一株西瓜专化型尖孢镰刀菌抑制菌的分离鉴定及其抑菌活性
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摘要
以黑龙江省扎龙自然保护区的植物根际土壤为实验材料,采用对峙实验筛选抑制西瓜专化型尖孢镰刀菌(Fusarium oxysporum f. sp. niveum,FON)的菌株,并采用菌丝生长、孢子萌发和扫描电镜等方法研究其抑菌特性。结果表明,分离获得了一株能抑制FON的菌株MLY01,根据其菌落形态特征和16S rDNA碱基序列同源性结果,鉴定MLY01为地衣芽孢杆菌Bacillus licheniformis,将其命名为Bacillus licheniformis MLY01。当培养基与无菌滤液比例为1∶1时,菌丝生长抑制率为71. 85%,生物量抑制率为79. 55%,36 h孢子萌发抑制率为76. 81%。将FON孢子悬液与无菌滤液按1∶1比例混合,培养12 h,孢子数目降低,表面粗糙,菌丝畸形扭曲,表面有多处凹陷。菌株MLY01对FON的生长具有明显的抑制作用,在生物菌剂的研发方面具有潜力。
To isolate a Fusarium oxyxporum f. sp. niveum (FON)-inhibiting strain,the plant rhizosphere soil which was located in Zhalong Nature Reserve,Heilongjiang Province was collected and used. The FON-inhibiting strain was screened out by antagonistic experiment,and was identified by morphological test and 16S rDNA sequence analysis. Moreover,the inhibition characteristics were studied using mycelial growth,spore germination and scanning electron microscope methods. The results showed that the FON-inhibiting strain was classified as Bacillus licheniformis,and was named as MLY01. When the ratio of filtrate to medium was 1∶ 1,the inhibition rate of FON mycelial growth was 71. 85%,the inhibition rate of biomass was 79. 55%,and the inhibition rate of spore germination was 76. 81% at 36 h. The number of spore was decreased and surface was rough,and the hyphae of FON was deformed after 12 h treatment by cell-free cultural filtrate of MLY01. These results indicated that MLY01 was a high-efficiency FON-inhibiting strain,and it could be used to develop antibacterial agent in future.
To isolate a Fusarium oxyxporum f. sp. niveum (FON)-inhibiting strain,the plant rhizosphere soil which was located in Zhalong Nature Reserve,Heilongjiang Province was collected and used. The FON-inhibiting strain was screened out by antagonistic experiment,and was identified by morphological test and 16S rDNA sequence analysis. Moreover,the inhibition characteristics were studied using mycelial growth,spore germination and scanning electron microscope methods. The results showed that the FON-inhibiting strain was classified as Bacillus licheniformis,and was named as MLY01. When the ratio of filtrate to medium was 1∶ 1,the inhibition rate of FON mycelial growth was 71. 85%,the inhibition rate of biomass was 79. 55%,and the inhibition rate of spore germination was 76. 81% at 36 h. The number of spore was decreased and surface was rough,and the hyphae of FON was deformed after 12 h treatment by cell-free cultural filtrate of MLY01. These results indicated that MLY01 was a high-efficiency FON-inhibiting strain,and it could be used to develop antibacterial agent in future.
引文
[1]黄春艳,卜元卿,单正军,等.西瓜连作病害机理及生物防治研究进展[J].生态学杂志,2016,35(6):1670-1676.HUANG C Y,BU Y Q,SHAN Z J,et al.Research advances in mechanisms of watermelon continuous cropping diseases and its biological control[J].Chinese Journal of Ecology,2016,35(6):1670-1676.(in Chinese with English abstract)
[2]雷白时,王笑颖,郭晓军,等.西瓜枯萎病拮抗细菌HD-5菌株的筛选及鉴定[J].湖北农业科学,2012,51(11):2236-2238.LEI B S,WANG X Y,GUO X J,et al.The screening and identification of antagonistic bacterium strain HD-5 against watermelon Fusarium wilt[J].Hubei Agricultural Sciences,2012,51(11):2236-2238.(in Chinese with English abstract)
[3]朱育菁,车建美,肖荣凤,等.尖孢镰刀菌(Fusarium oxysporum Schl.)的生长特性[J].中国农学通报,2007,23(8):373-376.ZHU Y J,CHE J M,XIAO R F,et al.Growth characteristics of Fusarium oxysporum Schl.[J].Chinese Agricultural Science Bulletin,2007,23(8):373-376.(in Chinese with English abstract)
[4]吕桂云,郭绍贵,张海英,等.西瓜与枯萎病菌非亲和互作的表达序列标签分析[J].中国农业科学,2010,43(9):1883-1894.LG Y,GUO S G,ZHANG H Y,et al.Analysis of expressed sequence tags in the incompatible interaction between watermelon and Fusarium oxysporum[J].Scientia Agricultura Sinica,2010,43(9):1883-1894.(in Chinese with English abstract)
[5]徐敬华,黄丹枫,支月娥.PAL活性与嫁接西瓜枯萎病抗性传递的相关性[J].上海交通大学学报(农业科学版),2004,22(1):12-16.XU J H,HUANG D F,ZHI Y E.Relationship of PAL activity and transfer of resistance to Fusarium oxysporum f.sp.niveum in grafted watermelon[J].Journal of Shanghai Jiaotong University(Agricultural Science),2004,22(1):12-16.(in Chinese with English abstract)
[6]王汉荣,方丽,任海英,等.西瓜枯萎病防治药剂筛选[J].植物保护,2011,37(1):150-152.WANG H R,FANG L,REN H Y,et al.Fungicide selection for controlling Fusarium wilt of watermelon[J].Plant Protection,2011,37(1):150-152.(in Chinese with English abstract)
[7]吴学宏,卢志军,王品品,等.西瓜枯萎病综合防治研究进展[J].植物保护,2011,37(4):27-32.WU X H,LU Z J,WANG P P,et al.Current advances in integrated management of watermelon Fusarium wilt[J].Plant Protection,2011,37(4):27-32.(in Chinese with English abstract)
[8]WANG H,NG T B,LIU Q.Isolation of a new heterodimeric lectin with mitogenic activity from fruiting bodies of the mushroom Agrocybe cylindracea[J].Life Sciences,2002,70(8):877-885.
[9]张雪花,李琳玲,程华,等.解淀粉芽孢杆菌CMN1308抗真菌特性研究[J].北方园艺,2016(2):117-121.ZHANG X H,LI L L,CHENG H,et al.Characterizing a novel strain of Bacillus amylolique faciens CMN1308 for potential biological control in pathogenic bacterium[J].Northern Horticulture,2016(2):117-121.(in Chinese with English abstract)
[10]武志江,李业燕,王亚军,等.春百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究[J].微生物学通报,2015,42(7):1307-1320.WU Z J,LI Y Y,WANG Y J,et al.Isolation and identification of an antagonistic bacterium against Fusarium on lily and its antifungal substances[J].Microbiology China,2015,42(7):1307-1320.(in Chinese with English abstract)
[11]钱英,汪琨,章小洪,等.解淀粉芽孢杆菌BW-13产生的抗真菌物质特性研究与初步分离纯化[J].浙江工业大学学报,2012,40(1):42-45.QIAN Y,WANG K,ZHANG X H,et al.Characterization and prefractionation of antifungal substance produced by Bacillus amyloliquefaciens BW-13[J].Journal of Zhejiang University of Technology,2012,40(1):42-45.(in Chinese with English abstract)
[12]方敦煌,李天飞,沐应祥,等.拮抗细菌GP13防治烟草黑胫病的田间应用[J].云南农业大学学报,2003,18(1):49-52.FANG D H,LI T F,MU Y X,et al.Field application of antagonistic bacteria GP13 to control tobacco black shank[J].Journal of Yunnan Agricultural University,2003,18(1):49-52.(in Chinese with English abstract)
[13]齐东梅,梁启美,惠明,等.棉花枯萎黄萎病拮抗芽孢杆菌的抗菌蛋白特性[J].微生物学通报,2005,32(4):42-46.QI D M,LIANG Q M,HUI M,et al.Characteristics of antagon istic proteins from Bacillus against cotton Fusarium wilt and Verticillium wilt[J].Microbiology China,2005,32(4):42-46.(in Chinese with English abstract)
[14]SID A,EZZIYYANI M,EGEA-GILABERT C,et al.Selecting bacterial strains for use in the biocontrol of diseases caused by Phytophthora capsici and Alternaria alternata in sweet pepper plants[J].Blologia Plantarum,2003,47:569-574.
[15]MORE S M,SHINDE A V,MORE B S,et al.Study of antagonistic action of antifungal compound produced by Bacillus licheniformis[J].Journal of Pure and Applied Microbiology,2013,7(2):1457-1462.
[16]CUI T B,CHAI H Y,JIANG L X.Isolation and partial characterization of an antifungal protein produced by Bacillus licheniformis BS-3[J].Molecules,2012,17(6):7336-7347.
[17]彭化贤,刘波微,陈小娟,等.水稻稻瘟病拮抗细菌的筛选与防治初探[J].中国生物防治,2002,18(1):25-27.PENG H X,LIU B W,CHEN X J,et al.Biological control of rice blast with Bacillus spp[J].Chinese Journal of Biological Control,2002,18(1):25-27.(in Chinese with English abstract)
[18]李玉峰,张笑宇,胡俊,等.地衣芽孢杆菌代谢物对马铃薯晚疫病菌的抑制作用[J].内蒙古农业大学学报,2009,30(1):10-13.LI Y F,ZHANG X Y,HU J,et al.Inhibitory effects of metabolite from Bacillus licheniformis against phytophthora infestans[J].Journalof Inner Mongolia Agricultural University,2009,30(1):10-13.(in Chinese with English abstract)
[19]罗秀俊,郭建峰,韩波波,等.一株香菇病原真菌的分离、鉴定与室内防治药剂筛选[J].云南农业大学学报(自然科学),2017,32(5):793-804.LUO X J,GUO J F,HAN B B,et al.2017.Isolation,identification of a Lentinula edodes pathogen and vitro screening of control medicament[J].Journal of Yunnan Agricultural University(Natural Science),2017,32(5):793-804.(in Chinese with English abstract)
[20]苑琳,郑雅心,李冠华.萎缩芽孢杆菌SF1对尖孢镰刀菌胡麻专化型抑菌效果研究[J].北方农业学报,2017,45(3):65-69.YUAN L,ZHENG Y X,LI G H.Study on the antibacterial effect of Bacllus atrophaeus SF1 on Fusarium oxysporum f.sp.lini[J].Journal of Northern Agriculture,2017,45(3):65-69.(in Chinese with English abstract)
[21]赵烨,杨恩东,牛晓伟,等.西瓜枯萎病内生拮抗细菌YGRE24的分离鉴定及其特性研究[J].安徽农业大学学报,2013,40(5):765-770.ZHAO Y,YANG E D,NIU X W,et al.Screening and identification of endophytic antagonistic bacteria strain YGRE24against watermelon Fusarium wilt and its characteristics[J].Journal of Anhui Agricultural University,2013,40(5):765-770.(in Chinese with English abstract)
[22]牛彦波,吴皓琼.Klebsilla ASP-15拮抗真菌病害能力与抗病机理的初步研究[J].生物技术,2009,19(3):55-58.NIU Y B,WU H Q.Preliminary study on the antifungal ability and resistance mechanism of Klebsilla ASP-15 into phytopathogenic fungi[J].Biotechnology,2009,19(3):55-58.(in Chinese with English abstract)
[23]吴石平,陆德清,刘世怡,等.三唑酮对豌豆根际木霉定殖和尖孢镰刀菌厚垣孢子萌发的影响[J].云南农业大学学报,2000,15(3):247-249.WU S P,LU D Q,LIU S Y,et al.Effects of tridemefon on colonization of Trichoderma harzianum and chlamydospore germination of Fusarium oxyporum in rhizosphere of pea seeding[J].Journal of Yunnan Agricultural University,2000,15(3):247-249.(in Chinese with English abstract)
[24]秦楠,郝林,李鑫.解淀粉芽胞杆菌HRH317抗菌蛋白的分离纯化及其抗菌作用[J].植物保护学报,2015,42(5):813-819.QIN N,HAO L,LI X.Isolation,purification and inhibitive effect of antifungal protein of Bacillus amyloliquefaciens HRH317[J].Journal of Plant Protection,2015,42(5):813-819.(in Chinese with English abstract)
[25]王芳,李静,张欢.青霉菌、放线菌株和石灰水对尖孢镰刀菌抑制作用的研究[J].中国农学通报,2013,29(12):185-189.WANG F,LI J,ZHANG H.The study on inhibition of penicillium,actinomycete and limewater to Fusarium oxysporum[J].Chinese Agricultural Science Bulletin,2013,29(12):185-189.(in Chinese with English abstract)
[26]ZHANG B,DONG C,SHANG Q,et al.Purification and partial characterization of bacillomycin L produced by Bacillus amyloliquefaciens K103 from lemon[J].Applied Biochemistry&Biotechnology,2013,171(8):2262-2272.
[27]EL-MOUNADI K,ISLAM K T,HERNANDEZ-ORTIZ P,et al.Antifungal mechanisms of a plant defensin Mt Def4 are not conserved between the ascomycete fungi Neurospora crassa and Fusarium graminearum[J].Molecular Microbiology,2016,100(3):542-559.
[2]雷白时,王笑颖,郭晓军,等.西瓜枯萎病拮抗细菌HD-5菌株的筛选及鉴定[J].湖北农业科学,2012,51(11):2236-2238.LEI B S,WANG X Y,GUO X J,et al.The screening and identification of antagonistic bacterium strain HD-5 against watermelon Fusarium wilt[J].Hubei Agricultural Sciences,2012,51(11):2236-2238.(in Chinese with English abstract)
[3]朱育菁,车建美,肖荣凤,等.尖孢镰刀菌(Fusarium oxysporum Schl.)的生长特性[J].中国农学通报,2007,23(8):373-376.ZHU Y J,CHE J M,XIAO R F,et al.Growth characteristics of Fusarium oxysporum Schl.[J].Chinese Agricultural Science Bulletin,2007,23(8):373-376.(in Chinese with English abstract)
[4]吕桂云,郭绍贵,张海英,等.西瓜与枯萎病菌非亲和互作的表达序列标签分析[J].中国农业科学,2010,43(9):1883-1894.LG Y,GUO S G,ZHANG H Y,et al.Analysis of expressed sequence tags in the incompatible interaction between watermelon and Fusarium oxysporum[J].Scientia Agricultura Sinica,2010,43(9):1883-1894.(in Chinese with English abstract)
[5]徐敬华,黄丹枫,支月娥.PAL活性与嫁接西瓜枯萎病抗性传递的相关性[J].上海交通大学学报(农业科学版),2004,22(1):12-16.XU J H,HUANG D F,ZHI Y E.Relationship of PAL activity and transfer of resistance to Fusarium oxysporum f.sp.niveum in grafted watermelon[J].Journal of Shanghai Jiaotong University(Agricultural Science),2004,22(1):12-16.(in Chinese with English abstract)
[6]王汉荣,方丽,任海英,等.西瓜枯萎病防治药剂筛选[J].植物保护,2011,37(1):150-152.WANG H R,FANG L,REN H Y,et al.Fungicide selection for controlling Fusarium wilt of watermelon[J].Plant Protection,2011,37(1):150-152.(in Chinese with English abstract)
[7]吴学宏,卢志军,王品品,等.西瓜枯萎病综合防治研究进展[J].植物保护,2011,37(4):27-32.WU X H,LU Z J,WANG P P,et al.Current advances in integrated management of watermelon Fusarium wilt[J].Plant Protection,2011,37(4):27-32.(in Chinese with English abstract)
[8]WANG H,NG T B,LIU Q.Isolation of a new heterodimeric lectin with mitogenic activity from fruiting bodies of the mushroom Agrocybe cylindracea[J].Life Sciences,2002,70(8):877-885.
[9]张雪花,李琳玲,程华,等.解淀粉芽孢杆菌CMN1308抗真菌特性研究[J].北方园艺,2016(2):117-121.ZHANG X H,LI L L,CHENG H,et al.Characterizing a novel strain of Bacillus amylolique faciens CMN1308 for potential biological control in pathogenic bacterium[J].Northern Horticulture,2016(2):117-121.(in Chinese with English abstract)
[10]武志江,李业燕,王亚军,等.春百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究[J].微生物学通报,2015,42(7):1307-1320.WU Z J,LI Y Y,WANG Y J,et al.Isolation and identification of an antagonistic bacterium against Fusarium on lily and its antifungal substances[J].Microbiology China,2015,42(7):1307-1320.(in Chinese with English abstract)
[11]钱英,汪琨,章小洪,等.解淀粉芽孢杆菌BW-13产生的抗真菌物质特性研究与初步分离纯化[J].浙江工业大学学报,2012,40(1):42-45.QIAN Y,WANG K,ZHANG X H,et al.Characterization and prefractionation of antifungal substance produced by Bacillus amyloliquefaciens BW-13[J].Journal of Zhejiang University of Technology,2012,40(1):42-45.(in Chinese with English abstract)
[12]方敦煌,李天飞,沐应祥,等.拮抗细菌GP13防治烟草黑胫病的田间应用[J].云南农业大学学报,2003,18(1):49-52.FANG D H,LI T F,MU Y X,et al.Field application of antagonistic bacteria GP13 to control tobacco black shank[J].Journal of Yunnan Agricultural University,2003,18(1):49-52.(in Chinese with English abstract)
[13]齐东梅,梁启美,惠明,等.棉花枯萎黄萎病拮抗芽孢杆菌的抗菌蛋白特性[J].微生物学通报,2005,32(4):42-46.QI D M,LIANG Q M,HUI M,et al.Characteristics of antagon istic proteins from Bacillus against cotton Fusarium wilt and Verticillium wilt[J].Microbiology China,2005,32(4):42-46.(in Chinese with English abstract)
[14]SID A,EZZIYYANI M,EGEA-GILABERT C,et al.Selecting bacterial strains for use in the biocontrol of diseases caused by Phytophthora capsici and Alternaria alternata in sweet pepper plants[J].Blologia Plantarum,2003,47:569-574.
[15]MORE S M,SHINDE A V,MORE B S,et al.Study of antagonistic action of antifungal compound produced by Bacillus licheniformis[J].Journal of Pure and Applied Microbiology,2013,7(2):1457-1462.
[16]CUI T B,CHAI H Y,JIANG L X.Isolation and partial characterization of an antifungal protein produced by Bacillus licheniformis BS-3[J].Molecules,2012,17(6):7336-7347.
[17]彭化贤,刘波微,陈小娟,等.水稻稻瘟病拮抗细菌的筛选与防治初探[J].中国生物防治,2002,18(1):25-27.PENG H X,LIU B W,CHEN X J,et al.Biological control of rice blast with Bacillus spp[J].Chinese Journal of Biological Control,2002,18(1):25-27.(in Chinese with English abstract)
[18]李玉峰,张笑宇,胡俊,等.地衣芽孢杆菌代谢物对马铃薯晚疫病菌的抑制作用[J].内蒙古农业大学学报,2009,30(1):10-13.LI Y F,ZHANG X Y,HU J,et al.Inhibitory effects of metabolite from Bacillus licheniformis against phytophthora infestans[J].Journalof Inner Mongolia Agricultural University,2009,30(1):10-13.(in Chinese with English abstract)
[19]罗秀俊,郭建峰,韩波波,等.一株香菇病原真菌的分离、鉴定与室内防治药剂筛选[J].云南农业大学学报(自然科学),2017,32(5):793-804.LUO X J,GUO J F,HAN B B,et al.2017.Isolation,identification of a Lentinula edodes pathogen and vitro screening of control medicament[J].Journal of Yunnan Agricultural University(Natural Science),2017,32(5):793-804.(in Chinese with English abstract)
[20]苑琳,郑雅心,李冠华.萎缩芽孢杆菌SF1对尖孢镰刀菌胡麻专化型抑菌效果研究[J].北方农业学报,2017,45(3):65-69.YUAN L,ZHENG Y X,LI G H.Study on the antibacterial effect of Bacllus atrophaeus SF1 on Fusarium oxysporum f.sp.lini[J].Journal of Northern Agriculture,2017,45(3):65-69.(in Chinese with English abstract)
[21]赵烨,杨恩东,牛晓伟,等.西瓜枯萎病内生拮抗细菌YGRE24的分离鉴定及其特性研究[J].安徽农业大学学报,2013,40(5):765-770.ZHAO Y,YANG E D,NIU X W,et al.Screening and identification of endophytic antagonistic bacteria strain YGRE24against watermelon Fusarium wilt and its characteristics[J].Journal of Anhui Agricultural University,2013,40(5):765-770.(in Chinese with English abstract)
[22]牛彦波,吴皓琼.Klebsilla ASP-15拮抗真菌病害能力与抗病机理的初步研究[J].生物技术,2009,19(3):55-58.NIU Y B,WU H Q.Preliminary study on the antifungal ability and resistance mechanism of Klebsilla ASP-15 into phytopathogenic fungi[J].Biotechnology,2009,19(3):55-58.(in Chinese with English abstract)
[23]吴石平,陆德清,刘世怡,等.三唑酮对豌豆根际木霉定殖和尖孢镰刀菌厚垣孢子萌发的影响[J].云南农业大学学报,2000,15(3):247-249.WU S P,LU D Q,LIU S Y,et al.Effects of tridemefon on colonization of Trichoderma harzianum and chlamydospore germination of Fusarium oxyporum in rhizosphere of pea seeding[J].Journal of Yunnan Agricultural University,2000,15(3):247-249.(in Chinese with English abstract)
[24]秦楠,郝林,李鑫.解淀粉芽胞杆菌HRH317抗菌蛋白的分离纯化及其抗菌作用[J].植物保护学报,2015,42(5):813-819.QIN N,HAO L,LI X.Isolation,purification and inhibitive effect of antifungal protein of Bacillus amyloliquefaciens HRH317[J].Journal of Plant Protection,2015,42(5):813-819.(in Chinese with English abstract)
[25]王芳,李静,张欢.青霉菌、放线菌株和石灰水对尖孢镰刀菌抑制作用的研究[J].中国农学通报,2013,29(12):185-189.WANG F,LI J,ZHANG H.The study on inhibition of penicillium,actinomycete and limewater to Fusarium oxysporum[J].Chinese Agricultural Science Bulletin,2013,29(12):185-189.(in Chinese with English abstract)
[26]ZHANG B,DONG C,SHANG Q,et al.Purification and partial characterization of bacillomycin L produced by Bacillus amyloliquefaciens K103 from lemon[J].Applied Biochemistry&Biotechnology,2013,171(8):2262-2272.
[27]EL-MOUNADI K,ISLAM K T,HERNANDEZ-ORTIZ P,et al.Antifungal mechanisms of a plant defensin Mt Def4 are not conserved between the ascomycete fungi Neurospora crassa and Fusarium graminearum[J].Molecular Microbiology,2016,100(3):542-559.