锰消除镰刀菌酸对枯草芽胞杆菌R31生物被膜形成的抑制
|
摘要
为了揭示芽胞杆菌类生防因子和镰刀菌病害的互作方式,对镰刀菌酸抑制枯草芽胞杆菌生物被膜形成及其消除开展了研究。首先利用24孔细胞培养板建立了镰刀菌酸抑制枯草芽胞杆菌R31生物被膜形成的生物测定体系,并筛选出抑制R31生物被膜形成所需的最低镰刀菌酸浓度。测定了在该镰刀菌酸浓度处理下的R31生长曲线,并利用显微镜观察了镰刀菌酸处理和对照在振荡培养和静置培养下的菌体形态。然后测定了不同浓度MnSO4添加消除镰刀菌酸抑制R31生物被膜形成的效果,并用高效液相色谱测定了各处理镰刀菌酸的残留量。结果显示,以24孔细胞培养板为培养容器,以BGM1为培养基的生物测定系统中,显著抑制R31生物被膜形成的最低镰刀菌酸用量为9μg/mL;该浓度的镰刀菌酸抑制了静置培养的R31菌体形成网状结构和漂浮在液面,并抑制了振荡培养的R31早期菌体增殖。但共培养体系中添加MnSO4可以恢复R31的生物被膜形成,其中200μg/mL的硫酸锰不仅能消除毒素抑制,还可显著促进R31的生物被膜形成。镰刀菌酸可能通过影响R31基质产生细胞的分化而抑制其生物被膜形成,硫酸锰可以作为钝化剂缓解镰刀菌酸对R31生物被膜形成的抑制。
In order to reveal the interaction mechanisms between Bacillus biocontrol agents and plant pathogen Fusarium, the inhibition of fusaric acid(FA) from Fusarium on the biofilm formation of Bacillus subtilis strain R31,and the elimination of the inhibition were investigated. Firstly, a bioassay system was established to test the inhibition of FA on the biofilm formation of B. subtilis R31 by 24-well cell culture plate, and the lowest concentration of FA on the biofilm formation of R31 was screened out. The growth curve of B. subtilis R31 was tested in the presence of the minimum concentration of FA, and the cells of R31 with or without the FA treatment under shaking or static cultivation were observed by microscopy. Then, different concentrations of MnSO4 were added into the co-cultural system to eliminate the inhibition of FA on the biofilm formation of R31, and the residue of FA in each treatment was determined by HPLC. The results showed that in this bioassay system, 9 μg/mL of FA could drastically inhibit the biofilm formation of R31 by inhibiting its reticular structure formation of R31 cells and reducing the cells floating on the liquid surface in static culture. 9 μg/mL of FA can also inhibit the growth of early-stage R31 in shaking culture. Adding MnSO4 into the co-culture system, however, can restore the biofilm formation of R31. More specially, 200 μg/mL of MnSO4 can not only eliminate the inhibition of FA on the biofilm formation of R31, but also significantly promote its formation. FA may inhibit the biofilm formation of R31 by affecting the differentiation of matrix-producing cells of R31, and MnSO4 can be used as a passivating agent to alleviate the inhibition of FA on the biofilm formation of R31.
In order to reveal the interaction mechanisms between Bacillus biocontrol agents and plant pathogen Fusarium, the inhibition of fusaric acid(FA) from Fusarium on the biofilm formation of Bacillus subtilis strain R31,and the elimination of the inhibition were investigated. Firstly, a bioassay system was established to test the inhibition of FA on the biofilm formation of B. subtilis R31 by 24-well cell culture plate, and the lowest concentration of FA on the biofilm formation of R31 was screened out. The growth curve of B. subtilis R31 was tested in the presence of the minimum concentration of FA, and the cells of R31 with or without the FA treatment under shaking or static cultivation were observed by microscopy. Then, different concentrations of MnSO4 were added into the co-cultural system to eliminate the inhibition of FA on the biofilm formation of R31, and the residue of FA in each treatment was determined by HPLC. The results showed that in this bioassay system, 9 μg/mL of FA could drastically inhibit the biofilm formation of R31 by inhibiting its reticular structure formation of R31 cells and reducing the cells floating on the liquid surface in static culture. 9 μg/mL of FA can also inhibit the growth of early-stage R31 in shaking culture. Adding MnSO4 into the co-culture system, however, can restore the biofilm formation of R31. More specially, 200 μg/mL of MnSO4 can not only eliminate the inhibition of FA on the biofilm formation of R31, but also significantly promote its formation. FA may inhibit the biofilm formation of R31 by affecting the differentiation of matrix-producing cells of R31, and MnSO4 can be used as a passivating agent to alleviate the inhibition of FA on the biofilm formation of R31.
引文
[1]陈楠,潘晓静,姚远,等.东北地区玉米茎腐病镰孢菌EF1区基因序列分析鉴定[J].玉米科学,2015,23(4):143-148.
[2]喻国辉,程萍,王燕鹂,等.一株香蕉枯萎病生防芽胞杆菌的鉴定、生物学特性和抗菌谱研究[J].中国农学通报,2010,26(12):216-220.
[3]黎永坚,程萍,喻国辉,等.枯草芽孢杆菌R31和TR21菌株防治香蕉枯萎病田间药效试验[J].广东农业科学,2012,39(23):70-72.
[4]陈川雁,王燕,喻国辉,等.枯草芽胞杆菌R31影响巴西蕉根系活性氧产生及对枯萎病的防治效果[J].中国生物防治学报,2017,33(2):226-233.
[5]李荣,程萍,喻国辉,等.枯草芽胞杆菌R31在香芽蕉根部的定殖能力测定[J].广东农业科学,2012,38(24):59-61.
[6]赵志国,李一平,喻国辉,等.枯草芽胞杆菌R31施用对中粉1号粉蕉根际微生物的影响[J].广东农业科学,2014(17):83-87,105.
[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]郭庆港,吴园园,李社增,等.ywb基因对枯草芽胞杆菌NCD-2菌株生物膜形成和根际定殖能力的影响[J].植物保护学报,2013,40(1):45-50.
[9]Rudrappa T,Czymmek K J,Pare P W,et al.Root-secreted malic acid recruits beneficial soil bacteria[J].Plant Physiology,2008,148(3):1547-1556.
[10]Beauregard P B,Chai Y,Vlamakis H,et al.Bacillus subtilis biofilm induction by plant polysaccharides[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(17):1621-1630.
[11]Rudrappa T,Quinn W J,Stanley-Wall N R,et al.Arabidopsis thaliana root surface chemistry regulates in planta biofilm formation of Bacillus subtilis[J].Plant Signal and Behavior,2007,2(5):349-350.
[12]Rudrappa T,Quinn W J,Stanley-Wall N R,et al.A degradation product of the salicylic acid pathway triggers oxidative stress resulting in down-regulation of Bacillus subtilis biofilm formation on Arabidopsis thaliana roots[J].Planta,2007,226(2):283-297.
[13]Stefanic P,Kraigher B,Lyons N A,et al.Kin discrimination between sympatric Bacillus subtilis isolates[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(45):14042-14047.
[14]Khezri M,Jouzani G S,Ahmadzadeh M.Fusarium culmorum affects expression of biofilm formation key genes in Bacillus subtilis[J].Brazilian Journal of Microbiology,2016,47(1):47-54.
[15]樊胜南,陈川雁,喻国辉,等.井冈霉素A对枯草芽胞杆菌R31生长和生物被膜形成的影响[J].广东农业科学,2018,45(9):96-102.
[16]姚艳平.枯萎病菌镰刀菌酸的产生和钝化及生物活性测定的研究[D].太原:山西农业大学,2002.
[17]刘开军,罗少波,王亚琴,等.镰刀菌毒素对植物形态和结构的影响[J].中国农学通报,2010,26(4):53-56.
[18]Wang H,Ng T B.Pharmacological activities of fusaric acid(5-butylpicolinic acid)[J].Life Sciences,1999,65(9):849-856.
[19]李梅婷,严琰,张绍升.香蕉枯萎病菌及其粗毒素对香蕉的致病性比较[J].热带作物学报,2010,31(3):446-452.
[20]许文耀,兀旭辉,林成辉.香蕉枯萎病菌粗毒素的毒性及其模型[J].热带作物学报,2004,25(4):25-29.
[21]曹永军,程萍,喻国辉,等.香蕉枯萎病菌菌株致病力分化及其原因研究[J].热带作物学报,2011,32(8):1532-1536.
[22]漆艳香,张欣,蒲金基,等.10种化合物对香蕉枯萎病菌的抑菌作用及对毒素钝化的效果[J].果树学报,2007,25(1):78-82.
[23]姬华伟,郑青松,董鲜,等.铜、锌元素对香蕉枯萎病的防治效果与机理[J].园艺学报,2012,39(6):1064-1072.
[24]Thangavelu R,Palaniswami A,Ramakrishnan G,et al.Involvement of fusaric acid detoxification by Pseudomonas fluorescens strain Pf10 in the biological control of Fusarium wilt of banana caused by Fusarium oxysporum f.sp.cubense[J].Journal of Plant Diseases and Protection,2001,108(5):433-445.
[25]黎永坚,杨紫红,陈远凤,等.香蕉枯萎病菌粗毒素对地衣芽胞杆菌生长和培养液上清蛋白组成的影响[J].微生物学通报,2009,36(12):1826-1831.
[26]Shemesh M,Chai Y.A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling[J].Journal of Bacteriology,2013,195(12):2747-2754.
[27]Mhatre E,Gallegos-Monterrosa R,Kuipers O P,et al.The impact of manganese on biofilm development of Bacillus subtilis[J].Microbiology,2016,162(8):1468-1478.
[28]李一平,沈汉国,喻国辉,等.一种芽胞杆菌生物被膜形成缺陷培养基的研究[J].广东农业科学,2016,43(9):98-104.
[29]Hamon M A,Lazazzera B A.The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis[J].Molecular Microbiology,2001,42(5):1199-1209.
[30]唐启义.DPS?数据处理系统:实验设计、统计分析及数据挖掘(第2版)[M].北京:科学出版社,2010.
[31]Lopez D,Vlamakis H,Kotler R.Generation of multiple cell types in Bacillus subtilis[J].FEMS Microbiology Reviews,2009,33(1):152-163.
[32]Murray E J,Kiley T B,Stanley-Wall N R.A pivotal role for the response regulator DegU in controlling multicellular behavior[J].Microbiology,2009,155(1):1-8.
[33]Kobayashi K,Iwano M.BslA(YuaB)forms a hydrophobic layer on the surface of Bacillus subtilis biofilms[J].Molecular Microbiology,2012,85(1):51-66.
[34]Eisenstadt E,Fisher S,Der C L,et al.Manganese transport in Bacillus subtilis W23 during growth and sporulation[J].Journal of Bacteriology,1973,113(3):1363-1372.
[35]Fisher S,Buxbaum L,Toth K,et al.Regulation of manganese accumulation and exchange in Bacillus subtilis W23[J].Journal of Bacteriology,1973,113(3):1373-1380.
[36]Oh Y K,Freese E.Manganese requirement of phosphoglycerate phosphomutase and its consequences for growth and sporulation of Bacillus subtilis[J].Journal of Bacteriology,1976,127(2):739-746.
[37]Akrigg A.Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis[J].The Biochemical Journal,1978,172(1):69-76.
[38]Vasantha N,Freese E.The role of manganese in growth and sporulation of Bacillus subtilis[J].Journal of General Microbiology,1979,112(2):329-336.
[39]Guedon E,Moore C M,Que Q,et al.The global transcriptional response of Bacillus subtilis to manganese involves the MntR,Fur,TnrA andσBregulons[J].Molecular Microbiology,2003,49(6):1477-1491.
[2]喻国辉,程萍,王燕鹂,等.一株香蕉枯萎病生防芽胞杆菌的鉴定、生物学特性和抗菌谱研究[J].中国农学通报,2010,26(12):216-220.
[3]黎永坚,程萍,喻国辉,等.枯草芽孢杆菌R31和TR21菌株防治香蕉枯萎病田间药效试验[J].广东农业科学,2012,39(23):70-72.
[4]陈川雁,王燕,喻国辉,等.枯草芽胞杆菌R31影响巴西蕉根系活性氧产生及对枯萎病的防治效果[J].中国生物防治学报,2017,33(2):226-233.
[5]李荣,程萍,喻国辉,等.枯草芽胞杆菌R31在香芽蕉根部的定殖能力测定[J].广东农业科学,2012,38(24):59-61.
[6]赵志国,李一平,喻国辉,等.枯草芽胞杆菌R31施用对中粉1号粉蕉根际微生物的影响[J].广东农业科学,2014(17):83-87,105.
[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]郭庆港,吴园园,李社增,等.ywb基因对枯草芽胞杆菌NCD-2菌株生物膜形成和根际定殖能力的影响[J].植物保护学报,2013,40(1):45-50.
[9]Rudrappa T,Czymmek K J,Pare P W,et al.Root-secreted malic acid recruits beneficial soil bacteria[J].Plant Physiology,2008,148(3):1547-1556.
[10]Beauregard P B,Chai Y,Vlamakis H,et al.Bacillus subtilis biofilm induction by plant polysaccharides[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(17):1621-1630.
[11]Rudrappa T,Quinn W J,Stanley-Wall N R,et al.Arabidopsis thaliana root surface chemistry regulates in planta biofilm formation of Bacillus subtilis[J].Plant Signal and Behavior,2007,2(5):349-350.
[12]Rudrappa T,Quinn W J,Stanley-Wall N R,et al.A degradation product of the salicylic acid pathway triggers oxidative stress resulting in down-regulation of Bacillus subtilis biofilm formation on Arabidopsis thaliana roots[J].Planta,2007,226(2):283-297.
[13]Stefanic P,Kraigher B,Lyons N A,et al.Kin discrimination between sympatric Bacillus subtilis isolates[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(45):14042-14047.
[14]Khezri M,Jouzani G S,Ahmadzadeh M.Fusarium culmorum affects expression of biofilm formation key genes in Bacillus subtilis[J].Brazilian Journal of Microbiology,2016,47(1):47-54.
[15]樊胜南,陈川雁,喻国辉,等.井冈霉素A对枯草芽胞杆菌R31生长和生物被膜形成的影响[J].广东农业科学,2018,45(9):96-102.
[16]姚艳平.枯萎病菌镰刀菌酸的产生和钝化及生物活性测定的研究[D].太原:山西农业大学,2002.
[17]刘开军,罗少波,王亚琴,等.镰刀菌毒素对植物形态和结构的影响[J].中国农学通报,2010,26(4):53-56.
[18]Wang H,Ng T B.Pharmacological activities of fusaric acid(5-butylpicolinic acid)[J].Life Sciences,1999,65(9):849-856.
[19]李梅婷,严琰,张绍升.香蕉枯萎病菌及其粗毒素对香蕉的致病性比较[J].热带作物学报,2010,31(3):446-452.
[20]许文耀,兀旭辉,林成辉.香蕉枯萎病菌粗毒素的毒性及其模型[J].热带作物学报,2004,25(4):25-29.
[21]曹永军,程萍,喻国辉,等.香蕉枯萎病菌菌株致病力分化及其原因研究[J].热带作物学报,2011,32(8):1532-1536.
[22]漆艳香,张欣,蒲金基,等.10种化合物对香蕉枯萎病菌的抑菌作用及对毒素钝化的效果[J].果树学报,2007,25(1):78-82.
[23]姬华伟,郑青松,董鲜,等.铜、锌元素对香蕉枯萎病的防治效果与机理[J].园艺学报,2012,39(6):1064-1072.
[24]Thangavelu R,Palaniswami A,Ramakrishnan G,et al.Involvement of fusaric acid detoxification by Pseudomonas fluorescens strain Pf10 in the biological control of Fusarium wilt of banana caused by Fusarium oxysporum f.sp.cubense[J].Journal of Plant Diseases and Protection,2001,108(5):433-445.
[25]黎永坚,杨紫红,陈远凤,等.香蕉枯萎病菌粗毒素对地衣芽胞杆菌生长和培养液上清蛋白组成的影响[J].微生物学通报,2009,36(12):1826-1831.
[26]Shemesh M,Chai Y.A combination of glycerol and manganese promotes biofilm formation in Bacillus subtilis via histidine kinase KinD signaling[J].Journal of Bacteriology,2013,195(12):2747-2754.
[27]Mhatre E,Gallegos-Monterrosa R,Kuipers O P,et al.The impact of manganese on biofilm development of Bacillus subtilis[J].Microbiology,2016,162(8):1468-1478.
[28]李一平,沈汉国,喻国辉,等.一种芽胞杆菌生物被膜形成缺陷培养基的研究[J].广东农业科学,2016,43(9):98-104.
[29]Hamon M A,Lazazzera B A.The sporulation transcription factor Spo0A is required for biofilm development in Bacillus subtilis[J].Molecular Microbiology,2001,42(5):1199-1209.
[30]唐启义.DPS?数据处理系统:实验设计、统计分析及数据挖掘(第2版)[M].北京:科学出版社,2010.
[31]Lopez D,Vlamakis H,Kotler R.Generation of multiple cell types in Bacillus subtilis[J].FEMS Microbiology Reviews,2009,33(1):152-163.
[32]Murray E J,Kiley T B,Stanley-Wall N R.A pivotal role for the response regulator DegU in controlling multicellular behavior[J].Microbiology,2009,155(1):1-8.
[33]Kobayashi K,Iwano M.BslA(YuaB)forms a hydrophobic layer on the surface of Bacillus subtilis biofilms[J].Molecular Microbiology,2012,85(1):51-66.
[34]Eisenstadt E,Fisher S,Der C L,et al.Manganese transport in Bacillus subtilis W23 during growth and sporulation[J].Journal of Bacteriology,1973,113(3):1363-1372.
[35]Fisher S,Buxbaum L,Toth K,et al.Regulation of manganese accumulation and exchange in Bacillus subtilis W23[J].Journal of Bacteriology,1973,113(3):1373-1380.
[36]Oh Y K,Freese E.Manganese requirement of phosphoglycerate phosphomutase and its consequences for growth and sporulation of Bacillus subtilis[J].Journal of Bacteriology,1976,127(2):739-746.
[37]Akrigg A.Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis[J].The Biochemical Journal,1978,172(1):69-76.
[38]Vasantha N,Freese E.The role of manganese in growth and sporulation of Bacillus subtilis[J].Journal of General Microbiology,1979,112(2):329-336.
[39]Guedon E,Moore C M,Que Q,et al.The global transcriptional response of Bacillus subtilis to manganese involves the MntR,Fur,TnrA andσBregulons[J].Molecular Microbiology,2003,49(6):1477-1491.