人参锈腐病生防用高效拮抗放线菌分离鉴定及抑菌作用
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摘要
为筛选和鉴定对人参锈腐病菌具有高效抑菌作用的拮抗放线菌,为生物防治人参锈腐病奠定基础。采用稀释涂布法和平板对峙法分离筛选高效拮抗放线菌,采用纸片法测定了菌株发酵浓缩液的抑菌活性,并采用培养特性、形态特征、生理生化特性和分子生物学方法鉴定了菌种。结果表明:采用稀释涂布法分离纯化出94株放线菌,其中20株为拮抗放线菌;通过复筛,筛选出2株高效拮抗放线菌YBUF5和YBUF7;这2株放线菌菌株及菌株发酵浓缩液对供试9种植物病原菌菌丝的生长均具有较强抑制作用; 2株放线菌菌株及菌株发酵浓缩液在温度15～25℃、pH为6时对人参锈腐病菌的抑制作用最强;根据培养特征、形态特征、生理生化特征及16S r DNA寡核苷酸序列分析结果,YBUF5菌株和YBUF7菌株初步鉴定为生暗灰链霉菌(Streptomyces caniferus)和湿链霉菌(Streptomyces humidus)。这说明筛选出的YBUF5和YBUF7菌株及发酵浓缩液不仅具有较宽的抑菌谱而且不同条件下对人参锈腐病菌均具有很强的抑制作用,在人参锈腐病生物防治方面具有较大的应用潜力。
The research aimed to screen and identify antagonistic actinomycetes with high-efficiency antagonistic to Cylindrocarpon destructans and lay a foundation for biological control of ginseng rust rot. Highly effective antagonistic actinomycetes were isolated and screened by dilution coating method and plate confrontation method; the antagonistic activity of the strains' fermentation concentrate was determined by paper method; the identification of strains was based on culture characteristics,morphological characteristics, physiological and biochemical characteristics, and molecular biology method. Ninety-four actinomycetes were isolated and purified by dilution coating method. Among them,20 strains were antagonistic actinomycetes; two highly effective antagonistic actinomycetes YBUF5 and YBUF7 were selected by rescreening; two actinomycete strains and their fermentation concentrate had strong antagonistic effect on the growth of nine tested plant pathogenic mycelia; two actinomycete strains and strains fermentation concentrate had the strongest antagonistic activity against Cylindrocarpon destructans in the temperature range of 15 ℃ to 25 ℃ and pH value of 6;based on culture characteristics,morphological characteristics,physiological and biochemical characteristics,and 16 S r DNA oligonucleotide sequence analysis results,YBUF5 strain was initially identified as Streptomyces caniferus,and YBUF7 strain was initially identified as Streptomyces humidus. The selected YBUF5 and YBUF7 strains and fermentation concentrate had not only a broad antagonistic spectrum,but also strong antagonistic activity against Cylindrocarpon destructans,and great potential for biological control of ginseng rust rot.
The research aimed to screen and identify antagonistic actinomycetes with high-efficiency antagonistic to Cylindrocarpon destructans and lay a foundation for biological control of ginseng rust rot. Highly effective antagonistic actinomycetes were isolated and screened by dilution coating method and plate confrontation method; the antagonistic activity of the strains' fermentation concentrate was determined by paper method; the identification of strains was based on culture characteristics,morphological characteristics, physiological and biochemical characteristics, and molecular biology method. Ninety-four actinomycetes were isolated and purified by dilution coating method. Among them,20 strains were antagonistic actinomycetes; two highly effective antagonistic actinomycetes YBUF5 and YBUF7 were selected by rescreening; two actinomycete strains and their fermentation concentrate had strong antagonistic effect on the growth of nine tested plant pathogenic mycelia; two actinomycete strains and strains fermentation concentrate had the strongest antagonistic activity against Cylindrocarpon destructans in the temperature range of 15 ℃ to 25 ℃ and pH value of 6;based on culture characteristics,morphological characteristics,physiological and biochemical characteristics,and 16 S r DNA oligonucleotide sequence analysis results,YBUF5 strain was initially identified as Streptomyces caniferus,and YBUF7 strain was initially identified as Streptomyces humidus. The selected YBUF5 and YBUF7 strains and fermentation concentrate had not only a broad antagonistic spectrum,but also strong antagonistic activity against Cylindrocarpon destructans,and great potential for biological control of ginseng rust rot.
引文
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[2]张亚玉.药用植物健康栽培新理念及技术体系[J].吉林农业大学学报,2018,40(4):440-443.
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[9]傅俊范,魏晓兵,周如军,等.哈茨木霉菌Tri41对人参锈腐病菌的抑制作用[J].吉林农业大学学报,2015,37(1):52-56.
[10]周如军,傅俊范,卯婷婷,等.人参锈腐病拮抗细菌BSO15最适发酵条件研究[J].吉林农业大学学报,2011,33(3):278-282.
[11]傅俊范,史会岩,周如军,等.人参锈腐病生防细菌的分离筛选与鉴定[J].吉林农业大学学报,2010,32(2):136-139.
[12]马贵龙,赫荣琳,毛永娜,等.人参锈腐病菌拮抗放线菌JY36发酵条件研究[J].吉林农业大学学报,2009,31(2):138-142.
[13] Qiu D. Current status and development strategy for biological control of plant diseases in China[J]. Plant Protection,2010,36(4):15-21.
[14]傅俊范,史会岩,周如军,等.人参锈腐病生防细菌的分离筛选与鉴定[J].吉林农业大学学报,2010,32(2):136-139.
[15] Jang Y,Sang G K,Kim Y H. Biocontrol Efficacies of Bacillus Species Against Cylindrocarpon destructans Causing Ginseng Root Rot[J]. Plant Pathology Journal,2011,27(4):333-341.
[16]王慧,傅俊范,周如军,等.木霉菌ECT-01-2对人参锈腐病菌的拮抗作用[J].河南农业科学,2008,37(2):66-69.
[17]丁万隆,程惠珍,陈君.应用木霉制剂防治几种药用植物病害的研究[J].中国中药杂志,2003,28(1):24-27.
[18]贾斌.人参黑斑病生防菌的分离鉴定及与杀菌剂混用可能性探讨[D].延吉:延边大学,2014.
[19]马廷会.人参锈腐病颉颃放线菌的筛选及鉴定[D].长春:吉林农业大学,2008.
[20]中国科学院微生物研究所放线菌分类组.链霉菌鉴定手册[M].北京:科学出版社,1975:13-665.
[21]董汉松.植病研究法[M].北京:中国农业出版社,2012:92-97.
[22]布坎南,吉本斯.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:16-1686.
[23]蔡妙英,东秀珠.常见细菌系统鉴定手册[M].北京:科学出版社,2001:9-418.
[24]阎逊初.放线菌的分类和鉴定[M].北京:科学出版社,1992:268-1048.
[25]赫荣琳.人参锈腐病颉颃菌的筛选及鉴定[D].长春:吉林农业大学,2006.
[26]史册,韩梅,张一鸣,等.人参病原菌拮抗放线菌的分离筛选与鉴定[J].生物技术通报,2014(9):102-108.
[27] Shim J O,Lee M W. Identification of Streptomyces species antagonistic to Fusarium solani or Cylindrocarpon destructans causing ginseng root rots[J]. Optics Letters,1991,16(19):324-326.
[28]于妍华.西洋参连作障碍微生态机制及生防放线菌的抗病作用[D].杨凌:西北农林科技大学,2011.
[29]贾斌,金海强,赵贞丽,等.人参锈腐病菌产孢条件研究[J].中国森林病虫,2013,32(1):1-3.
[30]金海强,朴光一,屈俊廷,等.解淀粉芽孢杆菌Y-S-Y12菌株对人参锈腐病的生防作用[J].中国森林病虫,2015,34(2):1-5.
[2]张亚玉.药用植物健康栽培新理念及技术体系[J].吉林农业大学学报,2018,40(4):440-443.
[3]徐中志.人参锈腐病研究概况[J].云南农业科技,1994(1):19-21.
[4]周如军,傅俊范,王秀宇,等.金属离子对人参锈腐病菌孢子萌发和菌落生长的影响[J].吉林农业大学学报,2010,32(6):612-615.
[5]姜云,陈长卿,王琳,等.人参锈腐病拮抗放线菌的鉴定及发酵条件[J].东北林业大学学报,2011,39(9):101-104.
[6]张鸿雁.人参连作根系病害发生的微生态机制及放线菌修复研究[D].杨凌:西北农林科技大学,2014.
[7]卢振兰,刘金海,马秀兰,等.栽参土壤中阿特拉津降解菌株筛选及影响生物降解的因素[J].东北林业大学学报,2007,35(1):58-59.
[8]李宝年,徐树辉,杨传波,等.樟子松枯梢病拮抗真菌的筛选[J].东北林业大学学报,2004,32(5):97-99.
[9]傅俊范,魏晓兵,周如军,等.哈茨木霉菌Tri41对人参锈腐病菌的抑制作用[J].吉林农业大学学报,2015,37(1):52-56.
[10]周如军,傅俊范,卯婷婷,等.人参锈腐病拮抗细菌BSO15最适发酵条件研究[J].吉林农业大学学报,2011,33(3):278-282.
[11]傅俊范,史会岩,周如军,等.人参锈腐病生防细菌的分离筛选与鉴定[J].吉林农业大学学报,2010,32(2):136-139.
[12]马贵龙,赫荣琳,毛永娜,等.人参锈腐病菌拮抗放线菌JY36发酵条件研究[J].吉林农业大学学报,2009,31(2):138-142.
[13] Qiu D. Current status and development strategy for biological control of plant diseases in China[J]. Plant Protection,2010,36(4):15-21.
[14]傅俊范,史会岩,周如军,等.人参锈腐病生防细菌的分离筛选与鉴定[J].吉林农业大学学报,2010,32(2):136-139.
[15] Jang Y,Sang G K,Kim Y H. Biocontrol Efficacies of Bacillus Species Against Cylindrocarpon destructans Causing Ginseng Root Rot[J]. Plant Pathology Journal,2011,27(4):333-341.
[16]王慧,傅俊范,周如军,等.木霉菌ECT-01-2对人参锈腐病菌的拮抗作用[J].河南农业科学,2008,37(2):66-69.
[17]丁万隆,程惠珍,陈君.应用木霉制剂防治几种药用植物病害的研究[J].中国中药杂志,2003,28(1):24-27.
[18]贾斌.人参黑斑病生防菌的分离鉴定及与杀菌剂混用可能性探讨[D].延吉:延边大学,2014.
[19]马廷会.人参锈腐病颉颃放线菌的筛选及鉴定[D].长春:吉林农业大学,2008.
[20]中国科学院微生物研究所放线菌分类组.链霉菌鉴定手册[M].北京:科学出版社,1975:13-665.
[21]董汉松.植病研究法[M].北京:中国农业出版社,2012:92-97.
[22]布坎南,吉本斯.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:16-1686.
[23]蔡妙英,东秀珠.常见细菌系统鉴定手册[M].北京:科学出版社,2001:9-418.
[24]阎逊初.放线菌的分类和鉴定[M].北京:科学出版社,1992:268-1048.
[25]赫荣琳.人参锈腐病颉颃菌的筛选及鉴定[D].长春:吉林农业大学,2006.
[26]史册,韩梅,张一鸣,等.人参病原菌拮抗放线菌的分离筛选与鉴定[J].生物技术通报,2014(9):102-108.
[27] Shim J O,Lee M W. Identification of Streptomyces species antagonistic to Fusarium solani or Cylindrocarpon destructans causing ginseng root rots[J]. Optics Letters,1991,16(19):324-326.
[28]于妍华.西洋参连作障碍微生态机制及生防放线菌的抗病作用[D].杨凌:西北农林科技大学,2011.
[29]贾斌,金海强,赵贞丽,等.人参锈腐病菌产孢条件研究[J].中国森林病虫,2013,32(1):1-3.
[30]金海强,朴光一,屈俊廷,等.解淀粉芽孢杆菌Y-S-Y12菌株对人参锈腐病的生防作用[J].中国森林病虫,2015,34(2):1-5.