生防菌YN201490在黄瓜植株体内的定殖能力及防病机制的初步研究
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摘要
为研究枯草芽孢杆菌YN201490在黄瓜体内的定殖能力及防病机制,采用平板对峙试验的方法测试该菌株对不同病原菌的抑制作用,利用灌根的方法研究其在黄瓜体内的定殖能力,并通过抑制孢子萌发对其发酵液进行了初步分离与活性检测.研究通过平板对峙试验证明了枯草芽孢杆菌YN201490对小麦雪腐病菌、番茄枯萎病菌、烟草赤星病菌、玉米拟轮枝镰孢菌、茶轮斑病菌和水稻叶鞘腐败病菌有很好的抑菌活性.用自然转化法以GFP标记,获得了荧光标记菌株YN90-P43GFPmut3a.通过灌根法,证明该菌株能在黄瓜植株体内定殖,其数量分布为根>叶>茎. YN201490发酵液的正丁醇萃取物能裂解小麦雪腐病菌孢子,使胞质外流,抑制91.72%孢子萌发,对黄瓜白粉病的预防效果达66.26%,并能诱导黄瓜产生明显抗性.研究结果表明枯草芽孢杆菌YN201490对多种病原真菌具有明显的抑制作用,并能够在黄瓜体内稳定定殖,具有作为生物菌剂开发利用的潜能.
To understand the colonization ability in cucumber plant and biocontrol mechanism of Bacillus subtilis YN201490, plate confrontation was proceeded to test the inhibitory effect of the strain on different pathogens and the method of drenching root was used to study the colonization ability in cucumber, and the isolation and activity of the fermentation suspension was tested by inhibiting spore germination. Antagonistic activity of YN201490 against Typhula incarnata, Fusarium oxysporum f. sp. lycopersici, Alternaria alternata, F.verticillioides, Pestalotiopsis theae and Sarocladium oryzae was confirmed by the confrontation test and a GFPlabeled strain respectively, YN90-P43GFPmut3 a was obtained to analyze the colonization ability. The results showed that strain could be colonize in cucumber plant with the colony numbers in root > leaf > stem based on drenching root experiments. The n-butanol extract from fermentation suspension could split the spores of T.incarnata to make the cytoplasm exudate and inhibit 91.72% of spores' germination. When it was sprayed on cucumber it also prevented 66.26% of cucumber powdery mildew and induced significant disease resistance. It is concluded that YN201490 has an obvious inhibitory effect on many pathogenic fungi and colonization ability in cucumber, which has the potential as a biological agent.
To understand the colonization ability in cucumber plant and biocontrol mechanism of Bacillus subtilis YN201490, plate confrontation was proceeded to test the inhibitory effect of the strain on different pathogens and the method of drenching root was used to study the colonization ability in cucumber, and the isolation and activity of the fermentation suspension was tested by inhibiting spore germination. Antagonistic activity of YN201490 against Typhula incarnata, Fusarium oxysporum f. sp. lycopersici, Alternaria alternata, F.verticillioides, Pestalotiopsis theae and Sarocladium oryzae was confirmed by the confrontation test and a GFPlabeled strain respectively, YN90-P43GFPmut3 a was obtained to analyze the colonization ability. The results showed that strain could be colonize in cucumber plant with the colony numbers in root > leaf > stem based on drenching root experiments. The n-butanol extract from fermentation suspension could split the spores of T.incarnata to make the cytoplasm exudate and inhibit 91.72% of spores' germination. When it was sprayed on cucumber it also prevented 66.26% of cucumber powdery mildew and induced significant disease resistance. It is concluded that YN201490 has an obvious inhibitory effect on many pathogenic fungi and colonization ability in cucumber, which has the potential as a biological agent.
引文
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[22]董汉松.植物抗病防卫基因表达调控与诱导抗性遗传的机制[J].植物病理学报,1996,26(4):289-293.Dong H S.Expressive regulation of plant disease defense genes in relation with hereditability of induced resistance[J].Acta Phytopathologica Sinica,1996,26(4):289-293.
[23]范志金,刘秀峰,刘凤丽,等.植物抗病激活剂诱导植物抗病性的研究进展[J].植物保护学报,2005,32(1):87-92.Fan Z J,Liu X F,Liu F L,et al.Advances in research on plant disease resistance induced by plant disease resistance activators[J].Journal of Plant Protection,2005,32(1):87-92.
[24]Sturz A,Matheson B.Populations of endophytic bacteria which influence host-resistance to Erwinia-induced bacterial soft rot in potato tubers[J].Plant and Soil,1996,184(2):265-271.
[2]贺字典,宋士清,高玉峰,等.棘孢木霉Trichoderma asperellum在土壤中定殖量的荧光定量PCR检测[J].植物保护学报,2016(4):552-558.He Z D,Song S Q,Gao Y F,et al.Detection of Trichoderma asperellum colonization in soils by real-time fluorescent quantitative PCR[J].Journal of Plant Protection,2016(4):552-558.
[3]贺字典,吴素霞,宋晓飞,等.生防菌与茄病镰刀菌在黄瓜根际动态变化[J].中国生物防治学报,2016,32(3):357-364.He Z D,Wu S X,Song X F,et al.Population dynamics of Trichoderma spp.,Bacillus subtillis and Fusarium solani f.sp.cucurbitae in cucumber rhizosphere[J].Chinese Journal of Biological,2016,32(3):357-364.
[4]张冬冬,刘涛,高同国,等.棉花黄萎病拮抗细菌Z-5菌株的定殖能力检测[J].棉花学报,2015,25(6):510-516.Zhang D D,Liu T,Gao T G,et al.Detection of the colonization ability of antagonistic bacteria strain Z-5against cotton verticillium wilt[J].Cotton Science,2015,25(6):510-516.
[5]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 D,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,2014,30(4):481-488.
[6]方正达.植病研究方法[M].北京:中国农业出版社,1998.Feng Z D.Research method of plant pathology[M].Beijing:China Agricultural Press,1998.
[7]李瑞芳,薛雯雯,黄亮,等.枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究[J].生物技术通报,2011,5:227-230.Li R F,Xue W W,Huang L,et al.Competent preparation and plasm id transformation of Bacillus subtili[J].Biotechnology Bulletin,2011,5:227-230.
[8]GB/T 17980.44-2000.农药田间药效试验准则(一):杀菌剂防治黄瓜白粉病[S].2000.GB/T 17980.44-2000.Pesticide field efficacy test guidelines(1):Fungicides against cucumber powdery mildew[S].2000.
[9]Cheng H R,Jiang N.Extremely rapid extraction of DNA from bacteria and yeasts[J].Biotechnology Letters,2006,28(1):55-59.
[10]Hsieh F C,Li M C,Lin T C,et al.Rapid detection and characterization of surfactin-producing Bacillus subtilis and closely related species based on PCR[J].Current Microbiology,2004,49(3):186-191.
[11]李卫利,何鹏飞,吴毅歆,等.黄瓜白粉病的生防细菌筛选及鉴定[J].中国农学通报,2015,31(16):160-165.Li W L,He P F,Wu Y X,et al.Screening and identification of bacterial biocontrol agents controlling cucumber powdery mildew[J].Chinese Agricultural Science Bulletin,2015,31(16):160-165.
[12]Kloepper J W,Beauchamp C J.A review of issues related to measuring colonization of plant roots by bacteria[J].Canadian Journal of Microbiology,1992,38(12):1 219-1 232.
[13]Compant S,Kaplan H,Sessitsch A,et al.Endophytic colonization of Vitis vinifera L.by Burkholderia phytofirmans strain PsJN:from the rhizosphere to inflorescence tissues[J].FEMS Microbiology Ecology,2008,63(1):84-93.
[14]杨瑞先,葛晓燕.植物内生细菌生物学作用研究进展及应用前景[J].洛阳大学学报,2006,21(4):78-81.Yang R X,Ge X Y.Advance in biological effects and application prospect of endophytic bacteria[J].Journal of Luoyang University,2006,21(4):78-81.
[15]Bull C T,Weller D M,Thomashow L S.Relationship between root colonization and suppression of Gaeumannomyces graminis var.tritici by Pseudomonas fluorescens strain 2-79[J].Phytopathology,1991,81(9):954-959.
[16]石晶盈,陈维信,刘爱媛.植物内生菌及其防治植物病害的研究进展[J].生态学报,2006,26(7):2 395-2 401.Shi J Y,Chen W X,Liu A Y.Advances in the study of endophytes and their efects on control of plant diseases[J].Acta Ecologica Sinica,2006,26(7):2 395-2 401.
[17]李洪连,王守正.植物诱导抗病性研究的现状与展望[J].河南农业大学学报,1994,28(3):219-223.Li H L,Wang S Z.Current status and prospects of plant induced disease resistance research[J].Journal of Henan Agricultural University,1994,28(3):219-223.
[18]陈颖潇,何胥,施洁君,等.黄瓜霜霉病生防菌株的筛选及防病促生研究[J].安徽农业科学,2015,43(23):121-124.Chen Y X,He X,Shi J J,et al.Screening of biocontrol strain against cucumber downy mildew and the preliminary study of disease prevention and hygiene pro-motion[J].Journal of Anhui Agricultural Sciences,2015,43(23):121-124.
[19]吴蔼民,顾本康,傅正擎,等.内生菌73a在不同抗性品种棉花体内的定殖和消长动态研究[J].植物病理学报,2001,31(4):289-294.Wu A M,Gu B K,Fu Z Q.Studies on the population fluctuation of entophytic bacteria 73a in cotton plant[J].Acta Phytopathologica Sinica,2001,31(4):289-294.
[20]Miller C,Miller R,Garton-Kenny D,et al.Ecomycins,unique antimycotics from Pseudomonas viridiflava[J].Journal of Applied Microbiology,1998,84(6):937-944.
[21]Han Q,Wu F,Wang X,et al.The bacterial lipopeptide iturins induce Verticillium dahliae cell death by affecting fungal signalling pathways and mediate plant defence responses involved in pathogen-associated molecular pattern-triggered immunity[J].Environmental Microbiology,2015,17(4):1166-1188.
[22]董汉松.植物抗病防卫基因表达调控与诱导抗性遗传的机制[J].植物病理学报,1996,26(4):289-293.Dong H S.Expressive regulation of plant disease defense genes in relation with hereditability of induced resistance[J].Acta Phytopathologica Sinica,1996,26(4):289-293.
[23]范志金,刘秀峰,刘凤丽,等.植物抗病激活剂诱导植物抗病性的研究进展[J].植物保护学报,2005,32(1):87-92.Fan Z J,Liu X F,Liu F L,et al.Advances in research on plant disease resistance induced by plant disease resistance activators[J].Journal of Plant Protection,2005,32(1):87-92.
[24]Sturz A,Matheson B.Populations of endophytic bacteria which influence host-resistance to Erwinia-induced bacterial soft rot in potato tubers[J].Plant and Soil,1996,184(2):265-271.