贝莱斯芽胞杆菌ZF2对多主棒孢病菌防治效果
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摘要
黄瓜棒孢叶斑病是近年来危害严重的黄瓜病害之一,严重时可导致黄瓜大面积减产。菌株ZF2是本实验室从黄瓜植株体内分离筛选得到的一株对多主棒孢菌有显著抑制效果的生防细菌。经生理生化测定、Biolog测定和多基因系统发育树综合分析,鉴定菌株ZF2为贝莱斯芽胞杆菌。酶学试验和光学显微观察结果表明,菌株ZF2在代谢过程中产生蛋白酶和纤维素酶,使多主棒孢菌菌丝生长受到抑制并形成连续的椭圆型膨胀,抑制率达60.10%。利用平板对峙法和双层培养法证明菌株ZF2具有广谱拮抗作用,对6种病原真菌和7种病原细菌具有显著拮抗效果。黄瓜棒孢叶斑病的防效测定结果显示,接种菌株ZF2的黄瓜发病明显减轻,防效为90.81%。综上,菌株ZF2生防性状优良,在植物病害生物防治中有一定应用潜力。
In recent years, Corynespora leaf spot caused by Corynespora cassiicola is one of the most destructive diseases of cucumber(Cucumis sativus), causing significant yield losses to cucumber production. A biocontrol bacterium ZF2 isolated from the cucumber plants had significant inhibitory effect on C. cassiicola. The strain ZF2 was identified as Bacillus velezensis based on 16 S r DNA sequence analysis, physiological and biochemical characteristics, biolog analysis, and multilocus sequence analysis. Enzymatic test and microscopic observation showed that strain ZF2 could produce protease and cellulase during the metabo lic process, which caused mycelium malformation and formed continuous elliptic expansion. The inhibition rate on mycelial growth was 60.10%. The antibacterial and antifungal activity of strain ZF2 was tested by double-layer culture method and plate confrontation method, respectively. The results showed that ZF2 exhibited significant and broad inhibitory spectrum against six pathogenic fungi and seven pathogenic bacteria. Meanwhile, the control effect of strain ZF2 on Corynespora leaf spot disease was examined using potted plant in greenhouse. The results indicated that the disease incidence of cucumber inoculated with strain ZF2 was significantly reduced and the control eff icacy was 90.81%. In summary, the strain ZF2 has excellent biocontrol characteristics and potential in biocontrol of plant diseases.
In recent years, Corynespora leaf spot caused by Corynespora cassiicola is one of the most destructive diseases of cucumber(Cucumis sativus), causing significant yield losses to cucumber production. A biocontrol bacterium ZF2 isolated from the cucumber plants had significant inhibitory effect on C. cassiicola. The strain ZF2 was identified as Bacillus velezensis based on 16 S r DNA sequence analysis, physiological and biochemical characteristics, biolog analysis, and multilocus sequence analysis. Enzymatic test and microscopic observation showed that strain ZF2 could produce protease and cellulase during the metabo lic process, which caused mycelium malformation and formed continuous elliptic expansion. The inhibition rate on mycelial growth was 60.10%. The antibacterial and antifungal activity of strain ZF2 was tested by double-layer culture method and plate confrontation method, respectively. The results showed that ZF2 exhibited significant and broad inhibitory spectrum against six pathogenic fungi and seven pathogenic bacteria. Meanwhile, the control effect of strain ZF2 on Corynespora leaf spot disease was examined using potted plant in greenhouse. The results indicated that the disease incidence of cucumber inoculated with strain ZF2 was significantly reduced and the control eff icacy was 90.81%. In summary, the strain ZF2 has excellent biocontrol characteristics and potential in biocontrol of plant diseases.
引文
[1]Li B J,Zhao Y J,Gao W,et al.First report of target leaf spot caused by Corynespora cassiiacola on balsam pear in China[J].Plant Disease,2010,94(1):127.
[2]李宝聚,高苇,石延霞,等.多主棒孢和棒孢叶斑病的研究进展[J].植物保护学报,2012,39(2):171-176.
[3]黄大野,向朝晖,周世位,等.壳寡糖对茄子棒孢叶斑病的防治效果研究[J].中国蔬菜,2017(9):46-49.
[4]孙冰冰,李伟,魏军,等.生防芽胞杆菌的研究进展[J].天津农业科学,2015,21(12):102-107.
[5]余小霞,田健,伍宁丰.枯草芽胞杆菌芽胞表面展示外源蛋白的研究进展[J].中国农业科技导报,2013,15(5):31-38.
[6]Palazzinia J M,Dunlapb C A,Bowmanc M J,et al.Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation:Genome sequencing and secondary metabolite cluster profiles[J].Microbiological Research,2016,192(1):30-36.
[7]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.
[8]王伟,李术娜,李红亚,等.大丽轮枝菌拮抗细菌菌株12-51的筛选鉴定与抗菌物性质分析[J].中国农学通报,2009,25(19):14-19.
[9]黄大野,贲海燕,曹春霞,等.死亡谷芽胞杆菌NBIF-001防治番茄棒孢叶斑病研究[J].植物保护,2017,43(6):192-195.
[10]黄大野,曹春霞,张亚妮,等.枯草芽胞杆菌NBF809防治番茄棒孢叶斑病研究[J].中国蔬菜,2018(12):40-44.
[11]Baiyye B,Pornsuriya C,Ito S,et al.Trichoderma spirale T76-1 displays biocontrol activity against leaf spot on lettuce(Lactuca sativa L.)caused by Corynespora cassiicola or Curvularia aeria[J].Biological Control,2019,129(2):195-200.
[12]程丽娟,薛泉宏.微生物学实验技术[M].西安:世界图书出版公司,2000.
[13]Chatterjee A,Cui Y,Liu Y,et al.Inactivation of rsm A leads to overproduction of extracellular pectinases,cellulases,and proteases in Erwinia carotovorasubsp.carotovora in the absence of the starvation/cell density-sensing signal,N-(3-oxohexanoyl)-L-homoserine lactone[J].Applied and Environmental Microbiology,1995,61(5):1959-1967.
[14]Weinert N,Meincke R,Gottwald C,et al.Bacterial diversity on the surface of potato tubers in soil and the influence of the plant genotype[J].FEMS Microbiology Ecology,2010,74(1):114-123.
[15]胥丽娜,徐亮,李磊,等.枯草芽胞杆菌对杨树水泡溃疡病菌菌丝生长的抑制作用[J].菌物研究,2007,5(3):165-168.
[16]Stonier T.Agrobacterium tumefaciens Conn.II.Production of an antibiotic substance[J].Journal of Bacteriology,1960,79(6):889-898.
[17]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.
[18]郭芳芳,谢镇,卢鹏,等.一株多粘类芽孢杆菌的鉴定及其生防促生效果初步测定[J].中国生物防治学报,2014,30(4):489-496.
[19]齐爱勇,魏东盛,刘大群,等.番茄灰霉病菌拮抗细菌B21的鉴定[J].中国农学通报,2006,22(9):360-363.
[20]张猛,王琼,冯发运,等.植物内生特基拉芽胞杆菌的分离、鉴定及防治西瓜枯萎病效果[J].中国生物防治学报,2017,33(3):371-377.
[21]阚琳娜,李宝聚,纪明山,等.黄瓜褐斑病防治药剂的活体筛选[J].中国蔬菜,2007(4):22-24.
[22]Ruiz G C,Béjar V,Martínez C F,et al.Bacillus velezensis sp.nov.,a surfactant-producing bacterium isolated from the river Vélez in Málaga,southern Spain[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(1):191-195.
[23]Kanjanamaneesathian M,Wiwattanapatapee R,Rotniam W,et al.Application of a suspension concentrate formulation of Bacillus velezensis to control root rot of hydroponically-grown vegetables[J].New Zealand Plant Protection,2013,66:229-234.
[24]罗文建,杨凡,史宣杰,等.黄瓜枯萎病拮抗菌的分离鉴定及其生物防效[J].华中农业大学学报,2018,37(3):32-38.
[25]姜乾坤,彭阁,王瑞,等.抗青枯内生细菌的筛选及其对烟草青枯病的防治效果[J].中国烟草科学,2017,38(5):13-17.
[26]马佳,李颖,胡栋,等.芽胞杆菌生物防治作用机理与应用研究进展[J].中国生物防治学报,2018,34(4):639-648.
[27]Gao Z F,Zhang B J,Liu H P,et al.Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinereal[J].Biological Control,2017,105(1):27-39.
[28]Cho S J,Lee S K,Cha B J,et al.Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03[J].FEMS Microbiology Letters,2003,223(1):47-51.
[29]Xu T,Zhu T H,Li S J.Β-1,3-1,4-glucanase gene form Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi[J].World Journal of Microbiology and Biotechnology,2016,32(2):1-9.
[30]于淑晶,王满意,田芳,等.黄瓜棒孢叶斑病的防治及抗药性研究进展[J].农药,2014,53(1):7-11.
[31]Miyamoto T,Ishii H,Seko T,et al.Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture,Japan[J].Plant Pathology,2009,58(6):1144-1151.
[2]李宝聚,高苇,石延霞,等.多主棒孢和棒孢叶斑病的研究进展[J].植物保护学报,2012,39(2):171-176.
[3]黄大野,向朝晖,周世位,等.壳寡糖对茄子棒孢叶斑病的防治效果研究[J].中国蔬菜,2017(9):46-49.
[4]孙冰冰,李伟,魏军,等.生防芽胞杆菌的研究进展[J].天津农业科学,2015,21(12):102-107.
[5]余小霞,田健,伍宁丰.枯草芽胞杆菌芽胞表面展示外源蛋白的研究进展[J].中国农业科技导报,2013,15(5):31-38.
[6]Palazzinia J M,Dunlapb C A,Bowmanc M J,et al.Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation:Genome sequencing and secondary metabolite cluster profiles[J].Microbiological Research,2016,192(1):30-36.
[7]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.
[8]王伟,李术娜,李红亚,等.大丽轮枝菌拮抗细菌菌株12-51的筛选鉴定与抗菌物性质分析[J].中国农学通报,2009,25(19):14-19.
[9]黄大野,贲海燕,曹春霞,等.死亡谷芽胞杆菌NBIF-001防治番茄棒孢叶斑病研究[J].植物保护,2017,43(6):192-195.
[10]黄大野,曹春霞,张亚妮,等.枯草芽胞杆菌NBF809防治番茄棒孢叶斑病研究[J].中国蔬菜,2018(12):40-44.
[11]Baiyye B,Pornsuriya C,Ito S,et al.Trichoderma spirale T76-1 displays biocontrol activity against leaf spot on lettuce(Lactuca sativa L.)caused by Corynespora cassiicola or Curvularia aeria[J].Biological Control,2019,129(2):195-200.
[12]程丽娟,薛泉宏.微生物学实验技术[M].西安:世界图书出版公司,2000.
[13]Chatterjee A,Cui Y,Liu Y,et al.Inactivation of rsm A leads to overproduction of extracellular pectinases,cellulases,and proteases in Erwinia carotovorasubsp.carotovora in the absence of the starvation/cell density-sensing signal,N-(3-oxohexanoyl)-L-homoserine lactone[J].Applied and Environmental Microbiology,1995,61(5):1959-1967.
[14]Weinert N,Meincke R,Gottwald C,et al.Bacterial diversity on the surface of potato tubers in soil and the influence of the plant genotype[J].FEMS Microbiology Ecology,2010,74(1):114-123.
[15]胥丽娜,徐亮,李磊,等.枯草芽胞杆菌对杨树水泡溃疡病菌菌丝生长的抑制作用[J].菌物研究,2007,5(3):165-168.
[16]Stonier T.Agrobacterium tumefaciens Conn.II.Production of an antibiotic substance[J].Journal of Bacteriology,1960,79(6):889-898.
[17]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.
[18]郭芳芳,谢镇,卢鹏,等.一株多粘类芽孢杆菌的鉴定及其生防促生效果初步测定[J].中国生物防治学报,2014,30(4):489-496.
[19]齐爱勇,魏东盛,刘大群,等.番茄灰霉病菌拮抗细菌B21的鉴定[J].中国农学通报,2006,22(9):360-363.
[20]张猛,王琼,冯发运,等.植物内生特基拉芽胞杆菌的分离、鉴定及防治西瓜枯萎病效果[J].中国生物防治学报,2017,33(3):371-377.
[21]阚琳娜,李宝聚,纪明山,等.黄瓜褐斑病防治药剂的活体筛选[J].中国蔬菜,2007(4):22-24.
[22]Ruiz G C,Béjar V,Martínez C F,et al.Bacillus velezensis sp.nov.,a surfactant-producing bacterium isolated from the river Vélez in Málaga,southern Spain[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(1):191-195.
[23]Kanjanamaneesathian M,Wiwattanapatapee R,Rotniam W,et al.Application of a suspension concentrate formulation of Bacillus velezensis to control root rot of hydroponically-grown vegetables[J].New Zealand Plant Protection,2013,66:229-234.
[24]罗文建,杨凡,史宣杰,等.黄瓜枯萎病拮抗菌的分离鉴定及其生物防效[J].华中农业大学学报,2018,37(3):32-38.
[25]姜乾坤,彭阁,王瑞,等.抗青枯内生细菌的筛选及其对烟草青枯病的防治效果[J].中国烟草科学,2017,38(5):13-17.
[26]马佳,李颖,胡栋,等.芽胞杆菌生物防治作用机理与应用研究进展[J].中国生物防治学报,2018,34(4):639-648.
[27]Gao Z F,Zhang B J,Liu H P,et al.Identification of endophytic Bacillus velezensis ZSY-1 strain and antifungal activity of its volatile compounds against Alternaria solani and Botrytis cinereal[J].Biological Control,2017,105(1):27-39.
[28]Cho S J,Lee S K,Cha B J,et al.Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03[J].FEMS Microbiology Letters,2003,223(1):47-51.
[29]Xu T,Zhu T H,Li S J.Β-1,3-1,4-glucanase gene form Bacillus velezensis ZJ20 exerts antifungal effect on plant pathogenic fungi[J].World Journal of Microbiology and Biotechnology,2016,32(2):1-9.
[30]于淑晶,王满意,田芳,等.黄瓜棒孢叶斑病的防治及抗药性研究进展[J].农药,2014,53(1):7-11.
[31]Miyamoto T,Ishii H,Seko T,et al.Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture,Japan[J].Plant Pathology,2009,58(6):1144-1151.