解淀粉芽孢杆菌FS6在人参体内的定殖特性及对人参诱导抗病性
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摘要
【目的】明确解淀粉芽孢杆菌(Bacillus amyloliquefaciens)FS6在人参体内的定殖规律及其在病原菌胁迫下对人参防御酶活性的影响,为揭示FS6诱导人参产生抗病性的诱导抗病机制奠定基础,进而为人参根部病害的绿色防控提供依据。【方法】采用抗生素标记法获得FS6菌株抗利福平的突变体菌株,采用灌根和叶面喷雾两种方法接种,研究FS6在人参植株体内及人参根际土壤中的定殖规律;采用灌根法,利用盆栽试验研究不同处理条件下FS6对人参抗病相关防御酶活性的影响。【结果】从含300μg/mL利福平抗性平板上得到标记菌株,该标记菌株能在NA培养基上稳定生长,抗性丢失率为0,且标记菌株与野生菌株的拮抗能力无明显差异。定殖试验结果表明,灌根和叶面喷雾两种接种方式下,FS6~(Rif)菌株均能在人参植株各个部位及根际土壤中定殖,并能在人参体内转移。其中,灌根后FS6~(Rif)在土壤中的定殖量在处理后第1天达到最大值,为4.8×10~3 CFU/g;叶面喷雾后FS6~(Rif)在叶片中的定殖量在处理后第1天达到最大值,为3.2×10~3 CFU/g。FS6发酵液处理后再接种人参根腐病菌,与其他3个处理相比,人参防御酶活性显著提高,CAT、POD和PPO分别在FS6发酵液灌根处理后第12,15和18天达到峰值。【结论】FS6菌株能够在人参植株体内稳定定殖并传导,且能在一定程度上诱导人参产生抗病性。
【Objective】 This study investigated the colonization and effects of Bacillus amyloliquefaciens FS6 on ginseng defense enzyme activities under pathogen stress to provide evidence for the further understanding induced resistance and green control of ginseng root disease.【Method】 Mutant strains of FS6 resistant to rifampin were screened and obtained using the antibiotic label method.The colonization of FS6 in ginseng was determined by the root irrigation and spraying methods.The effect of FS6 on activities of three defense enzymes of ginseng was then determined by pot experiment.【Result】 The marked strain was obtained from the resistant plating medium with rifampin of 300 μg/mL.The strain could grow steadily on the NA medium with the resistance loss rate of 0.The antagonism abilities of the marked strain had no significant difference from the wild strains.The colonization trials showed that the labeled strains of FS6~(Rif) could efficiently colonize and transmit in ginseng and soil with two inoculation methods.The maximum density in soil was 4.8×10~3 CFU/g 1 d after root irrigation,and the maximum density in leaf was 3.2×10~3 CFU/g 1 d after spraying.Defense enzyme activity assay test showed that the activities of polyphenol oxide(PPO),peroxidase(POD) and catalase(CAT) in ginseng leaves were significantly increased after inoculation with Fusarium solani and FS6,and the peak values appeared at 12,15 and 18 d after root irrigation,respectively.【Conclusion】 Bacillus amyloliquefaciens FS6 could efficiently colonize and transmit in ginseng and could induce disease resistance of ginseng.
【Objective】 This study investigated the colonization and effects of Bacillus amyloliquefaciens FS6 on ginseng defense enzyme activities under pathogen stress to provide evidence for the further understanding induced resistance and green control of ginseng root disease.【Method】 Mutant strains of FS6 resistant to rifampin were screened and obtained using the antibiotic label method.The colonization of FS6 in ginseng was determined by the root irrigation and spraying methods.The effect of FS6 on activities of three defense enzymes of ginseng was then determined by pot experiment.【Result】 The marked strain was obtained from the resistant plating medium with rifampin of 300 μg/mL.The strain could grow steadily on the NA medium with the resistance loss rate of 0.The antagonism abilities of the marked strain had no significant difference from the wild strains.The colonization trials showed that the labeled strains of FS6~(Rif) could efficiently colonize and transmit in ginseng and soil with two inoculation methods.The maximum density in soil was 4.8×10~3 CFU/g 1 d after root irrigation,and the maximum density in leaf was 3.2×10~3 CFU/g 1 d after spraying.Defense enzyme activity assay test showed that the activities of polyphenol oxide(PPO),peroxidase(POD) and catalase(CAT) in ginseng leaves were significantly increased after inoculation with Fusarium solani and FS6,and the peak values appeared at 12,15 and 18 d after root irrigation,respectively.【Conclusion】 Bacillus amyloliquefaciens FS6 could efficiently colonize and transmit in ginseng and could induce disease resistance of ginseng.
引文
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[3] 强薇,郭帅,张爱华,等.人参根际土壤中拮抗人参病害微生物的筛选 [J].华南农业大学学报,2014,35(5):76-81.Qiang W,Guo S,Zhang A H,et al.Screening antagonistic microorganisms against ginseng diseases from Panax ginseng rhizosphere soil [J].Journal of South China Agricultural University,2014,35(5):76-81.
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[8] 周如军,傅俊范,卯婷婷,等.人参锈腐病拮抗细菌BS015最适发酵条件研究 [J].吉林农业大学学报,2011,33(3):278-282.Zhou R J,Fu J F,Mao T T,et al.Optimal fermentation conditions of antagonistic bacterium strain BS015 against ginseng rust root rot [J].Journal of Jilin Agricultural University,2011,33(3):278-282.
[9] 孙卓,杨利民.人参锈腐病生防细菌的筛选及鉴定 [J].中国生物防治学报,2015,31(4):536-542.Sun Z,Yang L M.Screening and identification of antagonistic bacteria on Cylindrocarpon destructans (Zinss.) Scholtan [J].Chinese Journal of Biological Control,2015,31(4):536-542.
[10] 姜竹.人参土传真菌病害的生物防治 [D].哈尔滨:黑龙江大学,2010.Jiang Z.Biological control of ginseng soil-born fungi diseases [D].Harbin:Heilongjiang University,2010.
[11] Byung D L,Hoon P.枯草杆菌B-4228对人参根腐病的抑制作用 [J].人参研究,2005,20(4):48.Byung D L,Hoon P.Inhibition of Bacillus subtilis B-4228 to ginseng root rot [J].Ginseng Research,2005,20(4):48.
[12] 齐爱勇,赵绪生,刘大群.芽孢杆菌生物防治植物病害研究现状 [J].中国农学通报,2011,27(12):277.Qi A Y,Zhao X S,Liu D Q.Research of biological control in plant diseases by Bacillus spp.[J].Chinese Agricultural Science Bulletin,2011,27(12):277.
[13] 令利军,冯蕾,雷蕾,等.地衣芽孢杆菌TG116诱导黄瓜抗病性相关防御酶系的研究 [J].西北师范大学学报(自然科学版),2016,52(1):100-104.Ling L J,Feng L,Lei L,et al.Induction of defense-related enzymes in cucumber roots by Bacillus licheniformis strain TG116 [J].Journal of Northwest Normal University(Natural Science),2016,52(1):100-104.
[14] 齐希猛,易润华,曾涛,等.海洋细菌TC-1对香蕉防御酶活性的影响 [J].中国农学通报,2012,28(1):223-227.Qi X M,Yi R H,Zeng T,et al.Effect of antagonistic marine-born strain TC-1 on five major defensive enzymes in banana [J].Chinese Agricultural Science Bulletin,2012,28(1):223-227.
[15] 汤绍虎,罗充.植物生理学实验教程 [M].重庆:西南大学出版社,2012:126-131.Tang S H,Luo C.Experimental course on plant physiology [M].Chongqing:Southwest China University Press,2012:126-131.
[16] 李仕飞,刘世同,周建平,等.分光光度法测定植物过氧化氢酶活性的研究 [J].安徽农学通报,2007,13(2):72-73.Li S F,Liu S T,Zhou J P,et al.Measuration of catalase vigorin plants with spectrophotometry [J].Anhui Agri Sci Bull,2007,13(2):72-73.
[17] Tan S Y,Jiang Y,Song S,et al.Two Bacillus amyloliquefaciens strains isolated using the competitive tomato root enrichment method and their effects on suppressing Ralstonia solanacearum and promoting tomato plant growth [J].Crop Protection,2013,43:134-140.
[18] Weng J,Wang Y,Li J,et al.Enhanced root colonization and biocontrol activity of Bacillus amyloliquefaciens SQR9 by abrB gene disruption [J].Applied Microbiology & Biotechnology,2013,97(19):8823-8830.
[19] 杨洪凤,薛雅蓉,余向阳,等.内生解淀粉芽孢杆菌CC09菌株在小麦叶部的定殖能力及其防治白粉病效果研究 [J].中国生物防治学报,2014,30(4):481-488.Yang H F,Xue Y R,Yu X Y,et al.Colonization of Bacillus amyloliquefaciens CC09 in wheat leaf and its biocontrol effect on powdery mildew disease [J].Chinese Journal of Biological Control,2014,30(4):481-488.
[20] 谯天敏,张静,冉晓潇,等.解淀粉芽孢杆菌在山茶叶中的定殖及对山茶灰斑病的防效 [J].西北农林科技大学学报(自然科学版),2015,43(10):78-84.Qiao T M,Zhang J,Ran X X,et al.Colonization of Bacillus amyloliquefaciens in camellia leaves and its control efficacy against camellia gray spot [J].Journal of Northwest A&F University(Natural Science Edition),2015,43(10):78-84.
[21] 胡伟,赵兰凤,张亮,等.香蕉枯萎病生防菌AF11的鉴定及其定殖研究 [J].中国生物防治学报,2012,28(3):387-393.Hu W,Zhao L F,Zhang L,et al.Identification and colonization of bacteria AF11 antagonistic against Fusarium oxysporum f.sp.cubense Race 4,the pathogen of banana wilt disease [J].Chinese Journal of Biological Control,2012,28(3):387-393.
[22] 张楠,吴凯,沈怡斐,等.根际益生菌解淀粉芽孢杆菌SQR9在香蕉根表的定殖行为研究 [J].南京农业大学学报,2014,37(6):59-65.Zhang N,Wu K,Shen Y F,et al.Investigation of the colonization patterns of plant growth-promoting rhizobacteria Bacillus amyloliquefaciens SQR9 on banana roots [J].Journal of Nanjing Agricultural University,2014,37(6):59-65.
[23] Rahman A,Uddin W,Wenner N G.Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi-purified surfactin lipopeptides and live cells of Bacillus amyloliquefaciens [J].Molecular Plant Pathology,2015,16(6):546-558.
[24] Nihorimbere V,Fickers P,Thonart P,et al.Ecological fitness of Bacillus subtilis BGS3 regarding production of the surfactin lipopeptide in the rhizosphere [J].Environmental Microbiology Reports,2009,1(2):124-130.
[25] Bais H P,Fall R,Vivaneo J M.Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production [J].Plant Physiology,2004,134(1):307-319.
[26] 谷医林,王远宏,常若葵,等.解淀粉芽孢杆菌Lj1诱导黄瓜抗白粉病的研究 [J].农药学学报,2013,15(3):293-298.Gu Y L,Wang Y H,Chang R K,et al.Characterization of powdery mildew resistance induced by Bacillus amyloliquefaciens Lj1 in cucumber [J].Chinese Journal of Pesticide Science,2013,15(3):293-298.
[2] 黄小芳.人参根系分泌物及其自毒活性研究 [D].北京:中国协和医科大学,2009.Huang X F.Study on ginseng root exudates and their autotoxicity [D].Beijing:Peking Union Medical College,2009.
[3] 强薇,郭帅,张爱华,等.人参根际土壤中拮抗人参病害微生物的筛选 [J].华南农业大学学报,2014,35(5):76-81.Qiang W,Guo S,Zhang A H,et al.Screening antagonistic microorganisms against ginseng diseases from Panax ginseng rhizosphere soil [J].Journal of South China Agricultural University,2014,35(5):76-81.
[4] 杨依军,武侠,赵日丰.人参、西洋参根病的生物防治 [J].特产研究,1997(1):30-33.Yang Y J,Wu X,Zhao R F.Biological control of ginseng and American ginseng root disease [J].Special Wild Economic Animal and Plant Research,1997(1):30-33.
[5] 吴连举,关一鸣,逄世峰,等.利用拮抗微生物防治人参、西洋参土传病害研究进展 [J].安徽农业科学,2010,38(28):15630- 15631,15657.Wu L J,Guan Y M,Pang S F,et al.Research progress on biological control of soil-borne diseases in ginseng and American ginseng by anti-microorganism [J].Journal of Anhui Agri.Sci,2010,38(28):15630-15631,15657.
[6] Hartley C.Damping-off in forest nurseries [M].Washington D C:U S Dept of Agricuture,1921:934.
[7] 傅俊范,史会岩,周如军,等.人参锈腐病生防细菌的分离筛选与鉴定 [J].吉林农业大学学报,2010,32(2):136-139,144.Fu J F,Shi H Y,Zhou R J,et al.Isolation screening and identification of antagonistic bacteria against ginseng rusted root disease [J].Journal of Jilin Agricultural University,2010,32(2):136-139,144.
[8] 周如军,傅俊范,卯婷婷,等.人参锈腐病拮抗细菌BS015最适发酵条件研究 [J].吉林农业大学学报,2011,33(3):278-282.Zhou R J,Fu J F,Mao T T,et al.Optimal fermentation conditions of antagonistic bacterium strain BS015 against ginseng rust root rot [J].Journal of Jilin Agricultural University,2011,33(3):278-282.
[9] 孙卓,杨利民.人参锈腐病生防细菌的筛选及鉴定 [J].中国生物防治学报,2015,31(4):536-542.Sun Z,Yang L M.Screening and identification of antagonistic bacteria on Cylindrocarpon destructans (Zinss.) Scholtan [J].Chinese Journal of Biological Control,2015,31(4):536-542.
[10] 姜竹.人参土传真菌病害的生物防治 [D].哈尔滨:黑龙江大学,2010.Jiang Z.Biological control of ginseng soil-born fungi diseases [D].Harbin:Heilongjiang University,2010.
[11] Byung D L,Hoon P.枯草杆菌B-4228对人参根腐病的抑制作用 [J].人参研究,2005,20(4):48.Byung D L,Hoon P.Inhibition of Bacillus subtilis B-4228 to ginseng root rot [J].Ginseng Research,2005,20(4):48.
[12] 齐爱勇,赵绪生,刘大群.芽孢杆菌生物防治植物病害研究现状 [J].中国农学通报,2011,27(12):277.Qi A Y,Zhao X S,Liu D Q.Research of biological control in plant diseases by Bacillus spp.[J].Chinese Agricultural Science Bulletin,2011,27(12):277.
[13] 令利军,冯蕾,雷蕾,等.地衣芽孢杆菌TG116诱导黄瓜抗病性相关防御酶系的研究 [J].西北师范大学学报(自然科学版),2016,52(1):100-104.Ling L J,Feng L,Lei L,et al.Induction of defense-related enzymes in cucumber roots by Bacillus licheniformis strain TG116 [J].Journal of Northwest Normal University(Natural Science),2016,52(1):100-104.
[14] 齐希猛,易润华,曾涛,等.海洋细菌TC-1对香蕉防御酶活性的影响 [J].中国农学通报,2012,28(1):223-227.Qi X M,Yi R H,Zeng T,et al.Effect of antagonistic marine-born strain TC-1 on five major defensive enzymes in banana [J].Chinese Agricultural Science Bulletin,2012,28(1):223-227.
[15] 汤绍虎,罗充.植物生理学实验教程 [M].重庆:西南大学出版社,2012:126-131.Tang S H,Luo C.Experimental course on plant physiology [M].Chongqing:Southwest China University Press,2012:126-131.
[16] 李仕飞,刘世同,周建平,等.分光光度法测定植物过氧化氢酶活性的研究 [J].安徽农学通报,2007,13(2):72-73.Li S F,Liu S T,Zhou J P,et al.Measuration of catalase vigorin plants with spectrophotometry [J].Anhui Agri Sci Bull,2007,13(2):72-73.
[17] Tan S Y,Jiang Y,Song S,et al.Two Bacillus amyloliquefaciens strains isolated using the competitive tomato root enrichment method and their effects on suppressing Ralstonia solanacearum and promoting tomato plant growth [J].Crop Protection,2013,43:134-140.
[18] Weng J,Wang Y,Li J,et al.Enhanced root colonization and biocontrol activity of Bacillus amyloliquefaciens SQR9 by abrB gene disruption [J].Applied Microbiology & Biotechnology,2013,97(19):8823-8830.
[19] 杨洪凤,薛雅蓉,余向阳,等.内生解淀粉芽孢杆菌CC09菌株在小麦叶部的定殖能力及其防治白粉病效果研究 [J].中国生物防治学报,2014,30(4):481-488.Yang H F,Xue Y R,Yu X Y,et al.Colonization of Bacillus amyloliquefaciens CC09 in wheat leaf and its biocontrol effect on powdery mildew disease [J].Chinese Journal of Biological Control,2014,30(4):481-488.
[20] 谯天敏,张静,冉晓潇,等.解淀粉芽孢杆菌在山茶叶中的定殖及对山茶灰斑病的防效 [J].西北农林科技大学学报(自然科学版),2015,43(10):78-84.Qiao T M,Zhang J,Ran X X,et al.Colonization of Bacillus amyloliquefaciens in camellia leaves and its control efficacy against camellia gray spot [J].Journal of Northwest A&F University(Natural Science Edition),2015,43(10):78-84.
[21] 胡伟,赵兰凤,张亮,等.香蕉枯萎病生防菌AF11的鉴定及其定殖研究 [J].中国生物防治学报,2012,28(3):387-393.Hu W,Zhao L F,Zhang L,et al.Identification and colonization of bacteria AF11 antagonistic against Fusarium oxysporum f.sp.cubense Race 4,the pathogen of banana wilt disease [J].Chinese Journal of Biological Control,2012,28(3):387-393.
[22] 张楠,吴凯,沈怡斐,等.根际益生菌解淀粉芽孢杆菌SQR9在香蕉根表的定殖行为研究 [J].南京农业大学学报,2014,37(6):59-65.Zhang N,Wu K,Shen Y F,et al.Investigation of the colonization patterns of plant growth-promoting rhizobacteria Bacillus amyloliquefaciens SQR9 on banana roots [J].Journal of Nanjing Agricultural University,2014,37(6):59-65.
[23] Rahman A,Uddin W,Wenner N G.Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi-purified surfactin lipopeptides and live cells of Bacillus amyloliquefaciens [J].Molecular Plant Pathology,2015,16(6):546-558.
[24] Nihorimbere V,Fickers P,Thonart P,et al.Ecological fitness of Bacillus subtilis BGS3 regarding production of the surfactin lipopeptide in the rhizosphere [J].Environmental Microbiology Reports,2009,1(2):124-130.
[25] Bais H P,Fall R,Vivaneo J M.Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production [J].Plant Physiology,2004,134(1):307-319.
[26] 谷医林,王远宏,常若葵,等.解淀粉芽孢杆菌Lj1诱导黄瓜抗白粉病的研究 [J].农药学学报,2013,15(3):293-298.Gu Y L,Wang Y H,Chang R K,et al.Characterization of powdery mildew resistance induced by Bacillus amyloliquefaciens Lj1 in cucumber [J].Chinese Journal of Pesticide Science,2013,15(3):293-298.