棉花根系分泌物对枯草芽胞杆菌NCD-2菌株趋化性的影响
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摘要
枯草芽胞杆菌NCD-2菌株在不同棉花品种根际定殖能力表现不同,在感黄萎病品种冀棉11根际的定殖能力最强,而在耐病品种中棉所41根际的定殖能力最弱。本研究收集了5个不同棉花品种的根系分泌物,测定了NCD-2菌株对根系分泌物的趋化作用,结果表明NCD-2菌株对冀棉11根系分泌物的趋化作用最强,而对中棉所41根系分泌物的趋化作用最差。通过柱前衍生高效液相色谱法(AccQ-Tag-HPLC)测定了根系分泌物中氨基酸的成分及含量,结果表明不同氨基酸在不同棉花品种中含量不同。冀棉11的根系分泌物中氨基酸种类最多,可达17种。在中棉所41的根系分泌物中氨基酸种类最少且大多数氨基酸含量均为最低。测定了NCD-2菌株对14种氨基酸标准品的趋化性,结果表明NCD-2菌株对精氨酸(Arg)、丙氨酸(Ala)和赖氨酸(Lys)趋化性最强,但对甘氨酸(Gly)呈现负趋化性。RT-qPCR试验表明冀棉11的根系分泌物可提高cheA和cheD基因在NCD-2菌株中的表达量。室内防治试验表明,NCD-2菌株对冀棉11黄萎病的防效最好,防治效果可达到66.68%。本研究明确了棉花根系分泌物中氨基酸对NCD-2菌株趋化性的影响,该菌株在棉花根际定殖能力与棉花根系分泌物中氨基酸对本菌株的趋化性相一致。
Bacillus subtilis strain NCD-2 had different colonization abilities on the rhizosphere of different cotton cultivars,and the strongest colonization ability was achieved on cotton cultivar Jimian 11,but the weakest colonization was observed on cultivar Zhongmiansuo 41. In this study,the chemotaxis assays were performed to determine the chemotactic response of strain NCD-2 towards root exudates of the five cotton cultivars. Results showed that strain NCD-2 exhibited the strongest chemotactic response toward the root exudates of Jimian 11 and the weakest chemotactic response toward the root exudates of Zhongmiansuo 41. The amino acids from the five cotton cultivars root exudates were analyzed by high performance liquid chromatography method with AccQ-Tag precolumn derivatization( AccQ-Tag-HPLC). Results showed that the concentrations of certain amino acids varied significantly among the cotton cultivars. The root exudates of Jimian 11 have the most species of amino acids up to 17. However,the root exudates of Zhongmiansuo 41 have the least species of amino acids and the lowest concentration of amino acids. Chemotaxis assays were performed to determine the chemotactic responses of strain NCD-2 towards the standard samples of 14 amino acids. Results showed that strain NCD-2 exhibited the strongest chemotactic responses toward Arg,Ala and Lys and negative chemotactic responses toward Gly.RT-q PCR assay showed that the expression of the cheA and cheD gene increased significantly in response to root exudates of Jimian 11. Control effect of cotton verticillium wilt by strain NCD-2 in greenhouse indicated that the highest biocontrol efficacy of 66.68% was achieved on susceptible cultivar Jimian 11,while only 20.69%-25.58% biocontrol efficacy was achieved on resistant cultivar Pima 90. The results of this study showed that the amino acids in cotton root exudates affected the chemotaxis of strain NCD-2,and the colonization ability of strain NCD-2 was consistent with the chemotaxis towards to the amino acids.
Bacillus subtilis strain NCD-2 had different colonization abilities on the rhizosphere of different cotton cultivars,and the strongest colonization ability was achieved on cotton cultivar Jimian 11,but the weakest colonization was observed on cultivar Zhongmiansuo 41. In this study,the chemotaxis assays were performed to determine the chemotactic response of strain NCD-2 towards root exudates of the five cotton cultivars. Results showed that strain NCD-2 exhibited the strongest chemotactic response toward the root exudates of Jimian 11 and the weakest chemotactic response toward the root exudates of Zhongmiansuo 41. The amino acids from the five cotton cultivars root exudates were analyzed by high performance liquid chromatography method with AccQ-Tag precolumn derivatization( AccQ-Tag-HPLC). Results showed that the concentrations of certain amino acids varied significantly among the cotton cultivars. The root exudates of Jimian 11 have the most species of amino acids up to 17. However,the root exudates of Zhongmiansuo 41 have the least species of amino acids and the lowest concentration of amino acids. Chemotaxis assays were performed to determine the chemotactic responses of strain NCD-2 towards the standard samples of 14 amino acids. Results showed that strain NCD-2 exhibited the strongest chemotactic responses toward Arg,Ala and Lys and negative chemotactic responses toward Gly.RT-q PCR assay showed that the expression of the cheA and cheD gene increased significantly in response to root exudates of Jimian 11. Control effect of cotton verticillium wilt by strain NCD-2 in greenhouse indicated that the highest biocontrol efficacy of 66.68% was achieved on susceptible cultivar Jimian 11,while only 20.69%-25.58% biocontrol efficacy was achieved on resistant cultivar Pima 90. The results of this study showed that the amino acids in cotton root exudates affected the chemotaxis of strain NCD-2,and the colonization ability of strain NCD-2 was consistent with the chemotaxis towards to the amino acids.
引文
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[14]Scharf B E,Hynes M F,Alexandre G M.Chemotaxis signaling systems in model beneficial plant-bacteria associations[J].Plant Molecular Biology,2016,90(6):549-559.
[15]Parkinson J S,Hazelbauer G L,Falke J J.Signaling and sensory adaptation in Escherichia coli chemoreceptors:2015 update[J].Trends in Microbiology,2015,23(5):257-266.
[16]Yuan W,Glekas G D,Allen G M,et al.The importance of the interaction of CheD with CheC and the chemoreceptors compared to its enzymatic activity during chemotaxis in Bacillus subtilis[J].PLoS One,2012,7(12):e50689.
[17]Walukiewicz H E,Tohidifar P,Ordal G W,et al.Interactions among the three adaptation systems of Bacillus subtilis chemotaxis as revealed by an in vitro receptor-kinase assay[J].Molecular Microbiology,2014,93(6):1104-1118.
[18]Gao S,Wu H,Yu X,et al.Swarming motility plays the major role in migration during tomato root colonization by Bacillus subtilis SWR01[J].Biological Control,2016,98:11-17.
[19]Guo Q G,Wu Y Y,Li S Z,et al.Functional analysis of ywbB gene to the biofilm formation and rootcolonization in Bacillus subtilis strain NCD-2(in Chinese)[J].Journal of Plant Protection(植物保护学报),2013,40(1):45-50.
[20]Dong L H,Guo Q G,Zhang X Y,et al.Effects of cotton root exudates on the biofilm formation and root colonization of Bacillus subtilis strain NCD-2(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2015,45(5):541-547.
[21]Armenta J M,Cortes D F,Pisciotta J M,et al.Sensitive and rapid method for amino acid quantitation in malaria biological samples using AccQ.Tag ultra performance liquid chromatography-electrospray ionization-M S/M S with multiple reaction monitoring[J].Analytical Chemistry,2010,82(2):548-558.
[22]Wu C C,Jian G L,Wang A L,et al.Primary detection of QTL for verticillium wilt resistance in cotton[J].Molecular Plant Breeding,2010,8(4):680-686.
[23]Zhang N,Wu K,He X,et al.A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11[J].Plant and Soil,2011,344(1-2):87-97.
[24]Zhang N,Wang D D,Liu Y P,et al.Effects of different plant root exudates and their organic acid components on chemotaxis,biofilm formation and colonization by beneficial rhizosphere-associated bacterial strains[J].Plant and Soil,2013,374(1-2):689-700.
[25]Allard-Massicotte R,Tessier L,Lecuyer F,et al.Bacillus subtilis early colonization of Arabidopsis thaliana roots involves multiple chemotaxis receptors[J].M Bio,2016,7(6):e01664-16.
[26]Mark G L,Dow J M,Kiely P D,et al.Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(48):17454-17459.
[27]Wu K,Yuan S,Xun G,et al.Root exudates from two tobacco cultivars affect colonization of Ralstonia solanacearum and the disease index[J].European Journal of Plant Pathology,2014,141(4):667-677.
[28]Yang Y L,Abiola M P,Hofler C,et al.Relation between chemotaxis and consumption of amino acids in bacteria[J].Molecular Microbiology,2015,96(6):1272-1282.
[29]Rico-Jimenez M,Munoz-Martinez F,Garcia-Fontana C,et al.Paralogous chemoreceptors mediate chemotaxis tow ards protein amino acids and the non-protein amino acid gamma-aminobutyrate(GABA)[J].Molecular Microbiology,2013,88(6):1230-1243.
[2]Qiu M H,Zhang R F,Xue C,et al.Application of bio-organic fertilizer can control Fusarium wilt of cucumber plants by regulating microbial community of rhizosphere soil[J].Biology and Fertility of Soils,2012,48(7):807-816.
[3]Chen X H,Koumoutsi A,Scholz R,et al.Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42[J].Nature Biotechnology,2007,25(9):1007-1014.
[4]Fan B,Blom J,Klenk H P,et al.Bacillus amyloliquefaciens,Bacillus velezensis,and Bacillus siamensis form an"operational group B.amyloliquefaciens"within the B.subtilis species complex[J].Frontiers in Microbiology,2017,8:22.
[5]Bulgarelli D,Schlaeppi K,Spaepen S,et al.Structure and functions of the bacterial microbiota of plants[J].Annual Review of Plant Biology,2013,64:807-838.
[6]Liu Y P,Zhang N,Qiu M H,et al.Enhanced rhizosphere colonization of beneficial Bacillus amyloliquefaciens SQR9 by pathogen infection[J].FEM S Microbiology Letters,2014,353(1):49-56.
[7]Bais H P,Fall R,Vivanco J M.Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production[J].Plant Physiology,2004,134(1):307-319.
[8]Bais H P,Weir T L,Perry L G,et al.The role of root exudates in rhizosphere interactions with plants and other organisms[J].Annual Review of Plant Biology,2006,57:233-266.
[9]Gu Y,Hou Y,Huang D,et al.Application of biochar reduces Ralstonia solanacearum infection via effects on pathogen chemotaxis,sw arming motility,and root exudate adsorption[J].Plant and Soil,2016,415(1-2):269-281.
[10]Al-Ali A,Deravel J,Krier F,et al.Biofilm formation is determinant in tomato rhizosphere colonization by Bacillus velezensis FZB42[J].Environmental Science and Pollution Research International,2017,(5):1-11.
[11]Yaryura P M,Leon M,Correa O S,et al.Assessment of the role of chemotaxis and biofilm formation as requirements for colonization of roots and seeds of soybean plants by Bacillus amyloliquefaciens BNM 339[J].Current Microbiology,2008,56(6):625-632.
[12]Liu Y P,Chen L,Wu G W,et al.Identification of root-secreted compounds involved in the communication between cucumber,the beneficial Bacillus amyloliquefaciens,and the soil-borne pathogen Fusarium oxysporum[J].Molecular Plant-Microbe Interactions,2017,30(1):53-62.
[13]Rao C V,Glekas G D,Ordal G W.The three adaptation systems of Bacillus subtilis chemotaxis[J].Trends in Microbiology,2008,16(10):480-487.
[14]Scharf B E,Hynes M F,Alexandre G M.Chemotaxis signaling systems in model beneficial plant-bacteria associations[J].Plant Molecular Biology,2016,90(6):549-559.
[15]Parkinson J S,Hazelbauer G L,Falke J J.Signaling and sensory adaptation in Escherichia coli chemoreceptors:2015 update[J].Trends in Microbiology,2015,23(5):257-266.
[16]Yuan W,Glekas G D,Allen G M,et al.The importance of the interaction of CheD with CheC and the chemoreceptors compared to its enzymatic activity during chemotaxis in Bacillus subtilis[J].PLoS One,2012,7(12):e50689.
[17]Walukiewicz H E,Tohidifar P,Ordal G W,et al.Interactions among the three adaptation systems of Bacillus subtilis chemotaxis as revealed by an in vitro receptor-kinase assay[J].Molecular Microbiology,2014,93(6):1104-1118.
[18]Gao S,Wu H,Yu X,et al.Swarming motility plays the major role in migration during tomato root colonization by Bacillus subtilis SWR01[J].Biological Control,2016,98:11-17.
[19]Guo Q G,Wu Y Y,Li S Z,et al.Functional analysis of ywbB gene to the biofilm formation and rootcolonization in Bacillus subtilis strain NCD-2(in Chinese)[J].Journal of Plant Protection(植物保护学报),2013,40(1):45-50.
[20]Dong L H,Guo Q G,Zhang X Y,et al.Effects of cotton root exudates on the biofilm formation and root colonization of Bacillus subtilis strain NCD-2(in Chinese)[J].Acta Phytopathologica Sinica(植物病理学报),2015,45(5):541-547.
[21]Armenta J M,Cortes D F,Pisciotta J M,et al.Sensitive and rapid method for amino acid quantitation in malaria biological samples using AccQ.Tag ultra performance liquid chromatography-electrospray ionization-M S/M S with multiple reaction monitoring[J].Analytical Chemistry,2010,82(2):548-558.
[22]Wu C C,Jian G L,Wang A L,et al.Primary detection of QTL for verticillium wilt resistance in cotton[J].Molecular Plant Breeding,2010,8(4):680-686.
[23]Zhang N,Wu K,He X,et al.A new bioorganic fertilizer can effectively control banana wilt by strong colonization with Bacillus subtilis N11[J].Plant and Soil,2011,344(1-2):87-97.
[24]Zhang N,Wang D D,Liu Y P,et al.Effects of different plant root exudates and their organic acid components on chemotaxis,biofilm formation and colonization by beneficial rhizosphere-associated bacterial strains[J].Plant and Soil,2013,374(1-2):689-700.
[25]Allard-Massicotte R,Tessier L,Lecuyer F,et al.Bacillus subtilis early colonization of Arabidopsis thaliana roots involves multiple chemotaxis receptors[J].M Bio,2016,7(6):e01664-16.
[26]Mark G L,Dow J M,Kiely P D,et al.Transcriptome profiling of bacterial responses to root exudates identifies genes involved in microbe-plant interactions[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(48):17454-17459.
[27]Wu K,Yuan S,Xun G,et al.Root exudates from two tobacco cultivars affect colonization of Ralstonia solanacearum and the disease index[J].European Journal of Plant Pathology,2014,141(4):667-677.
[28]Yang Y L,Abiola M P,Hofler C,et al.Relation between chemotaxis and consumption of amino acids in bacteria[J].Molecular Microbiology,2015,96(6):1272-1282.
[29]Rico-Jimenez M,Munoz-Martinez F,Garcia-Fontana C,et al.Paralogous chemoreceptors mediate chemotaxis tow ards protein amino acids and the non-protein amino acid gamma-aminobutyrate(GABA)[J].Molecular Microbiology,2013,88(6):1230-1243.