烟草育苗基质典型腐生真菌的分离鉴定及其碳源代谢特征分析
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摘要
【目的】烤烟育苗基质在育苗期易出现腐生真菌生长,本研究旨在了解腐生真菌的种类及其碳源代谢特征。【方法】采用组织分离培养法对腐生真菌进行分离,采用形态学和rDNA-ITS分析的方法对其进行鉴定,并利用Biolog代谢对其碳源代谢特征进行分析。【结果】共分离纯化获得3株腐生真菌,经鉴定确定菌株JZ-1和JZ-2为Trichoderma harzianum,菌株JZ-3为Trichoderma asperellum。这些腐生真菌创伤接菌时均可使烟苗发病,基质接菌时均不能使烟苗发病。它们能利用Biolog FF代谢板上44种碳源,其中赤藓糖醇、L-海藻糖、乳果糖、麦芽三糖、D-甘露糖、D-松三糖和D-山梨醇能促进其产孢。【结论】"木霉属真菌"是育苗基质上的腐生菌之一,具有较强的碳源代谢能力,研究结果对烟草育苗基质腐生真菌的认识奠定了基础。
Flue-cured tobacco seedling substrate is prone to saprophytic fungi growth. The purpose of this study is to understand the species of saprophytic fungi and their carbon source metabolism. Saprophytic fungi were isolated by tissue culture method and identi?ed by morphology and rDNA-ITS analysis. Metabolism analysis on Biolog FF metabolic plates was performed to analyze carbon source metabolism characteristics of the pathogens. Three strains of saprophytic fungi were isolated and puri?ed. Strains JZ-1 and JZ-2 were identi?ed as Trichoderma harzianum, strain JZ-3 was Trichoderma asperellum. Saprophytic fungi can infect tobacco seedlings by wounded inoculation method on tobacco leaves, while not by inoculation directly on seedling substrate. These fungi metabolized the 44 carbon sources on Biolog FF metabolic plate, among which, erythritol, L-trehalose, lactulose, maltotriose, D-mannose, D-melezitose, and D-sorbitol can promote its conidia production. It is concluded that Trichoderma is one of the saprophytic fungi existing on tobacco seedling substrate,which has strong carbon source metabolism ability. Results of this study may help to enhance our understanding of saprophytic fungi on tobacco seedling substrate.
Flue-cured tobacco seedling substrate is prone to saprophytic fungi growth. The purpose of this study is to understand the species of saprophytic fungi and their carbon source metabolism. Saprophytic fungi were isolated by tissue culture method and identi?ed by morphology and rDNA-ITS analysis. Metabolism analysis on Biolog FF metabolic plates was performed to analyze carbon source metabolism characteristics of the pathogens. Three strains of saprophytic fungi were isolated and puri?ed. Strains JZ-1 and JZ-2 were identi?ed as Trichoderma harzianum, strain JZ-3 was Trichoderma asperellum. Saprophytic fungi can infect tobacco seedlings by wounded inoculation method on tobacco leaves, while not by inoculation directly on seedling substrate. These fungi metabolized the 44 carbon sources on Biolog FF metabolic plate, among which, erythritol, L-trehalose, lactulose, maltotriose, D-mannose, D-melezitose, and D-sorbitol can promote its conidia production. It is concluded that Trichoderma is one of the saprophytic fungi existing on tobacco seedling substrate,which has strong carbon source metabolism ability. Results of this study may help to enhance our understanding of saprophytic fungi on tobacco seedling substrate.
引文
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[18]张广志,杨合同,文成敬.木霉菌形态学分类检索与分子生物学鉴定[J].山东农业大学学报,2011,42(02):309-316.ZHANG Guangzhi,YANG Hetong,WEN Chengjing.The key to the morphological classification and the molecular identification of Trichoderma spp.[J].Journal of Shandong Agricultural University,2011,42(02):309-316.
[19]张广志,杨合同,张新建,等.木霉现有种类名录[J].菌物学报,2014,33(06):1210-1230.ZHANG Guangzhi,YANG Hetong,ZHANG Xinjian.A checklist of known species of Trichoderma[J].Mycosystema,2014,33(06):1210-1230.
[20]Lorito M,Woo SL,Harman GE,Monte E,et al.Translational research on Trichoderma:from'omics to the field[J].Ann Rev Phytopathol,2010,48:395-417.
[21]陈捷,朱洁伟,张婷,等.木霉菌生物防治作用机理与应用研究进展[J].中国生物防治学报,2011,27(02):145-151.CHEN Jie,ZHU Jiewei,ZHANG Ting,et al.Progress on mechanism and applications of Trichoderma as a biocontrol microbe[J].Chinese Journal of Biological Control,2011,27(02):145-151.
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[23]庄敬华,陈捷,杨长成,等.生防木霉菌生物安全性评价[J].中国农业科学,2006(04):715-720.ZHUANG Jinghua,CHEN Jie,YANG Changcheng,et al.Evaluation of biocontrol Trichoderma on biology security[J].Scientia Agricultura Sinica,2006(04):715-720.
[2]时向东,刘国顺,陈江华,等.烟草漂浮育苗系统中培养基质对烟苗生长发育影响的研究[J].中国烟草学报,2001(01):19-23.SHI Xiangdong,LIU Guoshun,CHEN Jianghua,et al.Effects of medium on tobacco transplant growth and development in floating system[J].Acta Tabacaria Sinica,2001(01):19-23.
[3]彭细桥,吴践志,陆中山,等.我国烟草漂浮育苗技术应用现状、研究进展及发展方向[J].中国烟草学报,2010,16(03):90-94.PENG Xiqiao,WU Jianzhi,LU Zhongshan,et al.Current status of application,research progress and future development in tobacco floating-bed seedling production technology in China[J].Acta Tabacaria Sinaca,2010,16(03):90-94.
[4]Wang Hancheng,Wang Jin,Li Licui,et al.Metabolic activities of five botryticides against Botrytis cinerea examined using the Biolog FF MicroPlate[J].Scientific Reports,2016,6:31025.
[5]汪汉成,张敏,张之矾,等.多菌灵等5种杀菌剂对烟草立枯病菌的生物活性[J].农药学学报,2017,19(5):569-575.WANG Hancheng,ZHANG Min,ZHANG Zhifan,et al.Bioactivities of carbendazim,etc.five fungicides against Rhizoctonia solani in tobacco[J]Chinese Journal of Pesticide Science,2017,19(5):569-575.
[6]李小杰,李淑君,李成军,等.鸢尾丝囊霉(Aphanomyces iridis)引起的烟草漂浮苗根腐病的初步报道[J].中国烟草学报,2018,24(2):125-128.LI Xiaojie,LI Shujun,LI Chengjun,et al.Preliminary report on tobacco seedling root rot induced by Aphanomyces iridis in fl oating bed[J]Acta Tabacaria Sinica,2018,24(2):125-128.
[7]封幸兵,冯柱安,唐斌,等.营养液深度对砂培漂浮育苗的影响[J].烟草科技,2009,9:59-61.FENG Xingbing,FENG Zhuan,TANG Bin,et al.Effects of liquid nutrient depth on float seedlings cultured in sand[J]Tobacco Science&Technology,2009,9:59-61.
[8]方中达.植病研究方法[M].北京:中国农业出版社,1998:122-125.FANG Zhongda.Methodology for Plant Pathology[M].Beijing:China Agriculture Press,1998:122-125.
[9]姚粟,程池,李金霞,等.Biolog微生物自动分析系统--丝状真菌鉴定操作规程的研究[J].食品与发酵工业,2006,(08):63-67.YAO Su,CHENG Chi,LI Jinxia,et al.Biolog microbial identification system-study on the operating regulation of filamentous fungi identification[J].Food and Fermentation Industries,2006,(08):63-67.
[10]林生,庄家强,陈婷,等.福建安溪不同年限茶树土壤养分与微生物Biolog功能多样性的差异分析[J].中国生态农业学报,2012,20(11):1471-1477.LIN Sheng,ZHUANG Jiaqiang,CHEN Ting,et al.Analysis of nutrient and microbial Biolog function diversity in tea soils with different planting years in Fujian Anxi[J].Chinese Journal of EcoAgriculture,2012,20(11):1471-1477.
[11]李运,盛慧,赵荣华.Biolog微生物鉴定系统在菌种鉴定中的应用[J].酿酒科技,2005(07):84-85.LI Yun,SHENG Hui,ZHAO Ronghua.Utilization of Biolog microbes identification system in the identification of microbial species[J].Liq-Mak Sci Technol,2005(07):84-85.
[12]燕勇,李卫平,高雯洁,等.rDNA-ITS序列分析在真菌鉴定中的应用[J].中国卫生检验杂志,2008,(10):1958-1961.YAN Yong,LI Weiping,GAO Weijie,et al.Application of rDNAITS sequence analysis in fungus identification[J].Chinese Journal of Health Laboratory Technology,2008,(10):1958-1961.
[13]秦健,陈振东,宋焕忠,等.广西苦瓜蔓枯病的病原分离与鉴定[J].植物病理学报,2018,48(02):280-284.QIN Jian,CHEN Zhendong,SONG Huanzhong,et al.Identification of causal organism of gummy stem blight of balsam pear in Guangxi[J].2018,48(02):280-284.
[14]周婧,黄丽云,李钢,等.一株红茄苳内生烟曲霉菌的分离鉴定及其发酵产物抗氧化活性[J].微生物学通报,2017,44(08):1958-1966.ZHOU Jing,HUANG Liyun,LI Gang.Isolation,identification and antioxidant activity Aspergillus fumigatus HQD24 endophytic in Rhizophora mucronata[J].Microbiology China,2017,44(08):1958-1966.
[15]Druzhinina IS,Kopchinskiy AG,KomońM,Bissett J,Szakacs G,Kubicek CP.An oligonucleotide barcode for species identification in Trichoderma,and Hypocrea[J].Fungal Genet Biol,2005,42:813-828.
[16]Kopchinskiy A.Tricho BLAST:a multiloci database of phylogenetic markers for Trichoderma and Hypocrea powered by sequence diagnosis and similarity search tools[J].Mycol Res,2005,109:658-660.
[17]孙军,段玉玺,吕国忠.木霉菌及其系统分类学研究回顾[J].菌物研究,2006(01):57-63.SUN Jun,DUAN Yuxi,LU Guozhong.A review of Trichoderma and its taxonomy[J].Journal of Fungal Research,2006(01):57-63.
[18]张广志,杨合同,文成敬.木霉菌形态学分类检索与分子生物学鉴定[J].山东农业大学学报,2011,42(02):309-316.ZHANG Guangzhi,YANG Hetong,WEN Chengjing.The key to the morphological classification and the molecular identification of Trichoderma spp.[J].Journal of Shandong Agricultural University,2011,42(02):309-316.
[19]张广志,杨合同,张新建,等.木霉现有种类名录[J].菌物学报,2014,33(06):1210-1230.ZHANG Guangzhi,YANG Hetong,ZHANG Xinjian.A checklist of known species of Trichoderma[J].Mycosystema,2014,33(06):1210-1230.
[20]Lorito M,Woo SL,Harman GE,Monte E,et al.Translational research on Trichoderma:from'omics to the field[J].Ann Rev Phytopathol,2010,48:395-417.
[21]陈捷,朱洁伟,张婷,等.木霉菌生物防治作用机理与应用研究进展[J].中国生物防治学报,2011,27(02):145-151.CHEN Jie,ZHU Jiewei,ZHANG Ting,et al.Progress on mechanism and applications of Trichoderma as a biocontrol microbe[J].Chinese Journal of Biological Control,2011,27(02):145-151.
[22]于新,田淑慧,徐文兴,等.木霉菌生防作用的生化机制研究进展[J].中山大学学报(自然科学版),2005(02):86-90.YU Xin,TIAN Shuhui,XU Wenxing,et al.Progress in the biochemical mechanisms of biocontrol effect research with Trichoderma[J].Acta Scientiarum Naturalium Universitatis Sunyatse,2005(02),86-90.
[23]庄敬华,陈捷,杨长成,等.生防木霉菌生物安全性评价[J].中国农业科学,2006(04):715-720.ZHUANG Jinghua,CHEN Jie,YANG Changcheng,et al.Evaluation of biocontrol Trichoderma on biology security[J].Scientia Agricultura Sinica,2006(04):715-720.