen high-efficiency biocontrol actinomycete strains. Morphological characteristics,physiological and biochemical characteristics,and 16 S rRNA DNA sequence analysis were used to identify strains. After that,a pot experiment was conducted to test the controlling effect on root rot of A. membranaceus. The results showed that among the total 244 tested strains of actinomycetes,11 strains had inhibitory rates of more than 50% against the root rot disease. After rescreening,the inhibitory rate was 7%-57%.Combined with the screening effect and the inter-strain affinity test,the strains DA4-3-12,DA8-4-10 and 221 were used as the research targets for the compound strains. The strains DA4-3-12 and 221 were identified as Streptomyces alboflavus and strain DA8-4-10 as Streptomyces alanosinicus. Single strains DA4-3-12,DA8-4-10 and 221 and their compound strains in different combinations( DA4-3-12 + DA8-4-10,DA4-3-12 + 221,DA8-4-10 + 221,DA4-3-12 + DA8-4-10 +221) all had controlling effects on root rot of A. membranaceus,and their control efficiencies were 60%,46%,70%,55%,70%,64%,and 72% respectively. Among them,the compound DA4-3-12+DA8-4-10+221 had the best controlling effect. The synergistic effects of various strains in the mixed strains were fully expressed,with wide antimicrobial spectrum.
引文
安登第,陈玉梅,李进,等.2010.银沙槐内生放线菌抗菌活性及其与内生细菌的拮抗关系.应用生态学报,21(4):1021-1025.
甘良,蓝星杰,戴蓬博,等.2015.放线菌混合菌剂对西瓜枯萎病的防治作用研究.中国生物防治学报,31(4):516-523.
高芬,郝锐,秦雪梅,等.2016.拮抗黄芪根腐病菌的根际促生菌的室内筛选与鉴定.中国中药杂志,41(22):4188-4193.
高芬,任小霞,王梦亮,等.2015.中草药根腐病及其微生物防治研究进展.中国中药杂志,40(21):4122-4126.
李威,肖熙鸥,李可,等.2018.茄子青枯病拮抗放线菌XL-6的筛选、鉴定及发酵条件优化.微生物学通报,45(2):357-367.
李海云,胡磊,牛世全,等.2015.河西走廊酒泉地区盐碱土壤中可培养放线菌多样性.生态学杂志,34(2):367-372.
李海云,姚拓,张榕,等.2018.红三叶根际促生菌中具生防效果菌株筛选、鉴定及特性研究[EB/OL].[2018-05-10].http://kns.cnki.net/kcms/detail/11.3996.S.20180424.1716.032.html.
李振高,骆永明,滕应.2008.土壤与环境生物研究法.北京:科学出版社.
刘彩云,许瑞瑞,季洪亮,等.2015.一株生防内生真菌的分离筛选、鉴定及抑菌特性.植物保护学报,42(5):806-812.
刘海龙,石培基,李生梅,等.2014.河西走廊生态经济系统协调度评价及其空间演化.应用生态学报,25(12):3645-3654.
罗光宏,陈叶,王振,等.2005.黄芪根腐病发生危害与防治.植物保护,31(4):75-76.
牛世全,耿晖,阎薇如,等.2016.黄芪根腐病生防放线菌筛选鉴定及其优化培养.植物保护学报,43(6):943-950.
秦雪梅,李震宇,孙海峰,等.2013.我国黄芪药材资源现状与分析.中国中药杂志,38(19):3234-3238.
申光辉,薛泉宏,张晶,等.2012.草莓根腐病拮抗真菌筛选鉴定及其防病促生作用.中国农业科学,45(22):4612-4626.
孙广宇,宗兆峰.2002.植物病理学实验技术.北京:中国农业出版社.
腾艳萍,梁宗锁,陈蓉.2006.木霉防治黄芪根腐病初步研究.西北农业学报,15(2):69-71.
辛中尧,徐红霞,陈秀蓉.2008.枯草芽孢杆菌(Bacillus subtilis)B1、B2菌株对当归、黄芪的防病促进生长效果.植物保护,34(6):142-144.
徐平,李文均,徐丽华,等.2003.微波法快速提取放线菌基因组DNA.微生物学通报,30(4):82-84.
薛应钰,范万泽,张树武,等.2016.苹果树腐烂病菌拮抗放线菌的筛选、鉴定及防效.应用生态学报,27(10):3379-3386.
张玲,李师翁,陈熙明,等.2014.青藏高原土壤中链霉菌的分离鉴定及其抗菌活性研究.冰川冻土,36(2):430-441
赵淑莉,任飞娥,刘金亮,等.2012.玉米大斑病生防放线菌的筛选鉴定及发酵条件优化.微生物学报,52(10):1228-1236.
Akila R,Rajendran L,Harish S,et al.2011.Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f.sp.cubense(Foc)causing Fusarium wilt in banana.Biological Control,57:175-183.
Ayari A,Morakchi H,Djamila KG.2012.Identification and antifungal activity of Streptomyces sp.S72 isolated from Lake Oubeira sediments in North-East of Algeria.African Journal of Biotechnology,11:305-311.
Crawford DL,Lynch JM,Whipps JM,et al.1993.Isolation and characterization of actinomycete antagonists of a fungal root pathogen.Applied&Environmental Microbiology,59:3899-3905.
Dubey SC,Singh V,Priyanka K,et al.2015.Combined application of fungal and bacterial bio-agents,together with fungicide and Mesorhizobium,for integrated management of Fusarium wilt of chickpea.Biocontrol,60:413-424.
Errakhi R,Lebrihi A,Barakate M.2009.In vitro and in vivo antagonism of actinomycetes isolated from Moroccan rhizospherical soils against Sclerotium rolfsii:A causal agent of root rot on sugar beet(Beta vulgaris L.).Journal of Applied Microbiology,107:672-681.
Hozzein WN,Rabie W,Ali MA.2011.Screening the Egyptian desert actinomycetes as candidates for new antimicrobial compounds and identification of a new desert Streptomyces strain.African Journal of Biotechnology,10:2295-2301.
Ji Z,Qiao G,Wei S,et al.2012.Isolation and characterization of two novel antibacterial cyclic hexapeptides from Streptomyces alboflavus 313.Chemistry&Biodiversity,9:1567-1578.
Murthy YKS,Thiemann JE,Coronelli C,et al.1996.Alanosine,a new antiviral and antitumour agent isolated from a Streptomyces.Nature,211:1198-1199.
Price NPJ,Weinstein SM.2014.Biosynthesis of 4-aminoheptose2-epimers,core structural components of the septacidins and spicamycins.Journal of Antibiotics,67:405-414.
Stach JEM,Maldonado LA,Goodfellow M,et al.2003.New primers for the class Actinobacteria:Application to marine and terrestrial environment.Environmental Microbiology,5:828-841.
Wang C,Wang Z,Qiao X,et al.2013.Antifungal activity of volatile organic compounds from Streptomyces alboflavus TD-1.FEMS Microbiology Letters,341:45-51.
甘良,蓝星杰,戴蓬博,等.2015.放线菌混合菌剂对西瓜枯萎病的防治作用研究.中国生物防治学报,31(4):516-523.
高芬,郝锐,秦雪梅,等.2016.拮抗黄芪根腐病菌的根际促生菌的室内筛选与鉴定.中国中药杂志,41(22):4188-4193.
高芬,任小霞,王梦亮,等.2015.中草药根腐病及其微生物防治研究进展.中国中药杂志,40(21):4122-4126.
李威,肖熙鸥,李可,等.2018.茄子青枯病拮抗放线菌XL-6的筛选、鉴定及发酵条件优化.微生物学通报,45(2):357-367.
李海云,胡磊,牛世全,等.2015.河西走廊酒泉地区盐碱土壤中可培养放线菌多样性.生态学杂志,34(2):367-372.
李海云,姚拓,张榕,等.2018.红三叶根际促生菌中具生防效果菌株筛选、鉴定及特性研究[EB/OL].[2018-05-10].http://kns.cnki.net/kcms/detail/11.3996.S.20180424.1716.032.html.
李振高,骆永明,滕应.2008.土壤与环境生物研究法.北京:科学出版社.
刘彩云,许瑞瑞,季洪亮,等.2015.一株生防内生真菌的分离筛选、鉴定及抑菌特性.植物保护学报,42(5):806-812.
刘海龙,石培基,李生梅,等.2014.河西走廊生态经济系统协调度评价及其空间演化.应用生态学报,25(12):3645-3654.
罗光宏,陈叶,王振,等.2005.黄芪根腐病发生危害与防治.植物保护,31(4):75-76.
牛世全,耿晖,阎薇如,等.2016.黄芪根腐病生防放线菌筛选鉴定及其优化培养.植物保护学报,43(6):943-950.
秦雪梅,李震宇,孙海峰,等.2013.我国黄芪药材资源现状与分析.中国中药杂志,38(19):3234-3238.
申光辉,薛泉宏,张晶,等.2012.草莓根腐病拮抗真菌筛选鉴定及其防病促生作用.中国农业科学,45(22):4612-4626.
孙广宇,宗兆峰.2002.植物病理学实验技术.北京:中国农业出版社.
腾艳萍,梁宗锁,陈蓉.2006.木霉防治黄芪根腐病初步研究.西北农业学报,15(2):69-71.
辛中尧,徐红霞,陈秀蓉.2008.枯草芽孢杆菌(Bacillus subtilis)B1、B2菌株对当归、黄芪的防病促进生长效果.植物保护,34(6):142-144.
徐平,李文均,徐丽华,等.2003.微波法快速提取放线菌基因组DNA.微生物学通报,30(4):82-84.
薛应钰,范万泽,张树武,等.2016.苹果树腐烂病菌拮抗放线菌的筛选、鉴定及防效.应用生态学报,27(10):3379-3386.
张玲,李师翁,陈熙明,等.2014.青藏高原土壤中链霉菌的分离鉴定及其抗菌活性研究.冰川冻土,36(2):430-441
赵淑莉,任飞娥,刘金亮,等.2012.玉米大斑病生防放线菌的筛选鉴定及发酵条件优化.微生物学报,52(10):1228-1236.
Akila R,Rajendran L,Harish S,et al.2011.Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f.sp.cubense(Foc)causing Fusarium wilt in banana.Biological Control,57:175-183.
Ayari A,Morakchi H,Djamila KG.2012.Identification and antifungal activity of Streptomyces sp.S72 isolated from Lake Oubeira sediments in North-East of Algeria.African Journal of Biotechnology,11:305-311.
Crawford DL,Lynch JM,Whipps JM,et al.1993.Isolation and characterization of actinomycete antagonists of a fungal root pathogen.Applied&Environmental Microbiology,59:3899-3905.
Dubey SC,Singh V,Priyanka K,et al.2015.Combined application of fungal and bacterial bio-agents,together with fungicide and Mesorhizobium,for integrated management of Fusarium wilt of chickpea.Biocontrol,60:413-424.
Errakhi R,Lebrihi A,Barakate M.2009.In vitro and in vivo antagonism of actinomycetes isolated from Moroccan rhizospherical soils against Sclerotium rolfsii:A causal agent of root rot on sugar beet(Beta vulgaris L.).Journal of Applied Microbiology,107:672-681.
Hozzein WN,Rabie W,Ali MA.2011.Screening the Egyptian desert actinomycetes as candidates for new antimicrobial compounds and identification of a new desert Streptomyces strain.African Journal of Biotechnology,10:2295-2301.
Ji Z,Qiao G,Wei S,et al.2012.Isolation and characterization of two novel antibacterial cyclic hexapeptides from Streptomyces alboflavus 313.Chemistry&Biodiversity,9:1567-1578.
Murthy YKS,Thiemann JE,Coronelli C,et al.1996.Alanosine,a new antiviral and antitumour agent isolated from a Streptomyces.Nature,211:1198-1199.
Price NPJ,Weinstein SM.2014.Biosynthesis of 4-aminoheptose2-epimers,core structural components of the septacidins and spicamycins.Journal of Antibiotics,67:405-414.
Stach JEM,Maldonado LA,Goodfellow M,et al.2003.New primers for the class Actinobacteria:Application to marine and terrestrial environment.Environmental Microbiology,5:828-841.
Wang C,Wang Z,Qiao X,et al.2013.Antifungal activity of volatile organic compounds from Streptomyces alboflavus TD-1.FEMS Microbiology Letters,341:45-51.
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