甲基营养型芽孢杆菌BH21对葡萄灰霉病菌的拮抗作用
|
摘要
【目的】甲基营养型芽孢杆菌(Bacillus methylotrophicus)BH21是一株对葡萄灰霉病菌(Botrytis cinerea)有较好拮抗作用的海洋源细菌,鉴定该菌株脂肽类抗菌物质合成基因,检测脂肽粗提物对葡萄灰霉病菌的拮抗作用,为应用该菌株防治葡萄灰霉病提供科学依据。【方法】通过特异引物的基因组PCR法检测甲基营养型芽孢杆菌BH21菌株合成脂肽的能力;盐酸沉淀和甲醇抽提法从无菌发酵液中提取脂肽粗提物;排油圈法检测脂肽粗提物的表面活性;采用菌丝生长速率法检测脂肽粗提物对灰霉病菌菌丝生长的抑制作用并计算有效中浓度EC50;利用高效液相色谱技术(HPLC)对脂肽粗提物洗脱分离,并采用菌丝生长速率法检测各组分对葡萄灰霉病菌的抑制能力;采用反相高效液相色谱(RP-HPLC)分析主要抑菌成分的脂肽类型。采用离体叶片接种法检测脂肽粗提物对葡萄灰霉病的防治效果。【结果】选取11对特异引物对菌株BH21基因组扩增,其中7对引物扩增出预期核酸片段;扩增产物经测序、BLAST比对分析,结果显示扩增产物与相关菌株脂肽基因相似度为96%—99%,扩增产物翻译的蛋白与相关菌株的脂肽合成蛋白相似度为96%—100%,表明甲基营养型芽孢杆菌BH21基因组中含有ituA、bamD、ituC、ituD、fenD、srfAB、yndJ,该菌株具有合成surfactin、iturin及fengycin等多种脂肽类抗菌物质的能力。盐酸沉淀和甲醇抽提从LB无菌发酵液中获得脂肽粗提物,得率为428 mg·L~(-1)。排油圈检测结果显示,脂肽粗提物使橄榄油膜形成排油圈,表明脂肽粗提物具有表面活性。脂肽粗提物对葡萄灰霉病菌菌丝生长具有显著的抑制作用,脂肽粗提物浓度为440μg·mL~(-1)时,对葡萄灰霉病菌菌丝生长的相对抑制率为82.8%。根据毒力方程计算,抑制葡萄灰霉病菌菌丝生长的脂肽粗提物EC50为144.39μg·mL~(-1)。HPLC分离纯化脂肽粗提物获得6个组分,只有组分BH21-2和BH21-3抑制葡萄灰霉病菌的生长,RP-HPLC色谱图分析表明组分BH21-2和BH21-3属于fengycin家族脂肽。葡萄灰霉病离体叶片试验结果表明,脂肽粗提物浓度为400μg·mL~(-1)时,对葡萄叶片灰霉病防病效果为100%;脂肽粗提物浓度为220μg·mL~(-1)时,对葡萄叶片病斑扩展相对抑制率为94.4%。【结论】甲基营养型芽孢杆菌菌株BH21具有合成surfactin、iturin及fengycin等多种脂肽类抗菌物质的基因,该菌株脂肽粗提物对葡萄灰霉病菌具有较强的拮抗作用,在葡萄灰霉病生物防治中具有应用潜力。
【Objective】Bacillus methylotrophicus strain BH21 is a marine derived bacterium that has a good antagonistic effect on Botrytis cinerea. The objective of this study is to identify the lipopeptide synthesis genes of the strain BH21 and investigate the antagonism of the crude extracts of lipopeptide to B. cinerea, and to provide a scientific basis for the prevention and control of B. cinerea. 【Method】 To determine the mechanisms of the antagonistic strain, PCR was used to screen strain BH21 for genes involved in biosynthesis of antimicrobial lipopeptide. Crude lipopeptide was extracted from the culture broth by hydrochloric acid precipitation and methanol extraction. Surface activity of the crude lipopeptide was determined by oil spreading method. The inhibition ability of the crude lipopeptide on mycelial growth of B. cinerea was investigated by mycelial growth rate method and EC50 was calculated. The crude lipopeptide was separated by liquid chromatography(HPLC) and the inhibition ability of each component to B. cinerea was detected by the mycelium growth rate method. Reversed phase high performance liquid chromatography(RP-HPLC) was used to analyze the types of the main antifungal components. The effect of the crude lipopeptide on the control of B. cinerea in grape was detected by tissue inoculation in vitro. 【Result】 A total of 11 pairs of specific primers were used for genomic amplification of strain BH21, and 7 gene fragments of the size expected to be correlated with biocontrol activities were efficiently amplified. After amplification, sequencing and BLAST analysis, the results showed that the similarity between the amplified products and the related lipopeptide genes was 96%-99%, the similarity between the protein produced by the nucleic acid fragment and the lipopeptide synthesized protein of the related strain was 96%-100%, which showed that the genome of strain BH21 contained ituA, bamD, ituC, ituD, fenD, srfAB and yndJ genes and the strain had the ability to synthesize antimicrobial lipopeptide such as surfactins, iturins and fengycins. Antifungal lipopeptide produced by BH21 was extracted by hydrochloric acid precipitation and methanol extraction, and the yield was 428 mg·L~(-1). The results of the oil spreading test showed that the crude lipopeptide had surface activity. The crude lipopeptide significantly inhibited mycelial growth of B. cinerea when the concentration was 440 μg·mL~(-1), the relative inhibition rate of mycelial growth of B. cinerea was 82.8%, and the effective medium concentration EC50 was 144.39 μg·mL~(-1). Six fractions were collected with elution time through HPLC, only BH21-2 and BH21-3 inhibited the growth of B. cinerea. RP-HPLC chromatogram analysis showed that the components BH21-2 and BH21-3 belong to the fengycin family. Grape leaf in vitro test results showed that when the concentration of crude lipopeptide was 440 μg·mL~(-1), the control effect against grape gray leaf spot was 100%, while the concentration was 220 μg·mL~(-1), the relative inhibitory rate of grape leaf lesion was 94.4%.【Conclusion】The strain BH21 has the genes for synthesizing antimicrobial lipopeptide such as surfactins, iturins and fengycins, and the lipopeptide extracted from this strain has strong antagonism to B. cinerea, so it has potential application in the biological control of B. cinerea.
【Objective】Bacillus methylotrophicus strain BH21 is a marine derived bacterium that has a good antagonistic effect on Botrytis cinerea. The objective of this study is to identify the lipopeptide synthesis genes of the strain BH21 and investigate the antagonism of the crude extracts of lipopeptide to B. cinerea, and to provide a scientific basis for the prevention and control of B. cinerea. 【Method】 To determine the mechanisms of the antagonistic strain, PCR was used to screen strain BH21 for genes involved in biosynthesis of antimicrobial lipopeptide. Crude lipopeptide was extracted from the culture broth by hydrochloric acid precipitation and methanol extraction. Surface activity of the crude lipopeptide was determined by oil spreading method. The inhibition ability of the crude lipopeptide on mycelial growth of B. cinerea was investigated by mycelial growth rate method and EC50 was calculated. The crude lipopeptide was separated by liquid chromatography(HPLC) and the inhibition ability of each component to B. cinerea was detected by the mycelium growth rate method. Reversed phase high performance liquid chromatography(RP-HPLC) was used to analyze the types of the main antifungal components. The effect of the crude lipopeptide on the control of B. cinerea in grape was detected by tissue inoculation in vitro. 【Result】 A total of 11 pairs of specific primers were used for genomic amplification of strain BH21, and 7 gene fragments of the size expected to be correlated with biocontrol activities were efficiently amplified. After amplification, sequencing and BLAST analysis, the results showed that the similarity between the amplified products and the related lipopeptide genes was 96%-99%, the similarity between the protein produced by the nucleic acid fragment and the lipopeptide synthesized protein of the related strain was 96%-100%, which showed that the genome of strain BH21 contained ituA, bamD, ituC, ituD, fenD, srfAB and yndJ genes and the strain had the ability to synthesize antimicrobial lipopeptide such as surfactins, iturins and fengycins. Antifungal lipopeptide produced by BH21 was extracted by hydrochloric acid precipitation and methanol extraction, and the yield was 428 mg·L~(-1). The results of the oil spreading test showed that the crude lipopeptide had surface activity. The crude lipopeptide significantly inhibited mycelial growth of B. cinerea when the concentration was 440 μg·mL~(-1), the relative inhibition rate of mycelial growth of B. cinerea was 82.8%, and the effective medium concentration EC50 was 144.39 μg·mL~(-1). Six fractions were collected with elution time through HPLC, only BH21-2 and BH21-3 inhibited the growth of B. cinerea. RP-HPLC chromatogram analysis showed that the components BH21-2 and BH21-3 belong to the fengycin family. Grape leaf in vitro test results showed that when the concentration of crude lipopeptide was 440 μg·mL~(-1), the control effect against grape gray leaf spot was 100%, while the concentration was 220 μg·mL~(-1), the relative inhibitory rate of grape leaf lesion was 94.4%.【Conclusion】The strain BH21 has the genes for synthesizing antimicrobial lipopeptide such as surfactins, iturins and fengycins, and the lipopeptide extracted from this strain has strong antagonism to B. cinerea, so it has potential application in the biological control of B. cinerea.
引文
[1]张玮,乔广行,黄金宝,王忠跃,李兴红.中国葡萄灰霉病菌对嘧霉胺的抗药性检测.中国农业科学,2013,46(6):1208-1212.ZHANG W,QIAO G X,HUANG J B,WANG Z Y,LI X H.Evaluation on resistance of grape gray mold pathogen Botrytis cinerea to pyrimethanil in China.Scientia Agricultura Sinica,2013,46(6):1208-1212.(in Chinese)
[2]严红,燕继晔,王忠跃,李亚宁,金桂华,李兴红.葡萄灰霉病菌对3种杀菌剂的多重抗药性检测.果树学报,2012,29(4):625-629.YAN H,YAN J Y,WANG Z Y,LI Y N,JIN G H,LI X H.Multiple fungicide resistance of Botrytis cinerea from grapevine to three fungicides.Journal of Fruit Science,2012,29(4):625-629.(in Chinese)
[3]MADHAIYAN M,POONGUZHALI S,KWON S W,SA T M.Bacillus methylotrophicus sp.nov.,a methanol-utilizing,plantgrowth-promoting bacterium isolated from rice rhizosphere soil.International Journal of Systematic and Evolutionary Microbiology,2010,60(10):2490-2495.
[4]王洪梅,吴云成,沈标.青枯病生防菌N5的特性及其生物学效应.土壤,2013,45(6):1082-1090.WANG H M,WU Y C,SHEN B.Characterization and biological effects of antagonistic N5 against Ralstonia solanacearum.Soils,2013,45(6):1082-1090.(in Chinese)
[5]刘伟,宋双,沈小英,安天赐,牛小义,安德荣.番茄灰霉病拮抗芽孢杆菌LW-6-1的筛选、鉴定及抑菌活性研究.西北农林科技大学学报(自然科学版),2013,41(11):73-79.LIU W,SONG S,SHEN X Y,AN T C,NIU X Y,AN D R.Screening,identification and antibacterial activity of antagonistic bacteria LW-6-1 against Botrytis cinerea.Journal of Northwest A&F University(Natural Science Edition),2013,41(11):73-79.(in Chinese)
[6]黄霄,陈波,周登博,谭昕,张锡炎.菌株BM-24的分离鉴定及对香蕉枯萎病菌的抑菌活性.植物保护学报,2013,40(2):121-127.HUANG X,CHEN B,ZHOU D B,TAN X,ZHANG X Y.Isolation,identification of BM-24 strains and its antifungal activity of antagonistic bacteria against Fusarium oxysporum f.sp.cubense.Acta Phytophylacica Sinica,2013,40(2):121-127.(in Chinese)
[7]伏波,姚娟妮,高小宁,黄丽丽,康振生,韩青梅.植物内生枯草芽孢杆菌Em7菌株对葡萄灰霉病菌的抑菌活性.农药学学报,2016,18(4):465-471.FU B,YAO J N,GAO X N,HUANG L L,KANG Z S,HAN Q M.Antifungal activity of plant endophytic Bacillus subtilis strain Em7against Botrytis cinerea.Chinese Journal of Pesticide Science,2016,18(4):465-471.(in Chinese)
[8]周泠璇,刘娅.红提葡萄内生细菌的分离鉴定及灰霉病拮抗菌的筛选.生物技术通报,2016,32(4):184-189.ZHOU L X,LIU Y.Isolation and identification of endophytic bacteria in red grape,and screening of antagonistic bacteria against Botrytis cinerea.Biotechnology Bulletin,2016,32(4):184-189.(in Chinese)
[9]GE B B,LIU B H,NWET T T,ZHAO W J,SHI L M,ZHANG K.Bacillus methylotrophicus strain NKG-1,isolated from Changbai Mountain,China,has potential applications as a biofertilizer or biocontrol agent.PLo S ONE,2016,11(11):e0166079.
[10]ONGENA M,JACQUES P.Bacillus lipopeptides:versatile weapons for plant disease biocontrol.Trends in Microbiology,2008,16(3):115-125.
[11]MORA I,CABREFIGA J,MONTESINOS E.Cyclic lipopeptide biosynthetic genes and products,and inhibitory activity of plantassociated Bacillus against phytopathogenic bacteria.PLo S ONE,2015,10(5):e0127738.
[12]向亚萍,陈志谊,罗楚平,周华飞,刘永锋.芽孢杆菌的抑菌活性与其产脂肽类抗生素的相关性.中国农业科学,2015,48(20):4064-4076.XIANG Y P,CHEN Z Y,LUO C P,ZHOU H F,LIU Y F.The antifungal activities of Bacillus spp.and its relationship with lipopeptide antibiotics produced by Bacillus spp..Scientia Agricultura Sinica,2015,48(20):4064-4076.(in Chinese)
[13]吕倩,胡江春,王楠,王雪梅,王书锦.南海深海甲基营养型芽孢杆菌SHB114抗真菌脂肽活性产物的研究.中国生物防治学报,2014,30(1):113-120.LüQ,HU J C,WANG N,WANG X M,WANG S J.Anti-fungal lipopeptides produced by Bacillus methylotrophicus SHB114 isolated from south China sea.Chinese Journal of Biological Control,2014,30(1):113-120.(in Chinese)
[14]LIU R F,ZHANG D J,LI Y G,LI M T,LI T.A new antifungal cyclic lipopeptide from Bacillus marinus B-9987.Helvetica Chimica Acta,2010,93(12):2419-2425.
[15]李德全,钱亚明,周鸣鸣,谈蓉,邓自发,袁素霞.海洋细菌NH-8防治草莓灰霉病机理及其抗菌物质分析.植物保护学报,2016,43(2):215-221.LI D Q,QIAN Y M,ZHOU M M,TAN R,DENG Z F,YUAN S X.The mechanism of biological control of strawberry gray mould using the marine bacterial NH-8 strain and analysis of the antifungal substances from the strain.Journal of Plant Protection,2016,43(2):215-221.(in Chinese)
[16]赵杨,苗则彦,李颖,白元俊.番茄灰霉病防治研究进展.中国植保导刊,2014,34(7):21-29.ZHAO Y,MIAO Z Y,LI Y,BAI Y J.Research progress on controlling against tomato gray mold.China Plant Protection,2014,34(7):21-29.(in Chinese)
[17]陈宇飞,文景芝,李立军.葡萄灰霉病研究进展.东北农业大学学报,2006,37(5):693-699.CHEN Y F,WEN J Z,LI L J.Research advance of grape grey mould.Journal of Northeast Agricultural University,2006,37(5):693-699.(in Chinese)
[18]方中达.植病研究方法.北京:中国农业出版社,1998.FANG Z D.Research Method for Plant Pathology.Beijing:China Agriculture Press,1998.(in Chinese)
[19]孙菽蔚,王子峰,岳海东,肖天.一株海洋几丁质酶产生菌的筛选及其产酶条件的初步研究.海洋科学,2007,31(5):10-16.SUN S W,WANG Z F,YUE H D,XIAO T.Screening of chitinase-producing marine bacterial strains and preliminary studies on chitinase-producing conditions.Marine Sciences,2007,31(5):10-16.(in Chinese)
[20]CAO Y,XU Z H,LING N,YUAN Y J,YANG X M,CHEN L H,SHEN B,SHEN Q R.Isolation and identification of lipopeptides produced by B.subtilis SQR 9 for suppressing Fusarium wilt of cucumber.Scientia Horticulturae,2012,135(1):32-39.
[21]曹小红,廖振宇,王春玲,哈志瑞,杨亚静,鲁梅芳,励建荣.Bacillus natto TK-1产脂肽的纯化、抑菌活性及其表面活性剂特性.中国生物工程杂志,2008,28(1):44-48.CAO X H,LIAO Z Y,WANG C L,HA Z R,YANG Y J,LU M F,LI J R.Purification and antimicrobial activity of lipopeptide produced by Bacillus natto TK-1 and its surfactant property.China Biotechnology,2008,28(1):44-48.(in Chinese)
[22]黄大野,周婷,姚经武,刘晓艳,曹春霞,杨妮娜,胡洪涛,龙同,杨自文.死亡谷芽孢杆菌NBIF-001防治灰霉病研究.中国蔬菜,2016(10):63-66.HUANG D Y,ZHOU T,YAO J W,LIU X Y,CAO C X,YANG N N,HU H T,LONG T,YANG Z W.Studies on controlling effect of Bacillus vallismortis NBIF-001 on gray mold.China Vegetables,2016(10):63-66.(in Chinese)
[23]DUNLAP C A,KIM S J,KWON S W,ROONEY A P.Phylogenomic analysis shows that Bacillus amyloliquefaciens subsp.plantarum is a later heterotypic synonym of Bacillus methylotrophicus.International Journal of Systematic and Evolutionary Microbiology,2015,65(7):2104-2109.
[24]TSUGE K,AKIYAMA T,SHODA M.Cloning,sequencing,and characterization of the iturin A operon.Journal of Bacteriology,2001,183(21):6265-6273.
[25]MOYNE A L,CLEVELAND T E,TUZUN S.Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D.FEMS Microbiology Letters,2004,234(1):43-49.
[26]KOUMOUTSI A,CHEN X H,HENNE A,LIESEGANG H,HITZEROTH G,FRANKE P,VATER J,BORRISS R.Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42.Journal of Bacteriology,2004,186(4):1084-1096.
[27]BLOM J,RUECKERT C,NIU B,WANG Q,BORRISS R.The complete genome of Bacillus amyloliquefaciens subsp.plantarum CAU B946 contains a gene cluster for nonribosomal synthesis of iturin A.Journal of Bacteriology,2012,194(7):1845-1846.
[28]ZHANG N,YANG D Q,WANG D D,MIAO Y Z,SHAO J H,ZHOU X,XU Z H,LI Q,FENG H C,LI S Q,SHEN Q R,ZHANG R F.Whole transcriptomic analysis of the plant-beneficial rhizobacterium Bacillus amyloliquefaciens SQR9 during enhanced biofilm formation regulated by maize root exudates.BMC Genomics,2015,16(1):685.
[29]JOSHI R,GARDENER B B M.Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis.Phytopathology,2006,96(2):145-154.
[30]ARREBOLA E,JACOBS R,KORSTEN L.Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens.Journal of Applied Microbiology,2010,108(2):386-395.
[31]刘淼,王继红,姜健,杨宝灵,温小红,陈玉飞,刘丽.海洋微生物应用于生物农药的研究进展.中国农学通报,2014,30(3):232-236.LIU M,WANG J H,JIANG J,YANG B L,WEN X H,CHEN Y F,LIU L.Advances in the studies on the application of marine microorganism in biological pesticides.Chinese Agricultural Science Bulletin,2014,30(3):232-236.(in Chinese)
[32]高伟,田黎,张久明,周俊英,郑立,崔志松,李元广.海洋芽孢杆菌B-9987菌株对番茄灰霉病和早疫病的作用机制初探.植物保护,2010,36(1):55-59.GAO W,TIAN L,ZHANG J M,ZHOU J Y,ZHENG L,CUI Z S,LI Y G.A primary study on the biocontrol mechanisms of Bacillus marinus B-9987 against the tomato gray mold and early blight.Plant Protection,2010,36(1):55-59.(in Chinese)
[33]巩文峰,李月飞,上官妮妮,赵新贝,王阳,马青.出芽短梗霉对苹果采后灰霉病的防治.中国生物防治学报,2016,32(2):251-257.GONG W F,LI Y F,SHANGGUAN N N,ZHAO X B,WANG Y,MA Q.Control of apple postharvest gray mold by Aureobasidium pullulans.Chinese Journal of Biological Control,2016,32(2):251-257.(in Chinese)
[34]申顺善,张莹莹,张维娜,吕雅悠,朱卓琳,朴凤植.绿针假单胞菌HL5-4对番茄灰霉菌的抑制活性及其定殖能力.园艺学报,2016,43(6):1195-1202.SHEN S S,ZHANG Y Y,ZHANG W N,LüY Y,ZHU Z L,PIAO F Z.Antifungal activity of Pseudomonas choloeaphtis HL5-4 against tomato gray mold and its colonization ability.Acta Horticulturae Sinica,2016,43(6):1195-1202.(in Chinese)
[35]HSIEH F C,LIN T C,MENG M,KAO S S.Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A.Current Microbiology,2008,56(1):1-5.
[2]严红,燕继晔,王忠跃,李亚宁,金桂华,李兴红.葡萄灰霉病菌对3种杀菌剂的多重抗药性检测.果树学报,2012,29(4):625-629.YAN H,YAN J Y,WANG Z Y,LI Y N,JIN G H,LI X H.Multiple fungicide resistance of Botrytis cinerea from grapevine to three fungicides.Journal of Fruit Science,2012,29(4):625-629.(in Chinese)
[3]MADHAIYAN M,POONGUZHALI S,KWON S W,SA T M.Bacillus methylotrophicus sp.nov.,a methanol-utilizing,plantgrowth-promoting bacterium isolated from rice rhizosphere soil.International Journal of Systematic and Evolutionary Microbiology,2010,60(10):2490-2495.
[4]王洪梅,吴云成,沈标.青枯病生防菌N5的特性及其生物学效应.土壤,2013,45(6):1082-1090.WANG H M,WU Y C,SHEN B.Characterization and biological effects of antagonistic N5 against Ralstonia solanacearum.Soils,2013,45(6):1082-1090.(in Chinese)
[5]刘伟,宋双,沈小英,安天赐,牛小义,安德荣.番茄灰霉病拮抗芽孢杆菌LW-6-1的筛选、鉴定及抑菌活性研究.西北农林科技大学学报(自然科学版),2013,41(11):73-79.LIU W,SONG S,SHEN X Y,AN T C,NIU X Y,AN D R.Screening,identification and antibacterial activity of antagonistic bacteria LW-6-1 against Botrytis cinerea.Journal of Northwest A&F University(Natural Science Edition),2013,41(11):73-79.(in Chinese)
[6]黄霄,陈波,周登博,谭昕,张锡炎.菌株BM-24的分离鉴定及对香蕉枯萎病菌的抑菌活性.植物保护学报,2013,40(2):121-127.HUANG X,CHEN B,ZHOU D B,TAN X,ZHANG X Y.Isolation,identification of BM-24 strains and its antifungal activity of antagonistic bacteria against Fusarium oxysporum f.sp.cubense.Acta Phytophylacica Sinica,2013,40(2):121-127.(in Chinese)
[7]伏波,姚娟妮,高小宁,黄丽丽,康振生,韩青梅.植物内生枯草芽孢杆菌Em7菌株对葡萄灰霉病菌的抑菌活性.农药学学报,2016,18(4):465-471.FU B,YAO J N,GAO X N,HUANG L L,KANG Z S,HAN Q M.Antifungal activity of plant endophytic Bacillus subtilis strain Em7against Botrytis cinerea.Chinese Journal of Pesticide Science,2016,18(4):465-471.(in Chinese)
[8]周泠璇,刘娅.红提葡萄内生细菌的分离鉴定及灰霉病拮抗菌的筛选.生物技术通报,2016,32(4):184-189.ZHOU L X,LIU Y.Isolation and identification of endophytic bacteria in red grape,and screening of antagonistic bacteria against Botrytis cinerea.Biotechnology Bulletin,2016,32(4):184-189.(in Chinese)
[9]GE B B,LIU B H,NWET T T,ZHAO W J,SHI L M,ZHANG K.Bacillus methylotrophicus strain NKG-1,isolated from Changbai Mountain,China,has potential applications as a biofertilizer or biocontrol agent.PLo S ONE,2016,11(11):e0166079.
[10]ONGENA M,JACQUES P.Bacillus lipopeptides:versatile weapons for plant disease biocontrol.Trends in Microbiology,2008,16(3):115-125.
[11]MORA I,CABREFIGA J,MONTESINOS E.Cyclic lipopeptide biosynthetic genes and products,and inhibitory activity of plantassociated Bacillus against phytopathogenic bacteria.PLo S ONE,2015,10(5):e0127738.
[12]向亚萍,陈志谊,罗楚平,周华飞,刘永锋.芽孢杆菌的抑菌活性与其产脂肽类抗生素的相关性.中国农业科学,2015,48(20):4064-4076.XIANG Y P,CHEN Z Y,LUO C P,ZHOU H F,LIU Y F.The antifungal activities of Bacillus spp.and its relationship with lipopeptide antibiotics produced by Bacillus spp..Scientia Agricultura Sinica,2015,48(20):4064-4076.(in Chinese)
[13]吕倩,胡江春,王楠,王雪梅,王书锦.南海深海甲基营养型芽孢杆菌SHB114抗真菌脂肽活性产物的研究.中国生物防治学报,2014,30(1):113-120.LüQ,HU J C,WANG N,WANG X M,WANG S J.Anti-fungal lipopeptides produced by Bacillus methylotrophicus SHB114 isolated from south China sea.Chinese Journal of Biological Control,2014,30(1):113-120.(in Chinese)
[14]LIU R F,ZHANG D J,LI Y G,LI M T,LI T.A new antifungal cyclic lipopeptide from Bacillus marinus B-9987.Helvetica Chimica Acta,2010,93(12):2419-2425.
[15]李德全,钱亚明,周鸣鸣,谈蓉,邓自发,袁素霞.海洋细菌NH-8防治草莓灰霉病机理及其抗菌物质分析.植物保护学报,2016,43(2):215-221.LI D Q,QIAN Y M,ZHOU M M,TAN R,DENG Z F,YUAN S X.The mechanism of biological control of strawberry gray mould using the marine bacterial NH-8 strain and analysis of the antifungal substances from the strain.Journal of Plant Protection,2016,43(2):215-221.(in Chinese)
[16]赵杨,苗则彦,李颖,白元俊.番茄灰霉病防治研究进展.中国植保导刊,2014,34(7):21-29.ZHAO Y,MIAO Z Y,LI Y,BAI Y J.Research progress on controlling against tomato gray mold.China Plant Protection,2014,34(7):21-29.(in Chinese)
[17]陈宇飞,文景芝,李立军.葡萄灰霉病研究进展.东北农业大学学报,2006,37(5):693-699.CHEN Y F,WEN J Z,LI L J.Research advance of grape grey mould.Journal of Northeast Agricultural University,2006,37(5):693-699.(in Chinese)
[18]方中达.植病研究方法.北京:中国农业出版社,1998.FANG Z D.Research Method for Plant Pathology.Beijing:China Agriculture Press,1998.(in Chinese)
[19]孙菽蔚,王子峰,岳海东,肖天.一株海洋几丁质酶产生菌的筛选及其产酶条件的初步研究.海洋科学,2007,31(5):10-16.SUN S W,WANG Z F,YUE H D,XIAO T.Screening of chitinase-producing marine bacterial strains and preliminary studies on chitinase-producing conditions.Marine Sciences,2007,31(5):10-16.(in Chinese)
[20]CAO Y,XU Z H,LING N,YUAN Y J,YANG X M,CHEN L H,SHEN B,SHEN Q R.Isolation and identification of lipopeptides produced by B.subtilis SQR 9 for suppressing Fusarium wilt of cucumber.Scientia Horticulturae,2012,135(1):32-39.
[21]曹小红,廖振宇,王春玲,哈志瑞,杨亚静,鲁梅芳,励建荣.Bacillus natto TK-1产脂肽的纯化、抑菌活性及其表面活性剂特性.中国生物工程杂志,2008,28(1):44-48.CAO X H,LIAO Z Y,WANG C L,HA Z R,YANG Y J,LU M F,LI J R.Purification and antimicrobial activity of lipopeptide produced by Bacillus natto TK-1 and its surfactant property.China Biotechnology,2008,28(1):44-48.(in Chinese)
[22]黄大野,周婷,姚经武,刘晓艳,曹春霞,杨妮娜,胡洪涛,龙同,杨自文.死亡谷芽孢杆菌NBIF-001防治灰霉病研究.中国蔬菜,2016(10):63-66.HUANG D Y,ZHOU T,YAO J W,LIU X Y,CAO C X,YANG N N,HU H T,LONG T,YANG Z W.Studies on controlling effect of Bacillus vallismortis NBIF-001 on gray mold.China Vegetables,2016(10):63-66.(in Chinese)
[23]DUNLAP C A,KIM S J,KWON S W,ROONEY A P.Phylogenomic analysis shows that Bacillus amyloliquefaciens subsp.plantarum is a later heterotypic synonym of Bacillus methylotrophicus.International Journal of Systematic and Evolutionary Microbiology,2015,65(7):2104-2109.
[24]TSUGE K,AKIYAMA T,SHODA M.Cloning,sequencing,and characterization of the iturin A operon.Journal of Bacteriology,2001,183(21):6265-6273.
[25]MOYNE A L,CLEVELAND T E,TUZUN S.Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D.FEMS Microbiology Letters,2004,234(1):43-49.
[26]KOUMOUTSI A,CHEN X H,HENNE A,LIESEGANG H,HITZEROTH G,FRANKE P,VATER J,BORRISS R.Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42.Journal of Bacteriology,2004,186(4):1084-1096.
[27]BLOM J,RUECKERT C,NIU B,WANG Q,BORRISS R.The complete genome of Bacillus amyloliquefaciens subsp.plantarum CAU B946 contains a gene cluster for nonribosomal synthesis of iturin A.Journal of Bacteriology,2012,194(7):1845-1846.
[28]ZHANG N,YANG D Q,WANG D D,MIAO Y Z,SHAO J H,ZHOU X,XU Z H,LI Q,FENG H C,LI S Q,SHEN Q R,ZHANG R F.Whole transcriptomic analysis of the plant-beneficial rhizobacterium Bacillus amyloliquefaciens SQR9 during enhanced biofilm formation regulated by maize root exudates.BMC Genomics,2015,16(1):685.
[29]JOSHI R,GARDENER B B M.Identification and characterization of novel genetic markers associated with biological control activities in Bacillus subtilis.Phytopathology,2006,96(2):145-154.
[30]ARREBOLA E,JACOBS R,KORSTEN L.Iturin A is the principal inhibitor in the biocontrol activity of Bacillus amyloliquefaciens PPCB004 against postharvest fungal pathogens.Journal of Applied Microbiology,2010,108(2):386-395.
[31]刘淼,王继红,姜健,杨宝灵,温小红,陈玉飞,刘丽.海洋微生物应用于生物农药的研究进展.中国农学通报,2014,30(3):232-236.LIU M,WANG J H,JIANG J,YANG B L,WEN X H,CHEN Y F,LIU L.Advances in the studies on the application of marine microorganism in biological pesticides.Chinese Agricultural Science Bulletin,2014,30(3):232-236.(in Chinese)
[32]高伟,田黎,张久明,周俊英,郑立,崔志松,李元广.海洋芽孢杆菌B-9987菌株对番茄灰霉病和早疫病的作用机制初探.植物保护,2010,36(1):55-59.GAO W,TIAN L,ZHANG J M,ZHOU J Y,ZHENG L,CUI Z S,LI Y G.A primary study on the biocontrol mechanisms of Bacillus marinus B-9987 against the tomato gray mold and early blight.Plant Protection,2010,36(1):55-59.(in Chinese)
[33]巩文峰,李月飞,上官妮妮,赵新贝,王阳,马青.出芽短梗霉对苹果采后灰霉病的防治.中国生物防治学报,2016,32(2):251-257.GONG W F,LI Y F,SHANGGUAN N N,ZHAO X B,WANG Y,MA Q.Control of apple postharvest gray mold by Aureobasidium pullulans.Chinese Journal of Biological Control,2016,32(2):251-257.(in Chinese)
[34]申顺善,张莹莹,张维娜,吕雅悠,朱卓琳,朴凤植.绿针假单胞菌HL5-4对番茄灰霉菌的抑制活性及其定殖能力.园艺学报,2016,43(6):1195-1202.SHEN S S,ZHANG Y Y,ZHANG W N,LüY Y,ZHU Z L,PIAO F Z.Antifungal activity of Pseudomonas choloeaphtis HL5-4 against tomato gray mold and its colonization ability.Acta Horticulturae Sinica,2016,43(6):1195-1202.(in Chinese)
[35]HSIEH F C,LIN T C,MENG M,KAO S S.Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A.Current Microbiology,2008,56(1):1-5.