European Journal of Plant Pathology

, Volume 140, Issue 4, pp 655–664 | Cite as

Genes clvA, clvF and clvG are unique to Clavibacter michiganensis subsp. michiganensis and highly conserved

  • Jarred Yasuhara-Bell
  • Glorimar Marrero
  • Anne M. Alvarez
Article

Abstract

Bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), occurs worldwide. This study focuses on three genes, clvA (clavicidin) and two associated genes (clvF and clvG, encoding a putative ABC-type multidrug transport system ATPase and permease component, respectively). Loop-mediated amplification of clvA and PCR of clvA, clvF and clvG showed that these genes were present only in Cmm and not in other Clavibacter species/subspecies, as well as other genera of plant-associated bacteria. Sequences of clvA, clvF and clvG from 48 geographically diverse strains of Cmm were analyzed phylogenetically to determine sequence variation. Maximum parsimony (MP), neighbour-joining (NJ) and maximum likelihood (ML) analyses placed strains into subgroups irrespective of their geographical origins. Multi-locus sequence analysis (MLSA) of these three genes produced the same results. In sum, all three clv genes are unique and conserved in Cmm, further supporting the use of the clvA gene for identification of Cmm, using either a previously designed LAMP assay or PCR with additional primers developed in this study. The genes investigated here are novel targets for specific detection of Cmm and have numerous molecular diagnostic applications.

Keywords

Clavibacter michiganensis Tomato Bacteria Pathogen Clavicidin 

Notes

Acknowledgments

This work was supported by USDA-NIFA SCRI-600-25320. We thank our colleagues for providing strains from their collections.

Supplementary material

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References

  1. Alvarez, A. M., & Kaneshiro, W. S. (1999). Detection and identification of Clavibacter michiganensis subsp. michiganensis in tomato seed. In J. W. Sheppard (Ed.), Proc. 3rd Intl. Seed Testing Association Seed Health Symposium (pp. 93–97). Zurich, Switzerland: Intl. Seed Testing Assn.Google Scholar
  2. Alvarez, A. M., Kaneshiro, W. S., & Vine, B. G. (2005). Diversity of Clavibacter michiganensis subsp. michiganensis populations in tomato seed: what is the significance? Acta Horticulturae, 695, 205–213.Google Scholar
  3. Baysal, Ö., Mercati, F., İkten, H., Yıldız, R. Ç., Carimi, F., Aysan, Y., et al. (2011). Clavibacter michiganensis subsp. michiganensis: tracking strains using genetic differentiations by ISSR markers in Southern Turkey. Physiological and Molecular Plant Pathology, 75, 113–119.CrossRefGoogle Scholar
  4. Davis, M. J., Gillespie, A. G. J., 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.CrossRefGoogle Scholar
  5. de Queiroz, A., Donoghue, M. J., & Kim, J. (1995). Seperate versus combined analysis of phylogenetic evidence. Annual Review of Ecology and Systematics, 26, 657–681.CrossRefGoogle Scholar
  6. Dreier, J., Bermpohl, A., & Eichenlaub, R. (1995). Southern hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis. Phytopathology, 85, 462–468.CrossRefGoogle Scholar
  7. Eichenlaub, R., & Gartemann, K. H. (2011). The Clavibacter michiganensis subspecies, molecular investigation of gram-positive bacterial plant pathogens. Annual Review of Phytopathology, 49, 7.1–7.20.CrossRefGoogle Scholar
  8. Eijsink, V. G. H., Axelsson, L., Diep, D. B., Havarstein, L. S., Holo, H., & Nes, I. F. (2002). Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie Van Leeuwenhoek, 81, 639–654.PubMedCrossRefGoogle Scholar
  9. EPPO. (2005). Clavibacter michiganensis subsp. michiganensis. OEPP/EPPO Bulletin, 35, 275–283.CrossRefGoogle Scholar
  10. Farris, J. S., Källersjö, M., Kluge, A. G., & Bult, C. (1994). Testing significance of incongruence. Cladistics, 10, 315–319.CrossRefGoogle Scholar
  11. Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783–791.CrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W., & Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology, 59, 307–321.PubMedCrossRefGoogle Scholar
  14. Holtsmark, I., Mantzilas, D., Eijsink, V. G. H., & Brurberg, M. B. (2006a). Purification, characterization, and gene sequence of Michiganin A, an actagardine-like lantibiotic produced by the tomato pathogen Clavibacter michiganensis subsp. michiganensis. Applied and Environmental Microbiology, 72, 5814–5821.CrossRefGoogle Scholar
  15. Holtsmark, I., Mantzilas, D., Eijsink, V. G. H., & Brurberg, M. B. (2006b). The tomato pathogen Clavibacter michiganensis ssp michiganensis: producer of several antimicrobial substances. Journal of Applied Microbiology, 102, 416–423.Google Scholar
  16. Jacques, M.-A., Durand, K., Ogreur, G., Balidas, S., Fricot, C., Bonneau, S., et al. (2012). Phylogenetic analysis and polyphasic characterization of Clavibacter michiganensis strains isolated from tomato seeds reveal that non-pathgenic strains are distinct from C. michiganensis subsp. michiganensis. Applied and Environmental Microbiology, 78(23), 8388–8402.PubMedCrossRefPubMedCentralGoogle Scholar
  17. Jenkins, D. M., Kubota, R., Dong, J., Li, Y., & Higashiguchi, D. (2011). Handheld device for real-time, quantitative, LAMP-based detection of Salmonella enterica using assimilating probes. Biosensors & Bioelectronics, 30(1), 255–260.CrossRefGoogle Scholar
  18. Jukes, T. H., & Cantor, C. R. (1969). Evolution of protein molecules. In H. N. Munro (Ed.), Mammalian protein metabolism (pp. 21–132). New York: Academic.CrossRefGoogle Scholar
  19. Kaneshiro, W. S. (2003). Detection and characterization of virulent, hypovirulent, and nonvirulent Clavibacter michiganensis subsp. michiganensis. Honolulu: University of Hawai’i at Mānoa.Google Scholar
  20. Kaneshiro, W. S., Mizumoto, C. Y., & Alvarez, A. M. (2006). Differentiation of Clavibacter michiganensis subsp. michiganensis from seed-borne saprophytes using ELISA, biolog and 16S DNA sequencing. European Journal of Plant Pathology, 16, 45–56.CrossRefGoogle Scholar
  21. Kawaguchi, A., Tanina, K., & Inoue, K. (2010). Molecular typing of Clavibacter michiganensis subsp. michiganensis in greenhouses in Japan. Plant Pathology, 59, 76–83.CrossRefGoogle Scholar
  22. Kleitman, F., Barash, I., Burger, A., Iraki, N., Falah, Y., Sessa, G., et al. (2008). Characterization of Clavibacter michiganensis subsp. michiganensis population in Israel. European Journal of Plant Pathology, 121, 463–475.CrossRefGoogle Scholar
  23. Kubota, R., Vine, B. G., Alvarez, A. M., & Jenkins, D. M. (2008). Detection of Ralstonia solanacaerum by loop-mediated isothermal amplification. Phytopathology, 98(9), 1045–1051.PubMedCrossRefGoogle Scholar
  24. Lee, I. M., Bartoszyk, I. M., Gunderson-Rindal, D. E., & Davis, R. E. (1997). Phylogeny and classification of bacteria in the genera Clavibacter and Rathayibacter on the basis of 16S rRNA gene sequence analyses. Applied and Environmental Microbiology, 63(7), 2631–2636.PubMedPubMedCentralGoogle Scholar
  25. Louws, F. J., Bell, J., Medina-Mora, C. M., Smart, C. D., Opgenorth, D., Ishimaru, C. A., et al. (1998). rep-PCR-mediated genomic fingerprinting: a rapid and effective method to identify Clavibacter michiganensis. Phytopathology, 88, 862–868.PubMedCrossRefGoogle Scholar
  26. Mickevich, M. F., & Farris, J. S. (1981). The implications of congruence in Menidia. Systematic Zoology, 30, 351–370.CrossRefGoogle Scholar
  27. Milijaevšić-Marčić, S., Gartemann, K. H., Frohwitter, J., Eichenlaub, R., Todorović, B., Rekanović, E., et al. (2012). Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia. European Journal of Plant Pathology, 134, 697–711.CrossRefGoogle Scholar
  28. Nei, M., & Kumar, S. (2000). Molecular evolution and phylogenetics. New York: Oxford University Press.Google Scholar
  29. Pastrik, K. H., & Rainey, F. A. (1999). Identification and differentiation of Clavibacter michiganensis subspecies by polymerase chain reaction-based techniques. Journal of Phytopathology, 147, 687–693.CrossRefGoogle Scholar
  30. Quesada-Ocampo, L. M., Landers, N. A., Lebeis, A. C., Fulbright, D. W., & Hausbeck, M. K. (2012). Genetic structure of Clavibacter michiganensis subsp. michiganensis populations in Michigan commercial tomato fields. Plant Disease, 96(6), 788–796.CrossRefGoogle Scholar
  31. Richert, K., Brambilla, E., & Stackebrandt, E. (2005). Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales. Journal of Microbiological Methods, 6(1), 115–123.CrossRefGoogle Scholar
  32. Rijlaarsdam, A., Woudt, B., Simons, G., Koenraadt, H., Oosterhof, J., Asma, M., et al. (2004). Development of specific primers for the molecular detection of Clavibacter michiganensis subsp. michiganensis. In EPPO Conference on Quality of Diagnosis and New Diagnostic Methods for Plant Pests, Noordwijkerhout, NL. Google Scholar
  33. Riley, M. A., & Gordon, D. M. (1999). The ecological role of bacteriocins in bacterial competition. Trends in Microbiology, 7, 129–133.PubMedCrossRefGoogle Scholar
  34. Saitou, N., & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406–425.PubMedGoogle Scholar
  35. Sarkar, I. N., Egan, M. G., Coruzzi, G., Lee, E. K., & DeSalle, R. (2008). Automated simultaneous phylogenetics (ASAP): an enabling tool for phylogenetics. BMC Bioinformatics, 9, 103.PubMedCrossRefPubMedCentralGoogle Scholar
  36. Schneider, K. L., Marrero, G., Alvarez, A. A., & Presting, G. G. (2011). Classification of plant associated bacteria using RIF, a computationally derived DNA marker. PLoS, 6(4), e18496.CrossRefGoogle Scholar
  37. Sousa Santos, M., Cruz, L., Norskov, P., & Rasmussen, O. F. (1997). A rapid and sensitive detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds by polymerase chain reaction. Seed Science and Technology, 25, 581–584.Google Scholar
  38. Stöver, B. C., & Müller, K. F. (2010). TreeGraph 2: combining and visualizing evidence from different phylogenetic analyses. BMC Bioinformatics, 11, 7. doi: 10.1186/1471-2105-11-7.PubMedCrossRefPubMedCentralGoogle Scholar
  39. Strider, D. L. (1969). Bacterial canker of tomato caused by Corynebacterium michiganense: a literature review and bibliography. North Carolina Agric. Exp. Stn., Tech Bull., 193.Google Scholar
  40. Swofford, J. L. (2002). PAUP*. Phylogenetic analysis using parsimony (*and other methods) (4th ed.). Sunderland: Sinauer Associates.Google Scholar
  41. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739.PubMedCrossRefPubMedCentralGoogle Scholar
  42. Vandamme, A.-M. (2003). Basic concepts of molecular evolution. In M. Salemi & A.-M. Vandamme (Eds.), The phylogenetics handbook: A practical approach to DNA and protein phylogeny (pp. 1–23). New York: Cambridge University Press.Google Scholar
  43. Waleron, M., Waleron, K., Kamasa, J., Przewodowski, W., & E., L. (2011). Polymorphism analysis of housekeeping genes for identification and differentiation of Clavibacter michiganensis subspecies. Eur. J. Plant Pathol., 131, 341–354.Google Scholar
  44. Yasuhara-Bell, J., & Alvarez, A. M. (2012). Loop-mediated amplification (LAMP) for specific detection of tomato phytopathogen Clavibacter michiganensis subsp. michiganensis (Cmm). Phytopathology, 102, S4.141.Google Scholar
  45. Yasuhara-Bell, J., & Alvarez, A. M. (2014). Differentiation of Clavibacter michiganensis subsp. michiganensis from other Clavibacter species found in seed and plant tissues. Acta Horticulturae. Google Scholar
  46. Yasuhara-Bell, J., Kubota, R., Jenkins, D. M., & Alvarez, A. M. (2013). Loop-mediated amplification of the Clavibacter michiganensis subsp. michiganensis micA gene is highly specific. Phytopathology, 103(12), 1220–1226.PubMedCrossRefGoogle Scholar
  47. Yim, K.-O., Lee, H.-I., Kim, J.-H., Lee, S.-D., Cho, J.-H., & Cha, J.-S. (2012). Characterization of phenotypis variants of Clavibacter michiganensis subsp. michiganensis isolated from Capisum annuum. European Journal of Plant Pathology, 133, 559–575.CrossRefGoogle Scholar
  48. Zaluga, J., Heylen, K., Van Hoorde, K., Hoste, B., Van Vaerenbergh, J., Maes, M., et al. (2011). GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter. Systematic and Applied Microbiology, 34, 400–407.PubMedCrossRefGoogle Scholar
  49. Zaluga, J., Stragier, P., Van Vaerenbergh, J., Maes, M., & De Vos, P. (2013a). Multilocus variable-number-tandem-repeats analysis (MLVA) distinguishes a clonal complex of Clavibacter michiganensis subsp. michiganensis strains isolated from recent outbreaks of bacterial wilt and canker in Belgium. BMC Microbiology, 13(1), 126. doi: 10.1186/1471-2180-13-126.CrossRefGoogle Scholar
  50. Zaluga, J., Van Vaerenbergh, J., Stragier, P., Maes, M., & De Vos, P. (2013b). Genetic diversity of non-pathogenic Clavibacter strains isolated from tomato seeds. Syst. Appl. Microbiol., 36(6), 426–435, doi:http://dx.doi.org/ 10.1016/j.syapm.2013.04.005.

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2014

Authors and Affiliations

  • Jarred Yasuhara-Bell
    • 1
  • Glorimar Marrero
    • 1
  • Anne M. Alvarez
    • 2
  1. 1.Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human ResourcesUniversity of Hawaii at MānoaHonoluluUSA
  2. 2.Department of Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human ResourcesUniversity of Hawaii at MānoaHonoluluUSA

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