拮抗匍枝根霉的生防菌R1B的筛选鉴定和抑菌活性分析
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摘要
为防治梨软腐病,从不同植物内生菌中筛选匍枝根霉拮抗菌,同时研究其生防活性。用对峙培养法筛选拮抗匍枝根霉的内生菌;采用果实打孔接种法检验R1B对梨软腐病的防治效果,初步探讨其抗菌活性物质。结果显示来自霍山石斛的菌株R1B对匍枝根霉具有较好的拮抗活性,生理生化和分子鉴定表明R1B与贝莱斯芽孢杆菌具有最近的亲缘关系。菌株R1B几乎完全抑制匍枝根霉菌丝的生长;接种匍枝根霉4 d后,R1B完全抑制梨软腐病的发生,而对照全部腐烂。R1B中含有抗菌肽合成基因bacA(溶杆菌素)、ituC(伊枯草菌素)、bmyB(杆菌抗霉素)及fenD(丰原素)。抗菌二肽溶杆菌素以及伊枯草菌素和丰原素可能是菌株R1B合成的抗菌物质。R1B具有防治梨软腐病的良好潜力。
In order to control the soft rot of pear fruit,the antagonistic bacteria against Rhizopus stolonifer were screened from endophytes isolated from different plants,and their biocontrol efficiency was investigated. Plate confrontation method was used to screen the endophytes against R. stolonifer,inoculation pathogen on punched fruit methods was adapted to investigate the efficacy of R1B against pear soft rot,and its antimicrobial substances were preliminarily examined. As results,strain R1B isolated from Dendrobium huoshanense presented antagonistic activities against R. stolonifer,and was revealed as the closest relatives of Bacillus velezensis based on the biochemical characteristics and16S rRNA gene sequence analysis. The growth of R. stolonifer treated with R1B was almost completely inhibited. The disease incidence of soft rot in the pears treated with strain R1B was totally inhibited,while the control ones completely rotted after 4 days of incubation. The genes of synthesizing antimicrobial peptide(AMP),bacA(bacylisin),ituC(iturin),bmyB(bacyllomicin),and fenD(fengycin),were detected in R1B. In sum,bacylisin,iturin and fengycin may be the antimicrobial substances produced by strain R1B. Results clearly suggest that strain R1B is a potential candidate for bio-controlling soft rot of pear fruit.
In order to control the soft rot of pear fruit,the antagonistic bacteria against Rhizopus stolonifer were screened from endophytes isolated from different plants,and their biocontrol efficiency was investigated. Plate confrontation method was used to screen the endophytes against R. stolonifer,inoculation pathogen on punched fruit methods was adapted to investigate the efficacy of R1B against pear soft rot,and its antimicrobial substances were preliminarily examined. As results,strain R1B isolated from Dendrobium huoshanense presented antagonistic activities against R. stolonifer,and was revealed as the closest relatives of Bacillus velezensis based on the biochemical characteristics and16S rRNA gene sequence analysis. The growth of R. stolonifer treated with R1B was almost completely inhibited. The disease incidence of soft rot in the pears treated with strain R1B was totally inhibited,while the control ones completely rotted after 4 days of incubation. The genes of synthesizing antimicrobial peptide(AMP),bacA(bacylisin),ituC(iturin),bmyB(bacyllomicin),and fenD(fengycin),were detected in R1B. In sum,bacylisin,iturin and fengycin may be the antimicrobial substances produced by strain R1B. Results clearly suggest that strain R1B is a potential candidate for bio-controlling soft rot of pear fruit.
引文
[1]孙平平,王文辉.2016/2017年世界苹果、梨、葡萄、桃及樱桃产量、市场及贸易情况[J].中国果树,2017,(2):91-100.
[2]Kwon JH,Lee CJ.Rhizopus Soft Rot on Pear(Pyrus serotina)Caused by Rhizopus stolonifer in Korea[J].Mycobiology 2006,34(3):151-153.
[3]Gutter Y,Littauer F.Antagonistic action of Bacillus subtilis against citrus fruit pathogens[J].Bulletin of the Research Council of Israel,1953,33(3):192-197
[4]Sharma RR,Singh D,Singh R.Biological control of postharvest diseases of fruits and vegetables by microbial antagonists:Areview[J].Biological Control,2009,50(3):205-221
[5]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[6]武利勤,顾海科,王青,等.石斛内生甲基营养芽胞杆菌的拮抗和促生作用研究[J].生物技术通报,2016,32(8):200-206.
[7]Mora I,Cabrefiga J,Montesinos E.Antimicrobial peptide genes in Bacillus strains from plant environments[J].International Microbiology,2011,14(4):213-223.
[8]Alvindia DG,Natsuaki,KT.Biocontrol activities of Bacillus amyloliquefaciens DGA14 isolated from banana fruit surface against banana crown rot-causing pathogens[J].Crop Protection,2009,28(3):236-242.
[9]Gálvez A,Abriouel H,Benomar,et al.Microbial antagonists to food borne pathogens and biocontrol[J].Curr Opin Biotechnol,2010,21(2):142-148.
[10]Lee JP,Lee SW,Kim CS,et al.Evaluation of formulations of Bacillus licheniformisfrom the biological control of tomato gray mold caused by Botrytis cinerea[J].Biological Control,2006,37(3):329-337.
[11]Lalloo R,Maharajh D,G?rgens J,et al.A downstream process for production of a viable and stable Bacillus cereus aquaculture biological agent[J].Applied Microbiology&Biotechnology,2010,86(2):499-508.
[12]孙平平,崔建潮,贾晓辉,等.贝莱斯芽孢杆菌L-1对梨灰霉和青霉病菌的抑制作用评价及全基因组分析[J].微生物学报,2018,58(9):1637-1646.
[13]张新刚,王海利,张晓慷.防治蔬菜灰霉病的生防菌SFJ1的鉴定及抑菌活性[J].农药,2018,57(3):181-183.
[14]杜淑涛,李术娜,朱宝成.白菜黑斑病拮抗细菌Bacillus velezensis DL-59的筛选鉴定及田间防效实验[J].河北农业大学学报,2010,33(6):51-56.
[15]陈中义,张杰,黄大昉.植物病害生防芽孢杆菌抗菌机制与遗传改良研究[J].植物病理学报,2003,33(2):97-103.
[16]Lugtenberg BJJ,Dekkers L,Bloemberg GV.Molecular determinants of rhizosphere colonization by Pseudomonas[J].Annual Review of Phytopathology,2001,39:461-490.
[17]Loeffler W,Tschen JSM,Vanittanakom N,et al.Antifungal effects of bacilysin and fengymycin from Bacillus subtilis F-29-3 A comparison with activities of other Bacillus antibiotics[J].Journal of Phytopathology,1986,115(3):204-213.
[18]Zheng G,Slavik MF.Isolation,partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain[J].Letters in Applied Microbiology,1999,28(5):363-367.
[19]饶正华,李丽蓓,高生.枯草菌素在饲料中的应用[J].中国饲料,2001,24:17-18.
[20]Abee T,Krockel L,Hill C.Bacteriocins:modes of action and potentials in food preservation and control of food poisoning[J].Int J Food Microbiol,1995,28(2):169-185.
[2]Kwon JH,Lee CJ.Rhizopus Soft Rot on Pear(Pyrus serotina)Caused by Rhizopus stolonifer in Korea[J].Mycobiology 2006,34(3):151-153.
[3]Gutter Y,Littauer F.Antagonistic action of Bacillus subtilis against citrus fruit pathogens[J].Bulletin of the Research Council of Israel,1953,33(3):192-197
[4]Sharma RR,Singh D,Singh R.Biological control of postharvest diseases of fruits and vegetables by microbial antagonists:Areview[J].Biological Control,2009,50(3):205-221
[5]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[6]武利勤,顾海科,王青,等.石斛内生甲基营养芽胞杆菌的拮抗和促生作用研究[J].生物技术通报,2016,32(8):200-206.
[7]Mora I,Cabrefiga J,Montesinos E.Antimicrobial peptide genes in Bacillus strains from plant environments[J].International Microbiology,2011,14(4):213-223.
[8]Alvindia DG,Natsuaki,KT.Biocontrol activities of Bacillus amyloliquefaciens DGA14 isolated from banana fruit surface against banana crown rot-causing pathogens[J].Crop Protection,2009,28(3):236-242.
[9]Gálvez A,Abriouel H,Benomar,et al.Microbial antagonists to food borne pathogens and biocontrol[J].Curr Opin Biotechnol,2010,21(2):142-148.
[10]Lee JP,Lee SW,Kim CS,et al.Evaluation of formulations of Bacillus licheniformisfrom the biological control of tomato gray mold caused by Botrytis cinerea[J].Biological Control,2006,37(3):329-337.
[11]Lalloo R,Maharajh D,G?rgens J,et al.A downstream process for production of a viable and stable Bacillus cereus aquaculture biological agent[J].Applied Microbiology&Biotechnology,2010,86(2):499-508.
[12]孙平平,崔建潮,贾晓辉,等.贝莱斯芽孢杆菌L-1对梨灰霉和青霉病菌的抑制作用评价及全基因组分析[J].微生物学报,2018,58(9):1637-1646.
[13]张新刚,王海利,张晓慷.防治蔬菜灰霉病的生防菌SFJ1的鉴定及抑菌活性[J].农药,2018,57(3):181-183.
[14]杜淑涛,李术娜,朱宝成.白菜黑斑病拮抗细菌Bacillus velezensis DL-59的筛选鉴定及田间防效实验[J].河北农业大学学报,2010,33(6):51-56.
[15]陈中义,张杰,黄大昉.植物病害生防芽孢杆菌抗菌机制与遗传改良研究[J].植物病理学报,2003,33(2):97-103.
[16]Lugtenberg BJJ,Dekkers L,Bloemberg GV.Molecular determinants of rhizosphere colonization by Pseudomonas[J].Annual Review of Phytopathology,2001,39:461-490.
[17]Loeffler W,Tschen JSM,Vanittanakom N,et al.Antifungal effects of bacilysin and fengymycin from Bacillus subtilis F-29-3 A comparison with activities of other Bacillus antibiotics[J].Journal of Phytopathology,1986,115(3):204-213.
[18]Zheng G,Slavik MF.Isolation,partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain[J].Letters in Applied Microbiology,1999,28(5):363-367.
[19]饶正华,李丽蓓,高生.枯草菌素在饲料中的应用[J].中国饲料,2001,24:17-18.
[20]Abee T,Krockel L,Hill C.Bacteriocins:modes of action and potentials in food preservation and control of food poisoning[J].Int J Food Microbiol,1995,28(2):169-185.