烟草青枯病菌在烟草根际的定殖及最适发病条件
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- 英文论文题名：Pathogenic factors of Ralstonia solanacearum to infect tobacco bacterial wilt and its Colonization
- 论文作者：李想 ; 刘艳霞 ; 石俊雄
- 英文论文作者：LI Xiang ; LIU Yanxia ; SHI Junxiong ; Guizhou Academy of Tobacco Science
- 年：2016
- 作者机构：贵州省烟科学研究院;
- 论文关键词：烟草青枯病 ; 茄科劳尔氏菌 ; 致病条件 ; 响应曲面法 ; 绿色荧光蛋白标记 ; 荧光定量PCR
- 英文论文关键词：Tobacco bacterial wilt ; Ralstonia solanacearum ; Pathogenic Factors ; Response Surface Methodology(RSM) ; Green fluorescent protein(gfp)-labeling ; Realtime-PCR
- 会议召开时间：2016-12-01
- 会议录名称：中国烟草学会2016年度优秀论文汇编——烟草农业主题
- 语种：中文
- 分类号：S435.72
- 学会代码：ZGYG
- 学会名称：中国烟草学会
- 页数：16
- 文件大小：794k
- 原文格式：D
摘要
【目的】烟草青枯病由茄科劳尔氏菌引起,由于烟草连作障碍非常严重,烟草青枯病成为影响贵州省烟叶发展的主要病害之一,研究青枯病的发病条件和病原菌在根际的定殖情况为防控青枯病提供重要的理论依据。【方法】采用响应曲面法(response surface methodology)Box-Behnken设计盆栽试验,探索病原菌致病的临界浓度、发病最适温湿度以及对烟株生长的影响;采用绿色荧光蛋白(gfp)标记病原菌,监测其在根际的定殖位置及数量动态变化,荧光定量PCR法测定病原菌在根表、根际和根圈土壤的数量。【结果】利用选择性培养基SMSA从贵州省烟草青枯病发病非常严重的田间土壤中分离到一株强致病力茄科劳尔氏菌菌株,经16S r RNA及回接鉴定为烟草青枯病病原菌(Ralstonia solanacearum);当根际土壤中的茄科劳尔氏菌数量达到10~7 cfu/g soil以上、温度达到30-35℃、湿度达75%以上时烟株易感染暴发青枯病,当茄科劳尔氏菌在根际的数量达到10~8 cfu/g soil、环境温度达到35℃、湿度为90%以上时,病情指数最大;在适宜条件下根际茄科劳尔氏菌病原菌数量显著高于根圈土中病原菌数量。【结论】烟草青枯病发病条件主要取决于病原菌浓度、温度以及湿度三项指标,其中病原菌浓度最为重要,同时病原菌易在根表富集定植,以上结果可为烟草青枯病的生物防控提供理论依据。
【Objective】Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most serious diseases due to the obstacle of succession mono-tobacco worldwide. 【Method】Pot experiments were carried out to evaluate the pathogenic threshold concentration of virulent R. solanacearum, the optimum temperature and humidity to induce tobacco bacterial wilt and effect on tobacco growth. Green fluorescent protein(gfp) labeling technique was applied to monitor the survival dynamics and locations of R. solanacearum on rhizoplane in different conditions. Quantitative data of the population of R. solanacearum on root surface, in rhizosphere and bulk soil were obtained by real-time PCR. 【Result】In this investigation, One virulent R. solanacearum was isolated on a semi-selective medium(SMSA) from a severely wilt-diseased soil in Guizhou province and identified by 16 S r RNA gene and re-inoculation. Tobacco plants were more susceptible to wilt disease when the population of R. solanacearum reached up to 107 colony forming units cfu /g soil, and the environmental temperature was 30-35 oC and the humidity kept 75 % above, and wilt disease reached highest at the population of R. solanacearum was 108 colony forming units cfu /g soil, and the environmental temperature was 35 oC and the humidity kept 90 % by using of Response Surface Methodology(RSM) with Box-Behnken Design. There were much more R. solanacearum colonized on root surface and in rhizosphere soil than that in bulk soil. 【Conclusion】 It was related to the population of the pathogen, the environmental temperature and the humidity in diseased soil that tobacco bacterial wilt occurred seriously or not, while the disease incidence was mainly determined by the population of the pathogen in plant rhizosphere soil. R. solanacearum colonized mostly on the root when it got to the optimum condition. These results would be served for the biological control of tobacco bacterial wilt.
【Objective】Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most serious diseases due to the obstacle of succession mono-tobacco worldwide. 【Method】Pot experiments were carried out to evaluate the pathogenic threshold concentration of virulent R. solanacearum, the optimum temperature and humidity to induce tobacco bacterial wilt and effect on tobacco growth. Green fluorescent protein(gfp) labeling technique was applied to monitor the survival dynamics and locations of R. solanacearum on rhizoplane in different conditions. Quantitative data of the population of R. solanacearum on root surface, in rhizosphere and bulk soil were obtained by real-time PCR. 【Result】In this investigation, One virulent R. solanacearum was isolated on a semi-selective medium(SMSA) from a severely wilt-diseased soil in Guizhou province and identified by 16 S r RNA gene and re-inoculation. Tobacco plants were more susceptible to wilt disease when the population of R. solanacearum reached up to 107 colony forming units cfu /g soil, and the environmental temperature was 30-35 oC and the humidity kept 75 % above, and wilt disease reached highest at the population of R. solanacearum was 108 colony forming units cfu /g soil, and the environmental temperature was 35 oC and the humidity kept 90 % by using of Response Surface Methodology(RSM) with Box-Behnken Design. There were much more R. solanacearum colonized on root surface and in rhizosphere soil than that in bulk soil. 【Conclusion】 It was related to the population of the pathogen, the environmental temperature and the humidity in diseased soil that tobacco bacterial wilt occurred seriously or not, while the disease incidence was mainly determined by the population of the pathogen in plant rhizosphere soil. R. solanacearum colonized mostly on the root when it got to the optimum condition. These results would be served for the biological control of tobacco bacterial wilt.
引文
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[2]Kamal K P,Brian M G.Biological control of plant pathogens[J].The plant Health Instructor,2006:1-25.
[3]Granada G A.Characteristics of colomdian isolates of pseudomonas solanacearum from tobacco[J].Phytopathology,1983,65:1004.
[4]Jansson J K.Marker reporter genes illuminating tools environmental microbiologists[J].Curr Opin Microbiol,2003,6:310-316.
[5]陈燕红,黎永坚,喻国辉,等.绿色荧光蛋白标记的枯草芽胞杆菌r31在西芹根际定殖研究[J].中国农学通报,2014,30(9):237-241.Chen Y H,Li Y J,Yu G H,et al.Colonization of Green Fluorescent Protein Gene Tagged Bacillus subtilis R31 in Celery Rhizosphere.Chinese Agricultural Science Bulletin,2014,30(9):237-241
[6]Cao Y,Zhang Z,Ling N,et al.Bacillus subtilis sqr9 can control fusarium wilt in cucumber by colonizing plant roots[J].Bio Fertil Soils,2011,47:495-506.
[7]Park K,Pau D,Kim K Y,et al.Induced systemic resistance by bacillus vallismortis extn-1 suppressed bacterial wilt in tomato caused by ralstonia solanacearum[J].Plant Pathology,2007,23:22-25.
[8]Roberts D P,Lohrke S M,Meyer S L F,et al.Biocontrol agents applied individually and in combination for suppression of soilborne diseases of cucumber[J].Crop Prot,2005,24:141-155.
[9]Schonfeld J,Heuer H,Van Elsas J D,et al.Specific and sensitive detection of ralstonia solanacearum in soil on the basis of pcr amplification of flic fragments[J].Appl Environ Microbiol,2003,69:7248-7256.
[10]何欣,黄启为,杨兴明,等.香蕉枯萎病致病菌筛选及致病菌浓度对香蕉枯萎病的影响[J].中国农业科学,2010,43(18):3809-3816.He X.,Huang Q W.,Yang X M.,et al.(2010)Screening and identification of pathogen causing banana Fusarium wilt and the relationship between spore suspension concentration and the incidence rate.Scientia Agricultura Sinica,2010,43:3809-3816.
[11]Englerbrecht M C.Modification of a semi-selective medium for the isolation and quantification of pseudomonas solanacearum[J].ACIAR Bact.Wilt News Lett,1994,10:3-5.
[12]Kelman A.The relationship of pathogenicity in pseudomonas solanacearum to colony appearance on a trtrazolium medium[J].Phytopathology,1954,44:693-695.
[13]Hsiao N H,Kirby R.Comparative genomics of streptomyces avermitilis,streptomyces cattleya,streptomyces maritimus and kitasatospora aureofaciens using a streptomyces coelicolor microarray system[J].Anton van Leeuw Int J G,2008,93:1-25.
[14]Zhang H,Yu Z,Huang Q,et al.Isolation,identification and characterization of phytoplankton-lytic bacterium ch-22 against microcystis aeruginosa[J].Limnologica,2011,41:70-77.
[15]Kumar S,Tamura K,Nei M.Mega3:Interaged software for molecular evolutionary genetics analysis and sequence alignment[J].Brief Bioinform,2004,5:150-163.
[16]Koch R.Uber den augenblicklichen stand der bakteriologischen choleradiagnose(in german)[M].Zeitschrift für Hygiene und Infectionskrankheiten,1893:319-333.
[17]肖文丽,关大伟,李俊,等.采用gfp和rfp基因标记评价大豆根瘤菌竞争结瘤能力[J].大豆科学,2010,29(3):366-369.Xiao W L,Guan D W,Li J,et al.Evaluation on the competitiveness of strains of soybean rhizobia marking with gfp and rfp genes.Soybean Science,2010,29:366-369.
[18]Ciampi-Panno L,Fernandez C,Bustamante P,et al.Biological control of bacterial wilt of potatoes caused by pseudomonas solanacearum[J].Am Potato J,1989,66:316-332.
[19]Joseph S,David W R,2002,Molecular cloning:A laboratory manual science press,
[20]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.中华人民共和国国家标准,gb/t 23222.2008General Administration of Quality Supervision,Inspection and Quarantine of the People's Republic of China,Standardization Administration of the People's Republic of China.Grade and investigation methods of tobacco diseases and insect pests,GB/T 23222.2008
[21]French E B,Gutarra L,Aley P,et al.Culture media for ralstonia solanacearum isolation,identification,and maintenace[J].Fitopatologia,1995,30:126-130.
[22]Aslan N,Cebeci Y.Application of box-behnken design and response surface methodology for modeling of some turkish coals[J].Fuel,2007,86:90-97.
[23]Kwak J S.Application of taguchi and response surface methodologies for geometric error in surface grinding process[J].International Journal of Machine Tools and Manufacture,2005,45(3):327-334.
[24]王永红,冉炜,张富国,等.混合菌种固体发酵菜粕生产氨基酸肥料的条件研究[J].中国农业科学,2009,43(10):3530-3540.Wang Y,Ran W,Zhang F G,et al.Study on conditions in solid-state fermentation of rapeseed meal by mixed strains for amino acid fertilizer.Scientia Agricultura Sinica,2009,42:3530-3540.
[25]葛慈斌,刘波,朱育菁,等.青枯雷尔氏菌致病力的番茄组培苗鉴定方法研究[J].武夷科学,2004,20:13-16.Ge C B,Liu B,Zhu Y Q,et al.Study on the methods on identifying the virulence of Ralstonia solanacearum using tissue cultured tomatoes.Wuyi Science Journal,2004,20:13-16.
[26]Yoshimochi T,ZHang Y,Kiba A,et al.Expression of hrpg and activation of response regulator hrpg are controlled by distinct signal cascades in ralstonia solanacearum[J].J Gen Plant Pathol,2009,75:196-204.
[27]Neveu B,Labbe C,Belanger R R.Gfp technology for the study of biocontrol agents in tritrophic interactions:A case study with pseudozyma flocculosa[J].J.Microbiol Meth,2007,68:275-281.
[28]von der Weid I,Artursson V,Seldin L,et al.Antifungal and root surface colonization properties of gfp-tagged paenibacillus brasilensis pb177[J].World Journal of Microbiology and Biotechnology,2005,21(8-9):1591-1597.
[29]Walker R,Rossall S,Ashe M.Colonization of the developing rhizosphere of sugar beet seedlings by potential biocontrol agents applied as seed treatments[J].J.Appl Microbiol,2002,92:228-237.
[30]Celine J,Francois V,Claude A.Soil health through soil disease suppression:Which strategy from descriptors to indicators[J].Soil Bio&Biochem,2007,39:1-23.
[31]Ren X,Zhang N,Cao M,et al.Biological control of tobacco black shank and colonization of tobacco roots by a paenibacillus polymyxa strain c5[J].Bio Fertil Soils,2012,48:613-620.
[32]Zhao Q,Dong C X,Yang X.M.,et al.Biocontrol of fusarium wilt disease for cucumis melo melon using bio-organic fertilized[J].Appl Soil Ecol,2011,47:67-75.
[33]陈宏宇,李晓鸣,王敬国.抗病性不同大豆品种根面及根际微生物区系的变化[J].植物营养与肥料学报,2005,11(6):804-809.Chen H Y,Li X M,Wang J G.Change of microflora in the rhizosplane and rhizosphere of different disease resistance soybean cultivars.Plant Nutrition and Fertilizer Science,2005,11:804-809.
[34]孔维栋,刘可星,廖宗文,等.不同腐熟程度有机物料对土壤微生物群落功能多样性的影响[J].生态学报,2005,25:2292-2296.
[2]Kamal K P,Brian M G.Biological control of plant pathogens[J].The plant Health Instructor,2006:1-25.
[3]Granada G A.Characteristics of colomdian isolates of pseudomonas solanacearum from tobacco[J].Phytopathology,1983,65:1004.
[4]Jansson J K.Marker reporter genes illuminating tools environmental microbiologists[J].Curr Opin Microbiol,2003,6:310-316.
[5]陈燕红,黎永坚,喻国辉,等.绿色荧光蛋白标记的枯草芽胞杆菌r31在西芹根际定殖研究[J].中国农学通报,2014,30(9):237-241.Chen Y H,Li Y J,Yu G H,et al.Colonization of Green Fluorescent Protein Gene Tagged Bacillus subtilis R31 in Celery Rhizosphere.Chinese Agricultural Science Bulletin,2014,30(9):237-241
[6]Cao Y,Zhang Z,Ling N,et al.Bacillus subtilis sqr9 can control fusarium wilt in cucumber by colonizing plant roots[J].Bio Fertil Soils,2011,47:495-506.
[7]Park K,Pau D,Kim K Y,et al.Induced systemic resistance by bacillus vallismortis extn-1 suppressed bacterial wilt in tomato caused by ralstonia solanacearum[J].Plant Pathology,2007,23:22-25.
[8]Roberts D P,Lohrke S M,Meyer S L F,et al.Biocontrol agents applied individually and in combination for suppression of soilborne diseases of cucumber[J].Crop Prot,2005,24:141-155.
[9]Schonfeld J,Heuer H,Van Elsas J D,et al.Specific and sensitive detection of ralstonia solanacearum in soil on the basis of pcr amplification of flic fragments[J].Appl Environ Microbiol,2003,69:7248-7256.
[10]何欣,黄启为,杨兴明,等.香蕉枯萎病致病菌筛选及致病菌浓度对香蕉枯萎病的影响[J].中国农业科学,2010,43(18):3809-3816.He X.,Huang Q W.,Yang X M.,et al.(2010)Screening and identification of pathogen causing banana Fusarium wilt and the relationship between spore suspension concentration and the incidence rate.Scientia Agricultura Sinica,2010,43:3809-3816.
[11]Englerbrecht M C.Modification of a semi-selective medium for the isolation and quantification of pseudomonas solanacearum[J].ACIAR Bact.Wilt News Lett,1994,10:3-5.
[12]Kelman A.The relationship of pathogenicity in pseudomonas solanacearum to colony appearance on a trtrazolium medium[J].Phytopathology,1954,44:693-695.
[13]Hsiao N H,Kirby R.Comparative genomics of streptomyces avermitilis,streptomyces cattleya,streptomyces maritimus and kitasatospora aureofaciens using a streptomyces coelicolor microarray system[J].Anton van Leeuw Int J G,2008,93:1-25.
[14]Zhang H,Yu Z,Huang Q,et al.Isolation,identification and characterization of phytoplankton-lytic bacterium ch-22 against microcystis aeruginosa[J].Limnologica,2011,41:70-77.
[15]Kumar S,Tamura K,Nei M.Mega3:Interaged software for molecular evolutionary genetics analysis and sequence alignment[J].Brief Bioinform,2004,5:150-163.
[16]Koch R.Uber den augenblicklichen stand der bakteriologischen choleradiagnose(in german)[M].Zeitschrift für Hygiene und Infectionskrankheiten,1893:319-333.
[17]肖文丽,关大伟,李俊,等.采用gfp和rfp基因标记评价大豆根瘤菌竞争结瘤能力[J].大豆科学,2010,29(3):366-369.Xiao W L,Guan D W,Li J,et al.Evaluation on the competitiveness of strains of soybean rhizobia marking with gfp and rfp genes.Soybean Science,2010,29:366-369.
[18]Ciampi-Panno L,Fernandez C,Bustamante P,et al.Biological control of bacterial wilt of potatoes caused by pseudomonas solanacearum[J].Am Potato J,1989,66:316-332.
[19]Joseph S,David W R,2002,Molecular cloning:A laboratory manual science press,
[20]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.中华人民共和国国家标准,gb/t 23222.2008General Administration of Quality Supervision,Inspection and Quarantine of the People's Republic of China,Standardization Administration of the People's Republic of China.Grade and investigation methods of tobacco diseases and insect pests,GB/T 23222.2008
[21]French E B,Gutarra L,Aley P,et al.Culture media for ralstonia solanacearum isolation,identification,and maintenace[J].Fitopatologia,1995,30:126-130.
[22]Aslan N,Cebeci Y.Application of box-behnken design and response surface methodology for modeling of some turkish coals[J].Fuel,2007,86:90-97.
[23]Kwak J S.Application of taguchi and response surface methodologies for geometric error in surface grinding process[J].International Journal of Machine Tools and Manufacture,2005,45(3):327-334.
[24]王永红,冉炜,张富国,等.混合菌种固体发酵菜粕生产氨基酸肥料的条件研究[J].中国农业科学,2009,43(10):3530-3540.Wang Y,Ran W,Zhang F G,et al.Study on conditions in solid-state fermentation of rapeseed meal by mixed strains for amino acid fertilizer.Scientia Agricultura Sinica,2009,42:3530-3540.
[25]葛慈斌,刘波,朱育菁,等.青枯雷尔氏菌致病力的番茄组培苗鉴定方法研究[J].武夷科学,2004,20:13-16.Ge C B,Liu B,Zhu Y Q,et al.Study on the methods on identifying the virulence of Ralstonia solanacearum using tissue cultured tomatoes.Wuyi Science Journal,2004,20:13-16.
[26]Yoshimochi T,ZHang Y,Kiba A,et al.Expression of hrpg and activation of response regulator hrpg are controlled by distinct signal cascades in ralstonia solanacearum[J].J Gen Plant Pathol,2009,75:196-204.
[27]Neveu B,Labbe C,Belanger R R.Gfp technology for the study of biocontrol agents in tritrophic interactions:A case study with pseudozyma flocculosa[J].J.Microbiol Meth,2007,68:275-281.
[28]von der Weid I,Artursson V,Seldin L,et al.Antifungal and root surface colonization properties of gfp-tagged paenibacillus brasilensis pb177[J].World Journal of Microbiology and Biotechnology,2005,21(8-9):1591-1597.
[29]Walker R,Rossall S,Ashe M.Colonization of the developing rhizosphere of sugar beet seedlings by potential biocontrol agents applied as seed treatments[J].J.Appl Microbiol,2002,92:228-237.
[30]Celine J,Francois V,Claude A.Soil health through soil disease suppression:Which strategy from descriptors to indicators[J].Soil Bio&Biochem,2007,39:1-23.
[31]Ren X,Zhang N,Cao M,et al.Biological control of tobacco black shank and colonization of tobacco roots by a paenibacillus polymyxa strain c5[J].Bio Fertil Soils,2012,48:613-620.
[32]Zhao Q,Dong C X,Yang X.M.,et al.Biocontrol of fusarium wilt disease for cucumis melo melon using bio-organic fertilized[J].Appl Soil Ecol,2011,47:67-75.
[33]陈宏宇,李晓鸣,王敬国.抗病性不同大豆品种根面及根际微生物区系的变化[J].植物营养与肥料学报,2005,11(6):804-809.Chen H Y,Li X M,Wang J G.Change of microflora in the rhizosplane and rhizosphere of different disease resistance soybean cultivars.Plant Nutrition and Fertilizer Science,2005,11:804-809.
[34]孔维栋,刘可星,廖宗文,等.不同腐熟程度有机物料对土壤微生物群落功能多样性的影响[J].生态学报,2005,25:2292-2296.
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