Skip to main content
SpringerLink
Log in

Studies on the colonization of axenically grown tomato plants by a GFP-tagged strain of Clavibacter michiganensis subsp. michiganensis

  • Original Research
  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

In this study, colonization and disease development of axenically-grown tomato plants by Clavibacter michiganensis subsp. michiganensis (Cmm), the causative agent of bacterial wilt and canker, was investigated. For this, a spontaneous rifampicin resistant strain of Cmm was tagged with a marker that expressed a green fluorescent protein (GFP) in a stable way and which possessed a similar virulence to the parental strain. In vitro plants were drop-inoculated at the stem base and the population dynamics was determined by dilution pour-plating in a selective medium. At 3 h after inoculation, Cmm was already present in low densities in roots, stems and leaves. At 16 dpi, Cmm was found throughout the entire plant in high densities of ca. 1010 cfu g−1. Symptoms developed in the in vitro plants typical for Cmm, such as canker, wilting and growth reduction. The presence of Cmm in vascular and parenchymatic tissue of in vitro tomato plants was confirmed by epifluorescence stereo- and confocal laser scanning microscopy. This study showed that in vitro tomato plants can be effectively used for detailed studies on interactions between Cmm and its host, in particular if a GFP-tagged strain of the pathogen is used.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Anonymous. (1995). Commission Directive 95/4/EC amendment of 21 February 1995 to the European Community Plant Health Directive (77/93/EEC). Official Journal of the European Communities, L44, 56–60.

    Google Scholar 

  • Anonymous. (2011). ISF Method for the detection of Clavibacter michiganensis subsp. michiganensis on tomato seeds, Version 4. http://www.worldseed.org/cms/medias/file/TradeIssues/PhytosanitaryMatters/SeedHealthTesting/ISHI-Veg/Tomato_Cmm_010811.pdf. Acessed 10 march 2013.

  • Benhamou, N. (1991). Cell surface interactions between tomato and Clavibacter michiganense subsp. michiganense: localization of some polysaccharides and hydroxyproline-rich glycoproteins in infected host leaf tissues. Physiological Molecular Plant Pathology, 38, 15–38.

    Article  CAS  Google Scholar 

  • Bialczyk, J., Lechowski, Z., & Dziga, D. (2004). Composition of the xylem sap of tomato seedlings cultivated on media with HCO3 − and nitrogen source as NO3 − or NH4 +. Plant and Soil, 263, 265–272.

    Article  CAS  Google Scholar 

  • Chalupowicz, L., Cohen-Kandli, M., Dror, O., Eichenlaub, R., Gartemann, K.-H., Sessa, G., Barash, I., & Manulis-Sasson, S. (2010). Sequential expression of bacterial virulence and plant defense genes during infection of tomato with Clavibacter michiganensis subsp. michiganensis. Phytopathology, 100, 252–261.

    Article  PubMed  CAS  Google Scholar 

  • Chalupowicz, L., Zellermann, E.-M., Fluegel, M., Dror, O., Eichenlaub, R., Gartemann, K.-H., Savidor, A., Sessa, G., Iraki, N., Barash, I., & Manulis-Sasson, S. (2012). Colonization and movement of GFP-labeled Clavibacter michiganensis subsp. michiganensis during tomato infection. Phytopathology, 102, 23–31.

    Article  PubMed  CAS  Google Scholar 

  • Chang, R. J., Ries, S. M., & Pataky, J. K. (1991). Dissemination of Clavibacter michiganensis subsp. michiganensis by practices used to produce tomato transplants. Phytopathology, 81, 1276–1281.

    Article  Google Scholar 

  • Chang, R. J., Ries, S. M., & Pataky, J. K. (1992). Effects of temperature, plant age, inoculum concentration, and cultivar on the incubation period and severity of bacterial canker of tomato. Plant Disease, 76, 1150–1155.

    Article  Google Scholar 

  • Czajkowski, R., de Boer, W. J., Velvis, H., & van der Wolf, J. M. (2010). Systemic colonization of potato plants by a soilborne, green fluorescent protein-tagged strain of Dickeya sp. biovar 3. Phytopathology, 100, 134–142.

    Article  PubMed  CAS  Google Scholar 

  • Davis, M. J., Gillaspie, A. G., Jr., Vidaver, A. K., & Harris, R. W. (1984). Clavibacter: a new genus containing some phytopathogenic coryneform bacteria, including Clavibacter xyli subsp. xyli sp. nov., subsp. nov. and Clavibacter xyli subsp. cynodontis subsp. nov., pathogens that cause ratoon stunting disease of sugarcane and bermudagrass stunting disease. International Journal of Systematic Bacteriology, 34, 107–117.

    Article  Google Scholar 

  • Devi, C. S., & Srinivasan, V. M. (2006). Studies on various atmospheric microorganisms affecting the plant tissue culture explants. American Journal of Plant Physiology, 1, 205–209.

    Article  Google Scholar 

  • Dreier, J., Meletzus, D., & Eichenlaub, R. (1997). Characterization of the plasmid encoded virulence region pat-1 of the phytopathogenic Clavibacter michiganensis subsp. michiganensis. Molecular Plant-Microbe Interaction, 10, 195–206.

    Article  CAS  Google Scholar 

  • Eichenlaub, R., & Gartemann, K.-H. (2011). The Clavibacter michiganensis subspecies, molecular investigation of gram-positive bacterial plant pathogens. Annual Review of Phytopathology, 49, 445–464.

    Article  PubMed  CAS  Google Scholar 

  • Eichenlaub, R., Gartemann, K. H., & Burger, A. (2006). Clavibacter michiganensis, a group of Gram-positive phytopathogenic bacteria. In S. S. Gnanamanickam (Ed.), Plant-Associated Bacteria (pp. 385–421). Dordrecht, The Netherlands: Springer Netherlands.

    Chapter  Google Scholar 

  • Fatmi, M., & Schaad, N. W. (1988). Semiselective agar medium for isolation of Clavibacter michiganensis subsp. michiganensis from tomato seed. Phytopathology, 78, 121–126.

    Article  Google Scholar 

  • Fatmi, M., Schaad, N. W., & Bolkan, H. A. (1991). Seed treatments for eradicating Clavibacter michiganensis subsp. michiganensis from naturally infected tomato seeds. Plant Disease, 75, 383–385.

    Article  CAS  Google Scholar 

  • Gartemann, K.-H., Kirchner, O., Engemann, J., Gräfen, I., Eichenlaub, R., & Burger, A. (2003). Clavibacter michiganensis subsp. michiganensis: first steps in the understanding of virulence of a Gram-positive phytopathogenic bacterium. Journal of Biotechnology, 106, 179–191.

    Article  PubMed  CAS  Google Scholar 

  • Gartemann, K.-H., Abt, B., Bekel, T., Burger, A., Engemann, J., Flügel, M., Gaigalat, L., Goesmann, A., Gräfen, I., Kalinowski, J., Kaup, O., Kirchner, O., Krause, L., Linke, B., McHardy, A., Meyer, F., Pohle, S., Rückert, C., Schneiker, S., Zellermann, E.-M., Pühler, A., Eichenlaub, R., Kaiser, O., & Bartels, D. (2008). The genome sequence of the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382 reveals a large island involved in pathogenicity. Journal of Bacteriology, 190, 2138–2149.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Gitaitis, R. D., Beaver, R. W., & Voloudakis, A. E. (1991). Detection of Clavibacter michiganensis subsp. michiganensis in symptomless tomato transplants. Plant Disease, 75, 834–838.

    Article  Google Scholar 

  • Hogenhout, S. A., & Loria, R. (2008). Virulence mechanisms of gram-positive plant pathogenic bacteria. Current Opinion in Plant Biology, 11, 1–8.

    Article  CAS  Google Scholar 

  • Huang, R., & Tu, J. C. (2001). Effects of nutrient solution pH on the survival and transmission of Clavibacter michiganensis ssp. michiganensis in hydroponically grown tomatoes. Plant Pathology, 50, 503–508.

    Article  CAS  Google Scholar 

  • Husted, S., Hebbern, C. A., Mattsson, M., & Schjoerring, J. K. (2000). A critical experimental evaluation of methods for determination of NH4 + in plant tissue, xylem sap and apoplastic fluid. Physiologia Plantarum, 109, 167–179.

    Article  CAS  Google Scholar 

  • Jahr, H., Dreier, J., Meletzus, D., Bahro, R., & Eichenlaub, R. (2000). The endo-β-1,4-glucanase of Clavibacter michiganensis subsp. michiganensis is a pathogenicity determinant required for the induction of bacterial wilt of tomato. Molecular Plant-Microbe Interactions, 13, 703–714.

    Article  PubMed  CAS  Google Scholar 

  • Kado, C. I. (2010). Plant Bacteriology. St. Paul, Minnesota: The American Phytopathological Society.

    Google Scholar 

  • Kawaguchi, A., Tanina, K., & Inoue, K. (2010). Molecular typing and spread of Clavibacter michiganensis subsp. michiganensis in greenhouses in Japan. Plant Pathology, 59, 76–83.

    Article  CAS  Google Scholar 

  • Kirchner, O., Gartemann, K.-H., Zellermann, E.-M., Eichenlaub, R., & Burger, A. (2001). A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis. Molecular Plant-Microbe Interactions, 14, 1312–1318.

    Article  PubMed  CAS  Google Scholar 

  • Leifert, C., Morris, C. E., & Waites, W. M. (1994). Ecology of microbial saprophytes and pathogens in tissue culture and field grow plants: reasons for contamination problems in vitro. Critical Reviews in Plants Science, 13, 139–83.

    Article  Google Scholar 

  • López, M. M., Bertolini, E., Caruso, P., Penyalver, R., Marco-Noales, E., Gorris, M. T., Morente, C., Salcedo, C., Cambra, M., & Llop, P. (2005). Advantages of an integrated approach for diagnosis of quarantine pathogenic bacteria in plant material. Phytopathologia Polonica, 35, 49–56.

    Google Scholar 

  • Meletzus, D., Bermpohl, A., Dreier, J., & Eichenlaub, R. (1993). Evidence for plasmid encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganense subsp. michiganense NCPPB382. Journal of Bacteriology, 175, 2131–2136.

    PubMed Central  PubMed  CAS  Google Scholar 

  • Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.

    Article  CAS  Google Scholar 

  • Payne, R. W., Murray, D. A., Harding, S. A., Baird, D. B., & Soutar, D. M. (2007). GenStat for Windows (9th Edition) Introduction.

  • Pine, T. S., Grogan, R. G., & Hewitt, W. B. (1955). Pathological anatomy of bacterial canker of young tomato plants. Phytopathology, 45, 267–271.

    Google Scholar 

  • Sen, Y., Feng, Z., Vandenbroucke, H., van der Wolf, J., Visser, R. G. F., & van Heusden, A. W. (2013). Screening for new sources of resistance to Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato. Euphytica, 190, 309–317.

    Article  CAS  Google Scholar 

  • Sharabani, G., Manulis-Sasson, S., Borenstein, M., Shulhani, R., Lofthouse, M., Chalupowicz, L., & Shtienberg, D. (2012). The significance of guttation in the secondary spread of Clavibacter michiganensis subsp. michiganensis in tomato greenhouses. Plant Pathology. doi:10.1111/j.1365-3059.2012.02673.x.

    Google Scholar 

  • Strider, D. L. (1969). Bacterial canker of tomato caused by Corynebacterium michiganense: a literature review and bibliography. North Carolina Agricultural Experiment Station Techical Bulletin, 193, 110.

    Google Scholar 

  • Subbarayan, K., Varadharajan, N., & Kalyanaraman, R. (2010). Indole-3-acetic acid from contaminant fungus and potential application for cell cultures of Alternanthera sessilis. International Journal of Pharma & Bio Sciences, 1, 257–262.

    CAS  Google Scholar 

  • Tancos, M. A., Chalupowicz, L., Barash, I., Manulis-Sasson, S., & Smart, C. D. (2013). Tomato fruit and seed colonization by Clavibacter michiganensis subsp. michiganensis through external and internal routes. Applied and Environmental Microbiology. doi:10.1128/AEM.02495-13.

    PubMed  Google Scholar 

  • Tans-Kersten, J., Huang, H., & Allen, C. (2001). Ralstonia solanacearum needs motility for invasive virulence on tomato. Journal of Bacteriology, 183, 3597–3605.

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Tsiantos, J. (1987). Transmission of Corynebacterium michiganense pv. michiganense by seeds. Journal of Phytopathology, 119, 142–46.

    Article  Google Scholar 

  • Unge, A., Tombolini, R., Mølbak, L., & Jansson, J. K. (1999). Simultaneous monitoring of cell number and metabolic activity of specific bacterial populations with a dual gfp-luxAB marker system. Applied and Environmental Microbiology, 65, 813–821.

    PubMed Central  PubMed  CAS  Google Scholar 

  • Van Steekelenburg, N. A. M. (1985). Resistance to Corynebacterium michiganense in tomato genotypes. Euphytica, 34, 245–250.

    Article  Google Scholar 

  • Wallis, F. M. (1977). Ultrastructural histopathology of tomato plants infected with Corynebacterium michiganense. Physiological Plant Pathology, 11, 333–342.

    Article  Google Scholar 

  • Xu, X., Rajashekara, G., Paul, P. A., & Miller, S. A. (2012). Colonization of tomato seedlings by bioluminescent Clavibacter michiganensis subsp. michiganensis under different humidity regimes. Phytopathology, 102, 177–184.

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. R. Eichenlaub (Bielefeld, Germany) for providing the gfp-plasmid and P. Kastelein, P. S. van der Zouwen, M. C. Krijger, B.C.E. Kronenburg-van de Ven and G.J.M. de Klerk for technical help and advices. W. de Boer for help with the statistical analysis. The editorial work of Mrs. L.J. Hyman (Dundee, Scotland, UK) is highly appreciated. Thanks are also indebted to the Capes Foundation, Ministry of Education of Brazil for providing a student travel grant. The project was financed by the Dutch Ministry of Economic Affairs via the program “Plant Propagation Material” (BO-04).

Author information

Author notes

  1. Robert Czajkowski

    Present address: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland

Authors and Affiliations

  1. Plant Research International, P.O. Box 69, 6700, AB, Wageningen, The Netherlands

    Flávia M. Vieira Lelis, Robert Czajkowski, Daniel H. Ribeiro & Jan M. van der Wolf

  2. Universidade Federal de Lavras, CP 3037, CEP 37200-000, Lavras, MG, Brazil

    Flávia M. Vieira Lelis, Ricardo Magela de Souza & Daniel H. Ribeiro

Authors
  1. Flávia M. Vieira Lelis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert Czajkowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ricardo Magela de Souza
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel H. Ribeiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jan M. van der Wolf
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jan M. van der Wolf.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Figure S1

Symptoms of in vitro tomato plants at 6 days after drop-inoculation with Clavibacter michiganensis subsp. michiganensis using a concentration of 108 cfu ml-1. a Water-inoculated plant (negative control). b Plant inoculated with a GFP-tagged strain of IPO3356 (IPO3525) showing yellowing of leaves. c Plant drop-inoculated with wild type strain IPO3356 showing yellowing of leaves and blackening of the midrib. (DOC 1028 kb)

Figure S2

Stability test of the GFP-expression by the transformant in plants at 31 dpi. Whole plants were macerated and plated on media supplemented with rifampicin and cycloheximide. Colonies were visualized using an epifluorescence stereo microscope. Colonies partially fluorescent were counted as non-fluorescent for the stability test. (DOC 240 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lelis, F.M.V., Czajkowski, R., de Souza, R.M. et al. Studies on the colonization of axenically grown tomato plants by a GFP-tagged strain of Clavibacter michiganensis subsp. michiganensis . Eur J Plant Pathol 139, 53–66 (2014). https://doi.org/10.1007/s10658-013-0362-9

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10658-013-0362-9

Keywords

Search

Navigation