贝莱斯芽孢杆菌3A3-15生防和促生机制
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摘要
为揭示贝莱斯芽孢杆菌(Bacillus velezensis)3A3-15生防和促生作用相关机理,采用9对特异性引物对其抑菌基因和促生长基因进行PCR检测,并分别通过Sackowski法、Schwyn和Neiland法检测其分泌吲哚乙酸(IAA)和嗜铁素能力,通过高效液相色谱-电喷雾质谱(HPLC-ESI-MS)分析其发酵液抑菌成分,采用平板对峙实验和孢子萌发抑制实验分析该菌株的抑菌机理.结果表明,贝莱斯芽孢杆菌3A3-15中的抑菌基因包括yndJ、srfAB、ituC、bamC和fenD,促生长基因包括ysnE和dhbC.该菌株分泌嗜铁素能力较强,A_s/A_r值达到0.238,鉴定其抑菌物质为C14～C15 surfactinA,且贝莱斯芽孢杆菌3A3-15的次生代谢物能导致病原菌(尖孢镰刀菌)菌丝扭曲、膨大、畸形,对其孢子萌发抑制作用较强,抑制率达93.2%.以上结果说明贝莱斯芽孢杆菌3A3-15具有较好的生防和促生潜力,是防治尖孢镰刀菌引起的大豆根腐病的潜在菌株.
In order to reveal the biocontrol and promotion mechanism of Bacillus velezensis 3 A3-15, PCR with 9 pairs of specific primers were used to detect genes related to inhibitory and growth promotion, and the ability to secrete indole-3-acetic acid(IAA) and siderophore was tested by the methods of Sackowski, Schwyn and Neiland, respectively. At the same time, the inhibitory substances of the fermentation broth were analyzed by high performance liquid chromatography-electrospray ionization mass spectrometry(HPLC-ESI-MS). Finally, the inhibitory mechanism of the strain was analyzed by plate dual-culture antagonistic test and spore germination inhibition test. Results showed that the inhibitory genes in B. velezensis 3 A3-15 include yndJ, srfAB, ituC, bamC and fenD, the growth-promoting genes include ysnE and dhbC. The strain had a strong ability to secrete siderophore with a A_s/A_r value of 0.238, and its inhibitory substance was C14～C15 SurfactinA. Moreover, the secondary metabolites production of B.velezensis 3 A3-15 could cause the hyphae of the pathogenic bacteria(Fusarium oxysporum)to distort,swellen,deform and had a strong inhibitory effect on the germination of spores,with the inhibition rate of 93.2%.These results indicated that B.velezensis 3 A3-15 had preferable biocontrol and growth promoting potential and was a potential strain to control soybean root rot caused by F.oxysporum.
In order to reveal the biocontrol and promotion mechanism of Bacillus velezensis 3 A3-15, PCR with 9 pairs of specific primers were used to detect genes related to inhibitory and growth promotion, and the ability to secrete indole-3-acetic acid(IAA) and siderophore was tested by the methods of Sackowski, Schwyn and Neiland, respectively. At the same time, the inhibitory substances of the fermentation broth were analyzed by high performance liquid chromatography-electrospray ionization mass spectrometry(HPLC-ESI-MS). Finally, the inhibitory mechanism of the strain was analyzed by plate dual-culture antagonistic test and spore germination inhibition test. Results showed that the inhibitory genes in B. velezensis 3 A3-15 include yndJ, srfAB, ituC, bamC and fenD, the growth-promoting genes include ysnE and dhbC. The strain had a strong ability to secrete siderophore with a A_s/A_r value of 0.238, and its inhibitory substance was C14～C15 SurfactinA. Moreover, the secondary metabolites production of B.velezensis 3 A3-15 could cause the hyphae of the pathogenic bacteria(Fusarium oxysporum)to distort,swellen,deform and had a strong inhibitory effect on the germination of spores,with the inhibition rate of 93.2%.These results indicated that B.velezensis 3 A3-15 had preferable biocontrol and growth promoting potential and was a potential strain to control soybean root rot caused by F.oxysporum.
引文
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[8] 杨胜清,张帆,马贵龙.贝莱斯芽孢杆菌S6拮抗物质分离纯化及抑菌机理[J].农药,2017,56(9):645-648.DOI:10.16820/j.cnki.1006-0413.2017.09.007.
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[11] 刘东岳,李敏,孙文献,等.拮抗尖孢镰刀菌的PGPR筛选与抑菌机制的初步研究[J].植物病理学报,2017,2(28):1-16.DOI:10.13926 /j.cnki.apps.000067.
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[13] 连彩,郭晓军,朱宝成,等.兰花枯萎病拮抗细菌的筛选与鉴定[J].华北农学报,2012,27(2):222-225.DOI:1000-7091(2012) 02-0222-04.
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[15] MORA I,CABREFIGA J,MONTESINOS E.Antimicrobial peptide genes in Bacillus strains from plant environments[J].International Microbiology,2011,14:213-223.DOI:10.2436/20.1501.01.151.
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[17] 余贤美,林超,郑服丛,等.枯草芽孢杆菌CAS15嗜铁素基因dhbC的克隆、表达及功能鉴定[J].生物工程学报,2009,25(6):819-825.DOI:10.3321/j.issn:1000-3061.2009.06.004.
[18] 王平,董飚,李阜棣,等.小麦根圈细菌铁载体的检测[J].微生物学报,1994,21(6):323-326.DOI:10.13344/j.microbiol.china.1994.06.001.
[19] 李振东,陈秀蓉,李鹏,等.珠芽蓼内生菌Z5产IAA和抑茵能力测定及其鉴定[J].草业学报,2010,19(2):61-68.DOI:1004-5759(2010) 02-0061-08.
[20] 赵翔,陈绍兴,谢志雄,等.高产铁载体荧光假单胞菌Pseudomonas fluorescens sp-f的筛选鉴定及其铁载体特性研究[J].微生物学报,2006,46(5):691-695.DOI:10.13343/j.cnki.wsxb.2006.05.002.
[21] 杨合同,王少杰,王建平,等.荧光假单胞菌噬铁素的性质研究[J].山东科学,1994,7(1):53-56.
[22] 陈华,王丽,袁成凌,等.高效液相色谱-电喷雾质谱法分离和鉴别枯草芽孢杆菌产生的脂肽类化合物[J].色谱,2008,26:343-347.DOI:10.3321/j.issn:1000-8713.2008.03.016.
[23] 赵长龙,赵慧妍.枯草芽孢杆菌YB5对菜豆根腐病菌的抑菌机制测定及应用[J].黑龙江农业科学,2016(8):54-57.DOI:10.11942/j.issn 1002-2767.2016.08.0054.
[24] 张彩铃,陆宗芳,王永全.环境因素对尖孢镰刀菌分生孢子萌发的影响[J].甘肃农业科技,2008,2(5):5-17.DOI:1001-1463(2008)02-0005-04.
[25] 姚锦爱,黄鹏,陈峰,等.建兰茎腐病原菌尖孢镰刀菌的生物学特性研究[J].福建农业学报,2018,33(2):190-194.DOI:10.19303/j.issn.1008-0384.2018.02.015.
[26] PERSMARK M,EXPERT D,NEILANDS J B.Isolation,characterization,and synthesis of chrysobactin,a compound with siderophore activity from Erwinia chrysanthemi[J].The Journal of Biological Chemistry,1989,264(6):3187-3193.DOI:10.1016/0014-5793(89)80590-3.
[27] TENDULKAR S R,SAIKUMARI Y K,PATEL V,et al.Isolation,purification and characterization of an antifungal molecule produced by Bacillus licheniformis BC98,and its effect on phytopathogen Magnaporthe grisea[J].Journal of Applied Microbiology,2007,103(6):2331-2339.DOI:10.1111/j.1365-2672.2007.03501.x.
[28] 陈梅春,王阶平,肖荣凤,等.地衣芽胞杆菌 FJAT-4脂肽结构鉴定及其对尖孢镰刀菌的抑制作用[J].微生物学报,2017,57(12):1924-1934.DOI:10.13343/j.cnki.wsxb.20170177.
[29] RADHAKRISHNAN R,SHIM K B,LEE B W,et al.IAA-producing Penicillium sp.NICS01 triggers plant growth and suppresses Fusarium sp.induced oxidative stress in sesame(Sesamum indicum L.)[J].Microbiol Biotechnol,2013,23:856-863.DOI:10.4014/jmb.1209.09045.
[30] 李法峰,平淑珍,苏宝林,等.粪产碱菌的Tn5转座诱变及吲哚乙酸生物合成特性的研究[J].微生物学报,2000,40(5):551-555.DOI:10.3321/j.issn:0001-6209.2000.05.018.
[31] 李鹏,陈秀蓉,李振东,等.乳白香青分泌吲哚乙酸内生细菌的16SrDNA鉴定[J].草原与草坪,2009,29(2):6-9.DOI:10.13817/ j.cnki.cyycp.2009.02.004.
[32] BEN J D,JAN W M,PETER A H,et al.Siderophore-mediated competition for iron and induced resistance in the suppression of Fusarium wilt of carnation by fluorescent Pseudomonas spp.[J].Netherlands Journal of Medicine,1993,99(5/6):277-289.DOI:10.1007/bf01974309.
[33] SCHWYN B,NEILANDS J B.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160:47-56.DOI:10.1016/0003-2697(87)90612-9.
[2] 魏巍,许艳丽,张思佳.大豆根腐病原镰孢菌种群多样性DGGE分析及其致病性研究[J].植物病理学报,2013,43(5):500-508.DOI:10.3969/j.issn.0412-0914.2013.05.007.
[3] BANAT I M,MAKKAR R S,CAMEOTRA S S.Potential commercial applications of microbial surfactants[J].Applied Microbiology and Biotechnology,2000,53(5):495-508.DOI:10.1007/s002530051648.
[4] CRISTINA R G,EMILIA Q,FERNANDO M C,et al.Bacillus axarquiensis sp.nov.and Bacillus malacitensis sp.nov.,isolated from river-mouth sediments in southern Spain[J].International Journal of Systematic and Evolutionary Microbiology,2005,55:1279-1285.DOI:10.1099/ijs.0.63567-0.
[5] WANG L T,LEE F L,TAI C J,et al.Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens[J].International Journal of Systermatic and Evolutionary Microbiology,2008,58(3):671-675.DOI:10.1016/j.diamond.2007.11.010.
[6] 宗英,赵月菊,刘阳.一株贝莱斯芽孢杆菌抑制禾谷镰刀菌的研究[J].核农学报,2018,32(2):0310-0317.DOI:10.11869/j.issn.100-8551.2018.02.0310.
[7] 杨可,郑柯斌,黄晓慧.海洋生境贝莱斯芽孢杆菌TCS001的鉴定及抑真菌活性[J].农药学学报,2018,20(3):333-339.DOI:10.16801/j.issn.1008-7303.2018.0041.
[8] 杨胜清,张帆,马贵龙.贝莱斯芽孢杆菌S6拮抗物质分离纯化及抑菌机理[J].农药,2017,56(9):645-648.DOI:10.16820/j.cnki.1006-0413.2017.09.007.
[9] LIU X Y,REN B,CHEN M,et al.Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3[J].Applied Microbiology Biotechnology,2010,87:1881-1893.DOI:10.1007/s00253-010-2653-9.
[10] MENG Q X,HE J,HAO J J.Effects of Bacillus velezensis strain BAC03 in promoting plant growth[J].Biological Control,2016,98:18-26.DOI:10.1016/j.biocontrol.2016.03.010.
[11] 刘东岳,李敏,孙文献,等.拮抗尖孢镰刀菌的PGPR筛选与抑菌机制的初步研究[J].植物病理学报,2017,2(28):1-16.DOI:10.13926 /j.cnki.apps.000067.
[12] FAN B,BLOM J,KLENKET H P,et al.Bacillus amyloliquefaciens,Bacillus velezensis,and Bacillus siamensis Form an “Operational Group B.amyloliquefaciens” within the B.subtilis Species Complex[J].Front Microbiol,2017(20):8-22.DOI:10.3389/fmicb.2017.00022.
[13] 连彩,郭晓军,朱宝成,等.兰花枯萎病拮抗细菌的筛选与鉴定[J].华北农学报,2012,27(2):222-225.DOI:1000-7091(2012) 02-0222-04.
[14] JOSHI R,MCSPADDEN GARDENER B B.Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis[J].Phytopathology,2006,96(2):145-154.DOI:10.1094/PHYTO-96-0145.
[15] MORA I,CABREFIGA J,MONTESINOS E.Antimicrobial peptide genes in Bacillus strains from plant environments[J].International Microbiology,2011,14:213-223.DOI:10.2436/20.1501.01.151.
[16] 周小江.2株海洋生境芽孢杆菌的抑菌促生长作用及有关控制基因[D].青岛:青岛科技大学,2015.
[17] 余贤美,林超,郑服丛,等.枯草芽孢杆菌CAS15嗜铁素基因dhbC的克隆、表达及功能鉴定[J].生物工程学报,2009,25(6):819-825.DOI:10.3321/j.issn:1000-3061.2009.06.004.
[18] 王平,董飚,李阜棣,等.小麦根圈细菌铁载体的检测[J].微生物学报,1994,21(6):323-326.DOI:10.13344/j.microbiol.china.1994.06.001.
[19] 李振东,陈秀蓉,李鹏,等.珠芽蓼内生菌Z5产IAA和抑茵能力测定及其鉴定[J].草业学报,2010,19(2):61-68.DOI:1004-5759(2010) 02-0061-08.
[20] 赵翔,陈绍兴,谢志雄,等.高产铁载体荧光假单胞菌Pseudomonas fluorescens sp-f的筛选鉴定及其铁载体特性研究[J].微生物学报,2006,46(5):691-695.DOI:10.13343/j.cnki.wsxb.2006.05.002.
[21] 杨合同,王少杰,王建平,等.荧光假单胞菌噬铁素的性质研究[J].山东科学,1994,7(1):53-56.
[22] 陈华,王丽,袁成凌,等.高效液相色谱-电喷雾质谱法分离和鉴别枯草芽孢杆菌产生的脂肽类化合物[J].色谱,2008,26:343-347.DOI:10.3321/j.issn:1000-8713.2008.03.016.
[23] 赵长龙,赵慧妍.枯草芽孢杆菌YB5对菜豆根腐病菌的抑菌机制测定及应用[J].黑龙江农业科学,2016(8):54-57.DOI:10.11942/j.issn 1002-2767.2016.08.0054.
[24] 张彩铃,陆宗芳,王永全.环境因素对尖孢镰刀菌分生孢子萌发的影响[J].甘肃农业科技,2008,2(5):5-17.DOI:1001-1463(2008)02-0005-04.
[25] 姚锦爱,黄鹏,陈峰,等.建兰茎腐病原菌尖孢镰刀菌的生物学特性研究[J].福建农业学报,2018,33(2):190-194.DOI:10.19303/j.issn.1008-0384.2018.02.015.
[26] PERSMARK M,EXPERT D,NEILANDS J B.Isolation,characterization,and synthesis of chrysobactin,a compound with siderophore activity from Erwinia chrysanthemi[J].The Journal of Biological Chemistry,1989,264(6):3187-3193.DOI:10.1016/0014-5793(89)80590-3.
[27] TENDULKAR S R,SAIKUMARI Y K,PATEL V,et al.Isolation,purification and characterization of an antifungal molecule produced by Bacillus licheniformis BC98,and its effect on phytopathogen Magnaporthe grisea[J].Journal of Applied Microbiology,2007,103(6):2331-2339.DOI:10.1111/j.1365-2672.2007.03501.x.
[28] 陈梅春,王阶平,肖荣凤,等.地衣芽胞杆菌 FJAT-4脂肽结构鉴定及其对尖孢镰刀菌的抑制作用[J].微生物学报,2017,57(12):1924-1934.DOI:10.13343/j.cnki.wsxb.20170177.
[29] RADHAKRISHNAN R,SHIM K B,LEE B W,et al.IAA-producing Penicillium sp.NICS01 triggers plant growth and suppresses Fusarium sp.induced oxidative stress in sesame(Sesamum indicum L.)[J].Microbiol Biotechnol,2013,23:856-863.DOI:10.4014/jmb.1209.09045.
[30] 李法峰,平淑珍,苏宝林,等.粪产碱菌的Tn5转座诱变及吲哚乙酸生物合成特性的研究[J].微生物学报,2000,40(5):551-555.DOI:10.3321/j.issn:0001-6209.2000.05.018.
[31] 李鹏,陈秀蓉,李振东,等.乳白香青分泌吲哚乙酸内生细菌的16SrDNA鉴定[J].草原与草坪,2009,29(2):6-9.DOI:10.13817/ j.cnki.cyycp.2009.02.004.
[32] BEN J D,JAN W M,PETER A H,et al.Siderophore-mediated competition for iron and induced resistance in the suppression of Fusarium wilt of carnation by fluorescent Pseudomonas spp.[J].Netherlands Journal of Medicine,1993,99(5/6):277-289.DOI:10.1007/bf01974309.
[33] SCHWYN B,NEILANDS J B.Universal chemical assay for the detection and determination of siderophores[J].Analytical Biochemistry,1987,160:47-56.DOI:10.1016/0003-2697(87)90612-9.
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