短密木霉菌株GAS1-1的分离鉴定、拮抗作用及其生物学特性
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摘要
为了解短密木霉Trichoderma brevicompactum对植物病害的生防作用及其生物学特性,利用稀释平板分离法从甘肃省景泰县马铃薯连作田植株根际土壤中分离获得1株木霉菌株GAS1-1,经形态观察、rDNA-ITS和EF-1α序列分析明确其分类地位;用生物学方法研究该菌的营养生长和产孢条件;采用对峙培养法测定该菌株对5种植物病原真菌的抑制作用。结果表明,基于形态学特征和基因序列分析结果,菌株GAS1-1被鉴定为短密木霉Trichoderma brevicompactum,为甘肃省木霉新记录种。该菌株对玉米镰孢茎腐病菌Fusarium graminearum、玉米穗腐病菌F. verticillioides、棉花枯萎病菌F. oxysporum、玉米腐霉茎腐病菌Pythium inflatum和灰葡萄孢霉Botrytis cinerea均具有较好的拮抗效果,尤其对玉米腐霉茎腐病菌的抑制作用最好,抑菌率达100.00%。该菌株营养生长和产孢的最适碳源和氮源分别为葡萄糖和酵母膏;其在15～35℃均可生长,菌丝最适生长温度为30℃,最佳产孢温度为25℃;在pH 5～12的培养基上菌丝均可生长,菌丝最适生长和产孢的pH均为5;24 h黑暗条件下菌丝营养生长最快,12 h光暗交替条件有利于产孢;孢子致死温度为69℃,10 min。表明短密木霉菌株GAS1-1具有较好的生防应用潜力。
In order to investigate the biological characteristics of Trichoderma brevicompactum and biocontrol of T. brevicompactum against plant diseases, a Trichoderma strain GAS1-1 was isolated from rhizosphere soils of continuously cropped potato in Jingtai County, Gansu Province. The taxonomic status of strain GAS1-1 was confirmed with morphologic observation, rDNA-ITS and EF-1α sequence analysis. Nutritional growth and sporulation conditions of strain GAS1-1 were ascertained by using biological method. The inhibitory effect of the strain GAS1-1 against five plant pathogenic fungi was determined by dual culture. The results of morphological and sequence analysis showed that the strain GAS1-1 was identified as T. brevicompactum, which is a firstly recorded Trichoderma species in Gansu. The strain showed strong antagonistic activity against Fusarium graminearum, F. verticillioides, F. oxysporum, Pythium inflatum and Botrytis cinerea. In particular, it produced the highest antifungal activity against P. inflatum with an inhibition ratio of 100.00%. The study of biological characteristics showed that the optimum carbon and nitrogen sources of the strain were glucose and yeast extract for mycelial growth and sporulation, respectively. The strain could grow at 15-35℃, and the optimal hyphal growth and sporulation temperatures were 30℃ and 25℃, respectively. The strain could grow at pH 5-12, and the optimal pH value for hyphal growth and sporulation was pH 5. The most favorable condition for mycelial growth was 24 h dark photoperiod, while the condition of alternative 12 h light and 12 h dark was the most favorable to its sporulation. The lethal temperature for spores was 69℃ for 10 min. It suggested that this strain of T. brevicompactum has biocontrol potential against plant diseases.
In order to investigate the biological characteristics of Trichoderma brevicompactum and biocontrol of T. brevicompactum against plant diseases, a Trichoderma strain GAS1-1 was isolated from rhizosphere soils of continuously cropped potato in Jingtai County, Gansu Province. The taxonomic status of strain GAS1-1 was confirmed with morphologic observation, rDNA-ITS and EF-1α sequence analysis. Nutritional growth and sporulation conditions of strain GAS1-1 were ascertained by using biological method. The inhibitory effect of the strain GAS1-1 against five plant pathogenic fungi was determined by dual culture. The results of morphological and sequence analysis showed that the strain GAS1-1 was identified as T. brevicompactum, which is a firstly recorded Trichoderma species in Gansu. The strain showed strong antagonistic activity against Fusarium graminearum, F. verticillioides, F. oxysporum, Pythium inflatum and Botrytis cinerea. In particular, it produced the highest antifungal activity against P. inflatum with an inhibition ratio of 100.00%. The study of biological characteristics showed that the optimum carbon and nitrogen sources of the strain were glucose and yeast extract for mycelial growth and sporulation, respectively. The strain could grow at 15-35℃, and the optimal hyphal growth and sporulation temperatures were 30℃ and 25℃, respectively. The strain could grow at pH 5-12, and the optimal pH value for hyphal growth and sporulation was pH 5. The most favorable condition for mycelial growth was 24 h dark photoperiod, while the condition of alternative 12 h light and 12 h dark was the most favorable to its sporulation. The lethal temperature for spores was 69℃ for 10 min. It suggested that this strain of T. brevicompactum has biocontrol potential against plant diseases.
引文
Bae H.2011.Trichoderma species as abiotic and biotic stress quenchers in plants.Research Journal of Biotechnology,6(3):73-79
Burmeister L,Hau B.2009.Control of the bean rust fungus Uromyces appendiculatus by means of Trichoderma harzianum:leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates.BioControl,54(4):575-585
Carbone I,Kone LM.1999.A method for designing primer sets for speciation studies in filamentous ascomycetes.Mycologia,91(3):553-556
Dawidziuk A,Popiel D,Kaczmarek J,Strakowska J,Jedryczka M.2016.Optimal Trichoderma strains for control of stem canker of brassicas:molecular basis of biocontrol propertiesand azole resistance.BioControl,61(6):755-768
Fang ZD.1998.Study method of plant disease(3rd edition).Beijing:China Agriculture Press,pp.122-137(in Chinese)[方中达.1998.植病研究方法(第3版).北京:中国农业出版社,pp.122-137]
Harman GE,Petzoldt R,Comis A,Chen J.2004.Interactions between Trichoderma harzianum strain T22 and maize inbred line Mo17and effects of these interactions on diseases caused by Pythium ultimum and Colletotrichum graminicola.Phytopathology,94(2):147-153
Ghisalberti EL.2002.Anti-infective agents produced by the hyphomycetes genera Trichoderma and Gliocladium.Current Medicinal Chemistry,1(4):343-374
Gu ZM,Bi H.2016.Influence factors of control efficacy against cucumber Fusarium wilt with Trichoderma longibrachiatum.Agrochemicals,55(1):58-60(in Chinese)[谷祖敏,毕卉.2016.长枝木霉THLJ菌株防治黄瓜枯萎病影响因素分析.农药,55(1):58-60]
Guo C,Zhang XR,Zhang JG,Li MQ,Xu SJ,Wang TY.2016.Biological characteristics of Trichoderma rossicum strain GAU 1-X-2.Pratacultural Science,33(12):2391-2397(in Chinese)[郭成,张新瑞,张军高,李敏权,徐生军,王天玉.2016.俄罗斯木霉菌株GAU 1-X-2的生物学特性.草业科学,33(12):2391-2397]
Kang PZ,Zhang LR,Shen RQ.2007.Antagonism of eleven Trichoderma species against Botrytis cinerea in grape.Chinese Agricultural Science Bulletin,23(8):392-395(in Chinese)[康萍芝,张丽荣,沈瑞清.2007.11种木霉菌对葡萄灰霉病菌的拮抗作用.中国农学通报,23(8):392-395]
Kopchinskiy A,Komon M,Kubicek CP,Druzhinina IS.2005.Tricho BLAST:a multilocus database for Trichoderma and Hypocrea identifications.Mycological Research,109(6):658-660
Krauss U,Soberanis W.2002.Effect of fertilization and biocontrol application frequency on cocoa pod disease.Biological Control,24(1):82-89
Li SP,Cui LL,Cheng JS,Chen T,Fu YP,Xie JT.2018.Assessment of two Trichoderma harzianum strains for biocontrol against rice sheath blight and growth promotion of rice.Acta Phytopathologica Sinica,48(1):98-107(in Chinese)[李松鹏,崔琳琳,程家森,陈桃,付艳苹,谢甲涛.2018.两株哈茨木霉菌株防治水稻纹枯病及促进水稻生长的潜力研究.植物病理学报,48(1):98-107]
Lin RZ.2016.Northeast farmland Trichoderma identification,evaluation and cellulose activity optimization of fermentation conditions.Master Thesis.Shenyang:Shenyang Agricultural University(in Chinese)[林润泽.2016.东北农田木霉菌的鉴定、酶活评价及其纤维素酶发酵工艺.硕士学位论文.沈阳:沈阳农业大学]
Liu LN,Tu YL,Zhang JZ.2010.Biocontrol potential of Trichoderma virens strain TY009 against rice sheath blight and other main fungal diseases.Scientia Agricultura Sinica,43(10):2031-2038(in Chinese)[刘路宁,屠艳拉,张敬泽.2010.绿木霉菌株TY009防治纹枯病等水稻主要真菌病害的潜力.中国农业科学,43(10):2031-2038]
Qin WT,Chen K,Zhuang WY.2016.Five Trichoderma species new to China and notes on two other widespread species.Mycosystema,35(8):994-1007(in Chinese)[秦文韬,陈凯,庄文颖.2016.木霉属5个中国新记录种及2种木霉在中国的新分布.菌物学报,35(8):994-1007]
Saravanar K,Yu CJ,Dou K,Wang M,Li YQ,Chen J.2016.Synergistic effect of Trichoderma-derived antifungal metabolites and cell wall degrading enzymes on enhanced biocontrol of Fusarium oxysporum f.sp.cucumerinum.Biological Control,94(23):37-46
Shi FY,Zhu YP,Yang WL.2005.Studies on antagonism of Trichoderma longibrachiatum against Rhizoctonia solania in rice.Chinese Agricultural Science Bulletin,21(2):264-265,271(in Chinese)[史凤玉,朱英波,杨文兰.2005.长枝木霉T8对水稻纹枯病菌拮抗的作用研究.中国农学通报,21(2):264-265,271]
Wang F.2006.Studies on the antagonistic mechanism,promotion and preparation of Trichoderma aureoviride.Master Thesis.Lanzhou:Gansu Agricultural University(in Chinese)[王芳.2006.深绿木霉的拮抗机理、促生作用及制剂加工研究.硕士学位论文.兰州:甘肃农业大学]
Wang Y,Wang WL,Huo JF,Liu CY,Hao YJ.2012.The screen of the antagonism of Trichoderma spp.against Botrytis cinerea and sequence analysis of translation elongation factor.Chinese Agricultural Science Bulletin,28(9):209-213(in Chinese)[王勇,王万立,霍建飞,刘春艳,郝永娟.2012.灰葡萄孢霉高效拮抗木霉菌株的筛选及其翻译延伸因子序列分析.中国农学通报,28(9):209-213]
Weindling R.1932.Trichoderma lignorum as a parasite of other soil fungi.Phytopathology,22:837-845
Wu ZY,Shen SL,Mi F,Mao WL.2017.Diversity and antagonizing effects of Trichoderma spp.in crop rhizosphere soils.Journal of Fungal Research,15(3):177-182,187(in Chinese)[吴紫燕,沈少力,糜芳,毛伟力.2017.作物根围土壤木霉菌物种多样性及其体外拮抗病原菌效应.菌物研究,15(3):177-182,187]
Yang HT.2009.Classification and identification of Trichoderma.Beijing:China Earth Press,pp.1-389(in Chinese)[杨合同.2009.木霉分类与鉴定.北京:中国大地出版社,pp.1-389]
Yang XT.2016.The identification,biological characteristics and inhibitive effect of soil-borne pathogenic fungi of three kinds of Trichoderma spp.and the growth-promoting on Impatiens balsamina.Master Thesis.Harbin:Northeast Forestry University(in Chinese)[杨兴堂.2016.3株木霉菌的鉴定、生物学特性和抑菌能力及其对凤仙花的促生作用.硕士学位论文.哈尔滨:东北林业大学]
Zhang SW,Gan YT,Xu BL.2014.Efficacy of Trichoderma longibrachiatum in the control of Heterodera avenae.BioControl,59(3):319-331
Zhu TH,Xing XP,Sun SD.2004.The antagonism mechanism and diseases control trials of Trichoderma strain T97against several plant fungal pathogens in greenhouse.Journal of Plant Protection,31(2):139-144(in Chinese)[朱廷恒,邢小平,孙顺娣.2004.木霉T97菌株对几种植物病原真菌的拮抗作用机制和温室防治试验.植物保护学报,31(2):139-144]
Zhu ZX,Zhuang WY.2014.Current understanding of the genus Trichoderma(Hypocreales,Ascomycota).Mycosystema,33(6):1136-1153(in Chinese)[朱兆香,庄文颖.2014.木霉属研究概况.菌物学报,33(6):1136-1153]
Burmeister L,Hau B.2009.Control of the bean rust fungus Uromyces appendiculatus by means of Trichoderma harzianum:leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates.BioControl,54(4):575-585
Carbone I,Kone LM.1999.A method for designing primer sets for speciation studies in filamentous ascomycetes.Mycologia,91(3):553-556
Dawidziuk A,Popiel D,Kaczmarek J,Strakowska J,Jedryczka M.2016.Optimal Trichoderma strains for control of stem canker of brassicas:molecular basis of biocontrol propertiesand azole resistance.BioControl,61(6):755-768
Fang ZD.1998.Study method of plant disease(3rd edition).Beijing:China Agriculture Press,pp.122-137(in Chinese)[方中达.1998.植病研究方法(第3版).北京:中国农业出版社,pp.122-137]
Harman GE,Petzoldt R,Comis A,Chen J.2004.Interactions between Trichoderma harzianum strain T22 and maize inbred line Mo17and effects of these interactions on diseases caused by Pythium ultimum and Colletotrichum graminicola.Phytopathology,94(2):147-153
Ghisalberti EL.2002.Anti-infective agents produced by the hyphomycetes genera Trichoderma and Gliocladium.Current Medicinal Chemistry,1(4):343-374
Gu ZM,Bi H.2016.Influence factors of control efficacy against cucumber Fusarium wilt with Trichoderma longibrachiatum.Agrochemicals,55(1):58-60(in Chinese)[谷祖敏,毕卉.2016.长枝木霉THLJ菌株防治黄瓜枯萎病影响因素分析.农药,55(1):58-60]
Guo C,Zhang XR,Zhang JG,Li MQ,Xu SJ,Wang TY.2016.Biological characteristics of Trichoderma rossicum strain GAU 1-X-2.Pratacultural Science,33(12):2391-2397(in Chinese)[郭成,张新瑞,张军高,李敏权,徐生军,王天玉.2016.俄罗斯木霉菌株GAU 1-X-2的生物学特性.草业科学,33(12):2391-2397]
Kang PZ,Zhang LR,Shen RQ.2007.Antagonism of eleven Trichoderma species against Botrytis cinerea in grape.Chinese Agricultural Science Bulletin,23(8):392-395(in Chinese)[康萍芝,张丽荣,沈瑞清.2007.11种木霉菌对葡萄灰霉病菌的拮抗作用.中国农学通报,23(8):392-395]
Kopchinskiy A,Komon M,Kubicek CP,Druzhinina IS.2005.Tricho BLAST:a multilocus database for Trichoderma and Hypocrea identifications.Mycological Research,109(6):658-660
Krauss U,Soberanis W.2002.Effect of fertilization and biocontrol application frequency on cocoa pod disease.Biological Control,24(1):82-89
Li SP,Cui LL,Cheng JS,Chen T,Fu YP,Xie JT.2018.Assessment of two Trichoderma harzianum strains for biocontrol against rice sheath blight and growth promotion of rice.Acta Phytopathologica Sinica,48(1):98-107(in Chinese)[李松鹏,崔琳琳,程家森,陈桃,付艳苹,谢甲涛.2018.两株哈茨木霉菌株防治水稻纹枯病及促进水稻生长的潜力研究.植物病理学报,48(1):98-107]
Lin RZ.2016.Northeast farmland Trichoderma identification,evaluation and cellulose activity optimization of fermentation conditions.Master Thesis.Shenyang:Shenyang Agricultural University(in Chinese)[林润泽.2016.东北农田木霉菌的鉴定、酶活评价及其纤维素酶发酵工艺.硕士学位论文.沈阳:沈阳农业大学]
Liu LN,Tu YL,Zhang JZ.2010.Biocontrol potential of Trichoderma virens strain TY009 against rice sheath blight and other main fungal diseases.Scientia Agricultura Sinica,43(10):2031-2038(in Chinese)[刘路宁,屠艳拉,张敬泽.2010.绿木霉菌株TY009防治纹枯病等水稻主要真菌病害的潜力.中国农业科学,43(10):2031-2038]
Qin WT,Chen K,Zhuang WY.2016.Five Trichoderma species new to China and notes on two other widespread species.Mycosystema,35(8):994-1007(in Chinese)[秦文韬,陈凯,庄文颖.2016.木霉属5个中国新记录种及2种木霉在中国的新分布.菌物学报,35(8):994-1007]
Saravanar K,Yu CJ,Dou K,Wang M,Li YQ,Chen J.2016.Synergistic effect of Trichoderma-derived antifungal metabolites and cell wall degrading enzymes on enhanced biocontrol of Fusarium oxysporum f.sp.cucumerinum.Biological Control,94(23):37-46
Shi FY,Zhu YP,Yang WL.2005.Studies on antagonism of Trichoderma longibrachiatum against Rhizoctonia solania in rice.Chinese Agricultural Science Bulletin,21(2):264-265,271(in Chinese)[史凤玉,朱英波,杨文兰.2005.长枝木霉T8对水稻纹枯病菌拮抗的作用研究.中国农学通报,21(2):264-265,271]
Wang F.2006.Studies on the antagonistic mechanism,promotion and preparation of Trichoderma aureoviride.Master Thesis.Lanzhou:Gansu Agricultural University(in Chinese)[王芳.2006.深绿木霉的拮抗机理、促生作用及制剂加工研究.硕士学位论文.兰州:甘肃农业大学]
Wang Y,Wang WL,Huo JF,Liu CY,Hao YJ.2012.The screen of the antagonism of Trichoderma spp.against Botrytis cinerea and sequence analysis of translation elongation factor.Chinese Agricultural Science Bulletin,28(9):209-213(in Chinese)[王勇,王万立,霍建飞,刘春艳,郝永娟.2012.灰葡萄孢霉高效拮抗木霉菌株的筛选及其翻译延伸因子序列分析.中国农学通报,28(9):209-213]
Weindling R.1932.Trichoderma lignorum as a parasite of other soil fungi.Phytopathology,22:837-845
Wu ZY,Shen SL,Mi F,Mao WL.2017.Diversity and antagonizing effects of Trichoderma spp.in crop rhizosphere soils.Journal of Fungal Research,15(3):177-182,187(in Chinese)[吴紫燕,沈少力,糜芳,毛伟力.2017.作物根围土壤木霉菌物种多样性及其体外拮抗病原菌效应.菌物研究,15(3):177-182,187]
Yang HT.2009.Classification and identification of Trichoderma.Beijing:China Earth Press,pp.1-389(in Chinese)[杨合同.2009.木霉分类与鉴定.北京:中国大地出版社,pp.1-389]
Yang XT.2016.The identification,biological characteristics and inhibitive effect of soil-borne pathogenic fungi of three kinds of Trichoderma spp.and the growth-promoting on Impatiens balsamina.Master Thesis.Harbin:Northeast Forestry University(in Chinese)[杨兴堂.2016.3株木霉菌的鉴定、生物学特性和抑菌能力及其对凤仙花的促生作用.硕士学位论文.哈尔滨:东北林业大学]
Zhang SW,Gan YT,Xu BL.2014.Efficacy of Trichoderma longibrachiatum in the control of Heterodera avenae.BioControl,59(3):319-331
Zhu TH,Xing XP,Sun SD.2004.The antagonism mechanism and diseases control trials of Trichoderma strain T97against several plant fungal pathogens in greenhouse.Journal of Plant Protection,31(2):139-144(in Chinese)[朱廷恒,邢小平,孙顺娣.2004.木霉T97菌株对几种植物病原真菌的拮抗作用机制和温室防治试验.植物保护学报,31(2):139-144]
Zhu ZX,Zhuang WY.2014.Current understanding of the genus Trichoderma(Hypocreales,Ascomycota).Mycosystema,33(6):1136-1153(in Chinese)[朱兆香,庄文颖.2014.木霉属研究概况.菌物学报,33(6):1136-1153]