荧光假单胞菌PEF-5#18防控番茄枯萎病的定殖机理

doi:10.16409/j.cnki.2095-039x.2017.05.012

摘要/Abstract

摘要： 通过电转法成功将EGFP表达质粒pEGFP20转入对番茄枯萎病具有优良防病效果的荧光假单胞菌PEF-5#18体内，并研究了该生防菌在番茄根际土壤与植株根、茎内部组织的定殖情况。结果表明，处理25 d后，标记型与野生型生防菌处理均能有效控制番茄枯萎病害并增加植株鲜、干重量。与病原菌阳性对照相比，野生型菌株的防治效果为76.92%，鲜重增加194.10%，干重增加564.71%；接种后的PEF-5#18能良好定殖于番茄根际土壤并扩展进入植物根茎内部生长，其分布数量呈现出根际土壤（1.05×10⁶ CFU/g） > 根（6.75×10⁴ CFU/g） > 茎（1.25×10² CFU/g）；与病原菌阳性对照相比，接种PEF-5#18对番茄根际土壤的可培养细菌总数影响差异不显著，但可显著提高根（增幅为8.42%）、茎（增幅为14.78%）内的可培养细菌总数以及根际土壤pH（增幅为1.61%）；接种PEF-5#18能显著降低根际土壤与根、茎内病原菌侵染数量；激光共聚焦镜检发现，接种25 d后，PEF-5#18能大量分布于番茄根系表面、根内木质部、根内皮层细胞、根内细胞间隙、茎内维管及其周围细胞。

关键词: 荧光假单胞菌, 番茄枯萎病, 标记, 定殖

Abstract: In order to study the colonization in rhizosphere soil as well as root-stem inside, plasmid pEGFP20 expressing green fluorescens protien was transferred into wild type Pseudomonas fluorescens PEF-5#18 with great effect against Fusarium wilt disease on tomato by electrophoretic transfer method. The results indicated that both wild type and marked P. fluorescens PEF-5#18 bio-control treatments could remarkably control Fusarium wilt disease on tomato with the efficiency of 76.92% and increase plant fresh (194.10%) or dry weight (564.71%) after 25 days, if compared with Forl positive treatment. Inoculated P. fluorescens PEF-5#18 could colonize in rhizosphere soil and extend into tomato root-stem inside, and the distribution amounts ranked as:rhizosphere soil (1.05×10⁶ CFU/g) > root (6.75×10⁴ CFU/g) > stem (1.25×10² CFU/g). Compared with pathogen positive control, the pathogen population was remarkably reduced in rhizosphere soil and root-stem inside, the cultivable bacteria population (increased 8.42% inside root and 14.78% inside stem) and pH value (increased 1.61%) were remarkably increased. Confocal microscopy observation demonstrated that P. fluorescens PEF-5#18 existed on root surface, root xylem, root endodermis cells, root intercellular space, stem vascular tissue cells and the cells around.

Key words: Pseudomonas fluorescens, tomato wilt, marker, colonization.

中图分类号:

S476

张亮, 盛浩, 袁红, 赵兰凤, 李华兴. 荧光假单胞菌PEF-5#18防控番茄枯萎病的定殖机理[J]. 中国生物防治学报, 2017, 33(5): 658-666.

ZHANG Liang, SHENG Hao, YUAN Hong, ZHAO Lanfeng, LI Huaxing. Colonization of Pseudomonas fluorescens PEF-5#18 to Control Fusarium Wilt Disease on Tomato[J]. journal1, 2017, 33(5): 658-666.

参考文献

[1] Colla P, Gilardi G, Gullino M L. A review and critical analysis of the European situation of soilborne disease management in the vegetable sector[J]. Phytoparasitica, 2012, 40(5):515-523.
[2] Kim J T, Park I H, Hahm Y I, et al. Crown and root rot of greenhouse tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici in Korea[J]. Plant Pathology Journal, 2001, 17(5):290-294.
[3] Fravel D R. Commercialization and implementation of biocontrol[J]. Annual Review of Phytopathology, 2005, 43:337-359.
[4] Larkin R P, Fravel D R. Efficacy of various fungal and bacterial biocontrol organisms for control of Fusarium wilt of tomato[J]. Plant Disease, 1998, 82(9):1022-1028.
[5] Weller D M, Raaijmakers J M, Gardener B B M, et al. Microbial populations responsible for specific soil suppressiveness to plant pathogens[J]. Annual Review of Phytopathology, 2002, 40:309-348.
[6] 郝晓娟, 刘波, 谢关林, 等. 短短芽孢杆菌JK-2菌株对番茄枯萎病的抑制作用及其小区防效[J]. 中国生物防治, 2007, 23(3):223-236.
[7] 刘邮洲, 陈志谊, 梁雪杰, 等. 番茄枯萎病和青枯病拮抗细菌的筛选、评价与鉴定[J]. 中国生物防治学报, 2012, 28(1):101-108.
[8] Gopalakrishnan S, Pande S, Sharma M, et al. Evaluation of actinomycete isolates obtained from herbal vermicompost for the biological control of Fusarium wilt of chickpea[J]. Crop Protection, 2011, 30(8):1070-1078.
[9] Gómezlama C C, Schilirò E, Valverdecorredor A, et al. The biocontrol endophytic bacterium Pseudomonas fluorescens PICF7 induces systemic defense responses in aerial tissues upon colonization of olive roots[J]. Frontiers in Microbiology, 2014, 5:427.
[10] Ramos-Solano B, Garcia-Villaraco A, Gutierrez-Mañero F J, et al. Annual changes in bioactive contents and production in field-grown blackberry after inoculation with Pseudomonas fluorescens[J]. Plant Physiology and Biochemistry, 2014, 74(1):1-8.
[11] Zhou J Y, Zhao X Y, Dai C C. Antagonistic mechanisms of endophytic Pseudomonas fluorescens against Athelia rolfsii[J]. Journal of Applied Microbiology, 2014, 117(4):1144-1158.
[12] Santoyo G, Orozco-Mosqueda M C, Govindappa M. Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas:a review[J]. Biocontrol Science and Technology, 2012, 22(8):855-872.
[13] Manikandan R, Raguchander T. Fusarium oxysporum f. sp. lycopersici retardation through induction of defensive response in tomato plants using a liquid formulation of Pseudomonas fluorescens (Pf1)[J]. European Journal of Plant Pathology, 2014, 140(3):469-480.
[14] Podile A R, Vukanti R, Sravani A, et al. Root colonization and quorum Sensing are the driving forces of plant growth promoting rhizobacteria (PGPR) for growth promotion[J]. Proceedings of the Indian National Science Academy, 2014, 80(2):407-413.
[15] 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.
[16] Zhang L, Khabbaz S E, Wang A, et al. Detection and characterization of broad-spectrum antipathogen activity of novel rhizobacterial isolates and suppression of Fusarium crown and root rot disease of tomato[J]. Journal of Applied Microbiology, 2015, 118(3):685-703.
[17] Komada H. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil[J]. Review of Plant Protection Research, 1975, 8:114-124.
[18] Runo S, Macharia S, Alakonya A, et al. Striga parasitizes transgenic hairy roots of Zea mays and provides a tool for studying plant-plant interactions[J]. Plant Methods, 2012, 8(1):20.
[19] Højberg O, Schnider U, Winteler H V, et al. Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil[J]. Applied and Environmental Microbiology, 1999, 65(9):4085-4093.
[20] Khabbaz S E, Abbasi P A. Isolation, characterization, and formulation of antagonistic bacteria for the management of seedlings damping-off and root rot disease of cucumber[J]. Canadian Journal of Microbiology, 2013, 60(1):25-33.
[21] Mihuta-Grimm L, Erb W A, Rowe R C. Fusarium crown and root rot of tomato in greenhouse rock wool systems:sources of inoculum and disease management with benomyl[J]. Plant Disease, 1990, 74(12):996-1002.
[22] Tenuta M, Lazarovits G. Ammonia and nitrous acid from nitrogenous amendments kill the microsclerotia of Verticillium dahliae[J]. Phytopathology, 2002, 92(3):255-264.
[23] Garcias-Bonet N, Arrieta J M, de Santana C N, et al. Endophytic bacterial community of a Mediterranean marine angiosperm (Posidonia oceanica)[J]. Frontiers in Microbiology, 2012, 3:342.
[24] Bloemberg G V, Wijfjes A H M, Lamers G E M, et al. Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere:new perspectives for studying microbial communities[J]. Molecular Plant-Microbe Interactions, 2000, 13(11):1170-1176.
[25] Whipps J M. Microbial interactions and biocontrol in the rhizosphere[J]. Journal of Experimental Botany, 2001, 52(1):487-511.
[26] Bais H P, Weir T L, Perry L G, et al. The role of root exudates in rhizosphere interactions with plants and other organisms[J]. Annual Review of Plant Biology, 2006, 57:233-266.
[27] Ryan R P, Germaine K, Franks A, et al. Bacterial endophytes:recent developments and applications[J]. FEMS Microbiology Letters, 2008, 278(1):1-9.
[28] Cavaglieri L, Orlando J, Etcheverry M. Rhizosphere microbial community structure at different maize plant growth stages and root locations[J]. Microbiological Research, 2009, 164(4):391-399.
[29] 郝变青, 马利平, 乔雄梧. GFP标记的植物促生菌B96-Ⅱ-gfp的定殖能力研究[J]. 中国生态农业学报, 2010, 18(4):861-865.
[30] 殷幼平, 袁训娥, 李强, 等. 生防菌枯草芽孢杆菌CQBS03的绿色荧光蛋白基因标记及其在柑橘叶片上的定殖[J]. 中国农业科学, 2010, 43(17):3555-3563.
[31] Long H H, Schmidt D D, Baldwin I T. Native bacterial endophytes promote host growth in a species-specific manner; phytohormone manipulations do not result in common growth responses[J]. PLoS ONE, 2008, 3(7):e2702.
[32] Prieto P, Schilirò E, Maldonado-González M M, et al. Root hairs play a key role in the endophytic colonization of olive roots by Pseudomonas spp. with biocontrol activity[J]. Microbial Ecology, 2011, 62(2):435-445.
[33] Mei C, Flinn B S. The use of beneficial microbial endophytes for plant biomass and stress tolerance improvement[J]. Recent Patents on Biotechnology, 2010, 4(1):81-95.