杜仲内生细菌拮抗小麦赤霉病菌研究
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摘要
为明确杜仲内生细菌对小麦赤霉病菌的抑菌活性,利用稀释涂布法对杜仲的内生细菌进行分离纯化;并以小麦赤霉病菌为指示菌,对杜仲内生细菌进行了对峙培养和盆栽防效等研究;通过分子鉴定确定拮抗菌株的分类地位,深入探讨拮抗菌株的抑菌机制。结果显示:杜仲中共分离获得104株内生细菌,其中内生细菌DZSG09、DZSJ16、DZSG23对小麦赤霉菌均具有较好的抑菌效果,且DZSG23对小麦赤霉病菌的抑制率达到69.04%;小麦盆栽试验结果表明,菌株DZSG23对小麦赤霉病的防治效果最佳,接种小麦赤霉病菌第10天,DZSG23处理组的防治效果达到40.89%;16S rDNA序列分析表明,DZSG23为芽孢杆菌属微生物,显微观察发现其可引起小麦赤霉病菌菌丝膨大、畸形;且DZSG23菌株的菌悬液、次级代谢产物对小麦赤霉病菌分生孢子萌发的影响较低,但致畸作用较明显,DZSG23菌悬液(1×10~6 CFU·mL~(-1))和次级代谢产物(10 mg·mL~(-1))处理36 h,小麦赤霉病菌分生孢子的畸形率分别达到57.70%和36.67%,均能造成小麦赤霉病菌分生孢子膨大、畸形,导致芽管肿胀、扭曲。本研究结果为拮抗菌株DZSG23的应用提供了科学依据。
This study was carried out to assess the inhibitory activities of endophytic bacteria from Eucommia ulmoides against Fusarium graminearum. The endophytic bacteria from E. ulmoides were isolated and purified, and the antimicrobial activities of endophytic bacteria against F. graminearum were examined using confront antibiotic culture experiment, disease-control pot experiment. Then, the antimicrobial strain was identified at molecular level and its resistance mechanism against F. graminearum was analyzed. The results indicated 104 endophytic bacteria were isolated from the leaves, stems and roots of E. ulmoides, and DZSG09, DZSJ16 and DZSG23 had strong inhibition against F. graminearum, inhibitory rate of DZSG23 on F. graminearum was 69.04%. Moreover, control efficacy of DZGS23 against F. graminearum was 40.89% by pot experiment after inoculation for 10 d. 16 S rDNA sequence analysis showed that DZGS23 belonged to Bacillus. DZGS23 could cause the abnormity and tortuosity of mycelial of phytopathogen and so on. Lastly, bacterial suspension and secondary metabolite of DZSG23 had strong teratogenic effect on conidia of F. graminearum conidia. Deformity rates of conidia were 57.70% and 36.67% after treated with suspension of DZSG23(1×10~6 CFU·mL~(-1)) and its secondary metabolites(10 mg·mL~(-1)) for 36 h, and they could cause abnormality of conidia and made tubes swollen and contort. However, the bacterial suspension and secondary metabolites of DZSG23 weakly affected germination of F. graminearum conidia. The results afforded scientific basis for DZSG23 application.
This study was carried out to assess the inhibitory activities of endophytic bacteria from Eucommia ulmoides against Fusarium graminearum. The endophytic bacteria from E. ulmoides were isolated and purified, and the antimicrobial activities of endophytic bacteria against F. graminearum were examined using confront antibiotic culture experiment, disease-control pot experiment. Then, the antimicrobial strain was identified at molecular level and its resistance mechanism against F. graminearum was analyzed. The results indicated 104 endophytic bacteria were isolated from the leaves, stems and roots of E. ulmoides, and DZSG09, DZSJ16 and DZSG23 had strong inhibition against F. graminearum, inhibitory rate of DZSG23 on F. graminearum was 69.04%. Moreover, control efficacy of DZGS23 against F. graminearum was 40.89% by pot experiment after inoculation for 10 d. 16 S rDNA sequence analysis showed that DZGS23 belonged to Bacillus. DZGS23 could cause the abnormity and tortuosity of mycelial of phytopathogen and so on. Lastly, bacterial suspension and secondary metabolite of DZSG23 had strong teratogenic effect on conidia of F. graminearum conidia. Deformity rates of conidia were 57.70% and 36.67% after treated with suspension of DZSG23(1×10~6 CFU·mL~(-1)) and its secondary metabolites(10 mg·mL~(-1)) for 36 h, and they could cause abnormality of conidia and made tubes swollen and contort. However, the bacterial suspension and secondary metabolites of DZSG23 weakly affected germination of F. graminearum conidia. The results afforded scientific basis for DZSG23 application.
引文
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[12] HUO X Q,CHEN J Y.Inhibition effect of antagonistic bacteria C23 on pathogens of damping-off and colonization in the rhizosphere of slash pine[J].Journal of Shanxi Agricultural Sciences,2009,37 (11):33-36.
[13] 李晶,杨谦.生防枯草芽孢杆菌的研究进展[J].安徽农业科学,2008,36(1):106-111,132.LI J,YANG Q.Research progress on biocontrol Bacillus subtilis[J].Journal of Anhui Agricultural Sciences,2008,36(1):106-111,132.(in Chinese with English abstract)
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[19] SIKES B A,HAWKES C V,FUKAMI T.Plant and root endophyte assembly history:interactive effects on native and exotic plants[J].Ecology,2016,97(2):484-493.
[20] NURBAILIS M,ADRIANSYAH H.Colonisation capability of Trichoderma viride(T1sk) on several banana cultivar roots and its effect against development of Fusarium wilt disease and plant growth[J].Journal of Biopesticides,2016,9 (2):196-203.
[21] KAVINO M,MANORANJITHAM S K.In vitro bacterization of banana (Musa spp.) with native endophytic and rhizospheric bacterial isolates:novel ways to combat Fusarium wilt[J].European Journal of Plant Pathology,2018,151(2):371-387.
[22] 龚凤娟,恩特马克·布拉提白,张宇凤,等.具有ACC脱氨酶活性的杜仲内生细菌的分离鉴定及其抗菌活性[J].微生物学通报,2011,38(10):1526-1532.GONG F J,ENTOMACK B,ZHANG Y F,et al.Isolation and antibacterial activity of ACC deaminase-containing endophytic bacteria from Eucommia ulmoides Oliver[J].Microbiology China,2011,38(10):1526-1532.(in Chinese with English abstract)
[23] 赵静.杜仲内生细菌的分离鉴定与活性菌株筛选[D].开封:河南大学,2017.ZHAO J.Isolation and screening of active endophytic bacteria from Eucommia[D].Kaifeng:Henan University,2017.(in Chinese with English abstract)
[24] GOND S K,BERGEN M S,TORRES M S,et al.Endophytic Bacillus spp.produce antifungal lipopeptides and induce host defence gene expression in maize[J].Microbiological Research,2015,172:79-87.
[25] SHRESTHA B K,KARKI H S,GROTH D E,et al.Biological control activities of rice-associated bacillus sp.strain s against sheath blight and bacterial panicle blight of rice[J].PLoS One,2016,11(1):e0146764.
[26] 杨瑞先,姬俊华,王祖华,等.牡丹根部内生细菌的分离鉴定及脂肽类物质的拮抗活性研究[J].微生物学通报,2015,42(6):1081-1088.YANG R X,JI J H,WANG Z H,et al.Isolation,indentification and inhibitory activity of lipopeptides of endophytic bacteria from the root of Paeonia suffruticosa[J].Microbiology China,2015,42(6):1081-1088.(in Chinese with English abstract)
[27] 姬婧媛,杨洁,高小宁,等.植物内生枯草芽孢杆菌E1R-j脂肽类化合物的分离鉴定及抑菌作用[J].农药学学报,2015,17(2):172-178.JI J Y,YANG J,GAO X N,et al.Isolation and identification of lipopeptides produced by endophytic bacteria Bacillus subtilis E1R-j and its anti-fungal mechanism studies[J].Chinese Journal of Pesticide Science,2015,17(2):172-178.(in Chinese with English abstract)
[28] LARSEN J C,HUNT J,PERRIN I,et al.Workshop on trichothecenes with a focus on DON:summary report[J].Toxicology Letters,2004,153(1):1-22.
[29] SCHNEIDER E,CURTUI V,SEIDLER C,et al.Rapid methods for deoxynivalenol and other trichothecenes[J].Toxicology Letters,2004,153(1):113-121.
[30] 张夕林,丁晓丽,钱允辉,等.水稻抽穗前后施用农药情况对稻米中农药残留量的影响研究[J].世界农药,2011,33(2):30-33.ZHANG X L,DING X L,QIAN Y H,et al.Effects of different pesticides,application rate and dosage used before and after rice heading on pesticide's residue in rice[J].World Pesticides,2011,33(2):30-33.(in Chinese with English abstract)
[31] ZHANG Y J,YU J J,ZHANG Y N,et al.Effect of carbendazim resistance on trichothecene production and aggressiveness of Fusarium graminearum[J].Molecular Plant-Microbe Interactions,2009,22(9):1143-1150.
[32] 管章玲,辛海峰,李建宏,等.小麦赤霉病拮抗菌的筛选及应用[J].江苏农业学报,2012,28(2):309-313.GUAN Z L,XIN H F,LI J H,et al.Screening and application of antagonistic bacteria against fusarium head blight of wheat[J].Jiangsu Journal of Agricultural Sciences,2012,28(2):309-313.(in Chinese with English abstract)
[33] 王建伟.小麦赤霉病菌拮抗菌的分离鉴定、发酵条件的优化及抗菌物质的研究[D].南京:南京农业大学,2010.WANG J W.Studies on identification,fermentation conditions and antifungal substance of the antagonistic bacterium against Fusarium graminearum[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese with English abstract)
[34] 何鹏飞.B9601-Y2菌株的基因组解析及部分功能验证[D].武汉:华中农业大学,2014.HE P F.Genomic analysis of B9601-Y2 strain and partial functions confirmation[D].Wuhan:Huazhong Agricultural University,2014.(in Chinese with English abstract)
[2] PALAZZINI J M,ALBERIONE E,TORRES A,et al.Biological control of Fusarium graminearum sensu stricto,causal agent of Fusarium head blight of wheat,using formulated antagonists under field conditions in Argentina[J].Biological Control,2016,94:56-61.
[3] VAN DER FELS-KLERX H J,DE RIJK T C,BOOIJ C J H,et al.Occurrence of Fusarium head blight species and Fusarium mycotoxins in winter wheat in the Netherlands in 2009[J].Food Additives & Contaminants:Part A,2012,29(11):1716-1726.
[4] 贾巧君,杨建明,朱靖环,等.不同类型大麦品种赤霉病抗性与DON毒素积累[J].浙江农业学报,2007,19(5):339-342.JIA Q J,YANG J M,ZHU J H,et al.Identification of different original barley lines for Fusarium head blight resistance and deoxynivalenol accumulation[J].Acta Agriculturae Zhejiangensis,2007,19(5):339-342.(in Chinese with English abstract)
[5] 马雪莉,赵京岚,耿忠义,等.禾谷镰孢菌对21种杀菌剂的敏感性测定[J].黑龙江农业科学,2017(7):30-34.MA X L,ZHAO J L,GENG Z Y,et al.Sensitivity of Fusarium graminearum to 21 fungicides[J].Heilongjiang Agricultural Sciences,2017(7):30-34.(in Chinese with English abstract)
[6] 管章玲,辛海峰,李建宏,等.小麦赤霉病的微生物防治研究进展[J].江苏农业科学,2012,40(2):94-96.GUAN Z L,XIN H F,LI J H,et al.Advances in microbial control of wheat scab disease[J].Jiangsu Agricultural Sciences,2012,40(2):94-96.(in Chinese)
[7] 袁善奎,周明国.玉蜀黍赤霉的营养亲和性及其对多菌灵的抗性在菌丝融合过程中的遗传[J].南京农业大学学报,2004,27(2):39-42.YUAN S K,ZHOU M G.Vegetative compatibility of Gibberella zeae and genetic study on its resistance to carbendazim in hyphal fusion[J].Journal of Nanjing Agricultural University,2004,27(2):39-42.(in Chinese with English abstract)
[8] 宋益民,丛国林,陈怀谷.多菌灵及其复配制剂防治小麦赤霉病的应用效果[J].植物保护学报,2018,45(2):352-358.SONG Y M,CONG G L,CHEN H G.Efficacy of carbendazim and its mixtures for controlling wheat scab[J].Journal of Plant Protection,2018,45(2):352-358.(in Chinese with English abstract)
[9] YUAN S K,ZHOU M G.A major gene for resistance to carbendazim,in field isolates of Gibberella zeae[J].Canadian Journal of Plant Pathology,2005,27(1):58-63.
[10] BACKMAN P A,SIKORA R A.Endophytes:an emerging tool for biological control[J].Biological Control,2008,46(1):1-3.
[11] MEJíA L C,ROJAS E I,MAYNARD Z,et al.Endophytic fungi as biocontrol agents of Theobroma cacao pathogens[J].Biological Control,2008,46(1):4-14.
[12] HUO X Q,CHEN J Y.Inhibition effect of antagonistic bacteria C23 on pathogens of damping-off and colonization in the rhizosphere of slash pine[J].Journal of Shanxi Agricultural Sciences,2009,37 (11):33-36.
[13] 李晶,杨谦.生防枯草芽孢杆菌的研究进展[J].安徽农业科学,2008,36(1):106-111,132.LI J,YANG Q.Research progress on biocontrol Bacillus subtilis[J].Journal of Anhui Agricultural Sciences,2008,36(1):106-111,132.(in Chinese with English abstract)
[14] DING T,SU B,CHEN X J,et al.An endophytic bacterial strain isolated from Eucommia ulmoides inhibits southern corn leaf blight[J].Frontiers in Microbiology,2017,8:903.
[15] 鲁素芸.植物病害生物防治学[M].北京:北京农业大学出版社,1993.
[16] 丁婷,孙微微,王帅,等.杜仲内生真菌中抗玉米纹枯病活性菌株的筛选[J].植物保护,2014,40(6):29-35.DING T,SUN W W,WANG S,et al.Screening of endophytic fungi isolated from Eucommia ulmoides Oliv.with antimicrobial activity against maize Rhizoctonia solani[J].Plant Protection,2014,40(6):29-35.(in Chinese with English abstract)
[17] 李洪连,徐敬友.农业植物病理学实验实习指导:第2版[M].北京:中国农业出版社,2007.
[18] CHELO I M,Zé-Zé L,TENREIRO R.Congruence of evolutionary relationships inside the Leuconostoc-Oenococcus-Weissella clade assessed by phylogenetic analysis of the 16S rRNA gene,dnaA,gyrB,rpoC and dnaK[J].International Journal of Systematic and Evolutionary Microbiology,2007,57 (2):276-286.
[19] SIKES B A,HAWKES C V,FUKAMI T.Plant and root endophyte assembly history:interactive effects on native and exotic plants[J].Ecology,2016,97(2):484-493.
[20] NURBAILIS M,ADRIANSYAH H.Colonisation capability of Trichoderma viride(T1sk) on several banana cultivar roots and its effect against development of Fusarium wilt disease and plant growth[J].Journal of Biopesticides,2016,9 (2):196-203.
[21] KAVINO M,MANORANJITHAM S K.In vitro bacterization of banana (Musa spp.) with native endophytic and rhizospheric bacterial isolates:novel ways to combat Fusarium wilt[J].European Journal of Plant Pathology,2018,151(2):371-387.
[22] 龚凤娟,恩特马克·布拉提白,张宇凤,等.具有ACC脱氨酶活性的杜仲内生细菌的分离鉴定及其抗菌活性[J].微生物学通报,2011,38(10):1526-1532.GONG F J,ENTOMACK B,ZHANG Y F,et al.Isolation and antibacterial activity of ACC deaminase-containing endophytic bacteria from Eucommia ulmoides Oliver[J].Microbiology China,2011,38(10):1526-1532.(in Chinese with English abstract)
[23] 赵静.杜仲内生细菌的分离鉴定与活性菌株筛选[D].开封:河南大学,2017.ZHAO J.Isolation and screening of active endophytic bacteria from Eucommia[D].Kaifeng:Henan University,2017.(in Chinese with English abstract)
[24] GOND S K,BERGEN M S,TORRES M S,et al.Endophytic Bacillus spp.produce antifungal lipopeptides and induce host defence gene expression in maize[J].Microbiological Research,2015,172:79-87.
[25] SHRESTHA B K,KARKI H S,GROTH D E,et al.Biological control activities of rice-associated bacillus sp.strain s against sheath blight and bacterial panicle blight of rice[J].PLoS One,2016,11(1):e0146764.
[26] 杨瑞先,姬俊华,王祖华,等.牡丹根部内生细菌的分离鉴定及脂肽类物质的拮抗活性研究[J].微生物学通报,2015,42(6):1081-1088.YANG R X,JI J H,WANG Z H,et al.Isolation,indentification and inhibitory activity of lipopeptides of endophytic bacteria from the root of Paeonia suffruticosa[J].Microbiology China,2015,42(6):1081-1088.(in Chinese with English abstract)
[27] 姬婧媛,杨洁,高小宁,等.植物内生枯草芽孢杆菌E1R-j脂肽类化合物的分离鉴定及抑菌作用[J].农药学学报,2015,17(2):172-178.JI J Y,YANG J,GAO X N,et al.Isolation and identification of lipopeptides produced by endophytic bacteria Bacillus subtilis E1R-j and its anti-fungal mechanism studies[J].Chinese Journal of Pesticide Science,2015,17(2):172-178.(in Chinese with English abstract)
[28] LARSEN J C,HUNT J,PERRIN I,et al.Workshop on trichothecenes with a focus on DON:summary report[J].Toxicology Letters,2004,153(1):1-22.
[29] SCHNEIDER E,CURTUI V,SEIDLER C,et al.Rapid methods for deoxynivalenol and other trichothecenes[J].Toxicology Letters,2004,153(1):113-121.
[30] 张夕林,丁晓丽,钱允辉,等.水稻抽穗前后施用农药情况对稻米中农药残留量的影响研究[J].世界农药,2011,33(2):30-33.ZHANG X L,DING X L,QIAN Y H,et al.Effects of different pesticides,application rate and dosage used before and after rice heading on pesticide's residue in rice[J].World Pesticides,2011,33(2):30-33.(in Chinese with English abstract)
[31] ZHANG Y J,YU J J,ZHANG Y N,et al.Effect of carbendazim resistance on trichothecene production and aggressiveness of Fusarium graminearum[J].Molecular Plant-Microbe Interactions,2009,22(9):1143-1150.
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