豇豆枯萎病拮抗木霉MA4菌株的分离与鉴定
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摘要
为筛选能有效抑制豇豆枯萎病菌(Fusarium oxysporum f. sp. tracheiphilum)的生防真菌,采用平板稀释法和平板对峙法从武汉设施蔬菜根际土壤中分离、纯化、筛选出一株优势拮抗真菌MA4,根据形态学和菌株18S rDNA序列对MA4菌株进行鉴定,采用平板对峙法对其抑菌活性和稳定性进行测定,并进行盆栽防效试验。结果表明,MA4对豇豆枯萎病菌的抑制率为75.69%;根据其形态学鉴定和18S rDNA序列分析,确定MA4菌株为哈茨木霉(Trichoderma harzianum),将该序列在GenBank中进行注册登录,登录号为MH539514。MA4菌株对番茄灰霉病菌、辣椒根腐病菌、西瓜枯萎病菌、黄瓜枯萎病菌、茄子褐纹病菌、番茄早疫病菌、茄子烂秆病菌和番茄果腐病菌等8种病原菌均具有良好的抑制作用,抑制率为62.12%～86.93%;MA4菌株经10代继代培养后,对豇豆枯萎病菌的抑制率仍为80.86%。盆栽试验结果表明,接种14 d后施加MA4发酵液的处理病情指数为11.85%,防效达85.75%,且对豇豆植株具有一定促生作用。
To screen microbial resources for biological control of cowpea wilt, a dominant antagonistic fungus MA4 strain against Fusarium oxysporum f. sp. tracheiphilum was isolated, purified and screened from rhizosphere soil in Wuhan by dilution plating and confront culture method. The MA4 strain was identified according to its morphology and 18 S r DNA sequence.The antifungal spectrum and stability of MA4 strain were studied by the plate confrontation method, and the pot experiment was also carried out. The results showed that the antifungal activity of MA4 against F. oxysporum f. sp. tracheiphilum was 75.69%. According to its morphology and 18 S r DNA sequence analysis, the MA4 strain was identified as Trichoderma harzianum, and registered the sequence in GenBank with a login number of MH539514. The antifungal activity of MA4 against Botrytis cinerea, F. oxysporum Schlecht, F. oxyporum f. sp. niveum, F. oxysporum. f. sp. cucumebrium, Phomopsis vexans, Alternaria solani, P. vexans and Rhizoctonia solani was from 62.12% to 86.93%. After the subculture of 10 generations, the antifungal activity of MA4 against F. oxysporum f. sp. tracheiphilum was 80.86%. The results of the potted experiment showed that the disease index of the treatment with MA4 fermentation liquor after 14 d inoculation was 11.85%, the prevention efficiency was 85.75%, and it had certain promoting effect on cowpea plants.
To screen microbial resources for biological control of cowpea wilt, a dominant antagonistic fungus MA4 strain against Fusarium oxysporum f. sp. tracheiphilum was isolated, purified and screened from rhizosphere soil in Wuhan by dilution plating and confront culture method. The MA4 strain was identified according to its morphology and 18 S r DNA sequence.The antifungal spectrum and stability of MA4 strain were studied by the plate confrontation method, and the pot experiment was also carried out. The results showed that the antifungal activity of MA4 against F. oxysporum f. sp. tracheiphilum was 75.69%. According to its morphology and 18 S r DNA sequence analysis, the MA4 strain was identified as Trichoderma harzianum, and registered the sequence in GenBank with a login number of MH539514. The antifungal activity of MA4 against Botrytis cinerea, F. oxysporum Schlecht, F. oxyporum f. sp. niveum, F. oxysporum. f. sp. cucumebrium, Phomopsis vexans, Alternaria solani, P. vexans and Rhizoctonia solani was from 62.12% to 86.93%. After the subculture of 10 generations, the antifungal activity of MA4 against F. oxysporum f. sp. tracheiphilum was 80.86%. The results of the potted experiment showed that the disease index of the treatment with MA4 fermentation liquor after 14 d inoculation was 11.85%, the prevention efficiency was 85.75%, and it had certain promoting effect on cowpea plants.
引文
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[11]朱萍萍,凌健,席亚东,等.蔬菜土传病害生防木霉菌株资源的筛选及其防治效果评价[J].中国蔬菜,2015,1(8):28-33.
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[13]方中达.植病研究法[M].第三版.北京:中国农业出版社,2001.
[14]起登风,邹良平,周登博,等.GA1-2菌株的分离鉴定及其对香蕉尖孢镰刀菌的抑菌效果[J].植物保护学报,2017,44(5):809-816.
[15]常媛,杨兴堂,姜传英,等.一株能拮抗3种土传病害病原真菌的长枝木霉[J].草业科学,2017,34(2):246-254.
[16]张丽荣,康萍芝,沈瑞清.木霉菌对土传病害病原真菌的拮抗作用[J].内蒙古农业科技,2007,1(5):48-50.
[17] ZIMAND G,ELAD Y,CHET I. Effect of Trichoderma harzianum on Botrytis cinerea pathogenicity[J].Phytopathology,1996,86(11):1255-1260.
[18] BERG G. Plant-microbe interactions promoting plant growth and health:Perspectives for controlled use of microorganisms in agriculture[J]. Applied Microbiology and Biotechnology,2009,84(1):11-18.
[19]朱双杰.哈茨木霉对植物的促生作用及其机制研究[D].合肥:安徽农业大学,2007.
[20]刘爱荣,陈双晨,陈凯,等.哈茨木霉对黄瓜尖孢镰刀菌的抑制作用和抗性相关基因表达[J].植物保护学报,2010,37(3):249-254.
[21]韩勇军.哈茨木霉对禾谷镰孢病原菌的抑菌活性研究[J].湖北农业科学,2010,49(2):356-358.
[22]高苇,王勇,张春祥.番茄灰霉病拮抗细菌的筛选与鉴定[J].植物保护导刊,2016,36(8):16-19,25.
[2]于海龙,古瑜,韩启厚,等.多菌灵对豇豆枯萎病病原菌抑菌效果及包衣后对种子生活力的影响[J].北方园艺,2013,1(12):113-115.
[3]吴仁锋,杨绍丽,万鹏,等.豇豆枯萎病病原分离鉴定[J].湖北大学学报(自然科学版),2012,34(1):100-104.
[4]姜素平,张健,秦盛,等.抗茄链格孢菌放线菌的筛选及鉴定[J].江苏农业学报,2017,33(3):543-549.
[5]高雪丽.侧钩木霉Y01的分离、鉴定与固态发酵产孢研究[D].杭州:浙江大学,2014.
[6]覃柳燕,郭成林,黄素梅,等.棘孢木霉菌株PZ6对香蕉促生效应及枯萎病室内防效的影响[J].南方农业学报,2017,48(2):277-283.
[7] KHAN B,AKASH Z,ASAD S,et al. Antagonistic potential of Trichoderma harzianum against Fusarium oxysporum f. sp.cubense associated with panama wilt of banana[J].Pakistan Journal of Phytopathology,2017,29(1):111-116.
[8]张鹤,杜国栋,宋亚楠,等.防治草莓根腐病的木霉菌筛选、鉴定及其防病效果[J].沈阳农业大学学报,2015,46(6):654-660.
[9] PRIYA S,UPADHYAY J P. Antagonistic potential of Trichoderma harzianum against Rhizoctonia solani causing banded leaf and sheath blight of maize[J].International Journal of Current Microbiology and Applied Sciences,2017,6(10):886-890.
[10]熊敏.拮抗黄瓜枯萎病菌的木霉菌株筛选及主要生防机理研究[D].长沙:湖南农业大学,2011.
[11]朱萍萍,凌健,席亚东,等.蔬菜土传病害生防木霉菌株资源的筛选及其防治效果评价[J].中国蔬菜,2015,1(8):28-33.
[12] KHALIQ A,KHAN H,MARIA,et al. In vitro efficacy of Trichoderma harzianum Rifai against various isolates of Alternaria sp[J].Journal of Entomology and Zoology Studies,2017,5(1):442-445.
[13]方中达.植病研究法[M].第三版.北京:中国农业出版社,2001.
[14]起登风,邹良平,周登博,等.GA1-2菌株的分离鉴定及其对香蕉尖孢镰刀菌的抑菌效果[J].植物保护学报,2017,44(5):809-816.
[15]常媛,杨兴堂,姜传英,等.一株能拮抗3种土传病害病原真菌的长枝木霉[J].草业科学,2017,34(2):246-254.
[16]张丽荣,康萍芝,沈瑞清.木霉菌对土传病害病原真菌的拮抗作用[J].内蒙古农业科技,2007,1(5):48-50.
[17] ZIMAND G,ELAD Y,CHET I. Effect of Trichoderma harzianum on Botrytis cinerea pathogenicity[J].Phytopathology,1996,86(11):1255-1260.
[18] BERG G. Plant-microbe interactions promoting plant growth and health:Perspectives for controlled use of microorganisms in agriculture[J]. Applied Microbiology and Biotechnology,2009,84(1):11-18.
[19]朱双杰.哈茨木霉对植物的促生作用及其机制研究[D].合肥:安徽农业大学,2007.
[20]刘爱荣,陈双晨,陈凯,等.哈茨木霉对黄瓜尖孢镰刀菌的抑制作用和抗性相关基因表达[J].植物保护学报,2010,37(3):249-254.
[21]韩勇军.哈茨木霉对禾谷镰孢病原菌的抑菌活性研究[J].湖北农业科学,2010,49(2):356-358.
[22]高苇,王勇,张春祥.番茄灰霉病拮抗细菌的筛选与鉴定[J].植物保护导刊,2016,36(8):16-19,25.