Netherlands Journal of Plant Pathology

, Volume 97, Issue 5, pp 321–334 | Cite as

Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry

  • N. J. M. Roozen
  • J. W. L. Van Vuurde


Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 108 colony forming units (cfu) per ml.

Plating efficiency of Cms in yeast glucose mineral medium (YGM) was 104% compared with yeast peptone glucose medium. Nalidixic acid, polymyxin B sulphate and the experimental disinfectant S-0208 inhibited colony growth of cattle slurry bacteria as compared with Cms in YGM. The optimal concentration of these inhibitors in combination was determined by modified agar diffusion tests and by pour plating in 24-well tissue culture plates. The semi-selective medium YGMI consisted of YGM supplemented with nalidixic acid (2 mg/l), polymyxin B sulphate (30 mg/l) and S-0208 (125 mg/l). Plating efficiency varied for Cms between 50.9 and 69.6%, for cattle slurry bacteria between 1.8 and 2.5% and for saprophytes from potato heel end extracts between 11.5 and 27.4%.

Differentiation of Cms colonies from other colonies was based on their small and bluish colony morphology in pour plates and on immunofluorescence colony-staining (IFC). IFC of a pure culture of micro colonies of Cms in YGM was possible after one day incubation (colonies c. 5 cells). Green background fluorescence in the agar gels was prevented by addition of Tween 20 (0.1%) to the washing buffer and the use of 1% agar gels. IFC of macro colonies of Cms in YGMI, visible with 4x objective magnification, was possible after 4 days. The detection level of the target organism in artificially inoculated cattle slurry in YGMI based on colony morphology varied between 1.4×103 and 2.3×104 cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (Φ=16 mm), proved suitable for detection, but was c. 30 times les sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R2=0.74).

Additional keywords

antibiotic screening colony differentiation cross-reaction detection level fluorescent background reduction micro colony assay miniaturized agar plating plating efficiency potato heel end 


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  1. Allen, E. & Kelman, A., 1977. Immunofluorescence stain procedures for detection and identification ofErwinia carotovora var.atroseptica. Phytopathology 67: 1305–1312.Google Scholar
  2. Barry, A.L., 1980. Procedure for testing antibiotics in agar media: Theoretical considerations. In: Lorian, V. (Ed.), Antibiotics in laboratory medicine. Williams & Wilkins, Baltimore: 1–23.Google Scholar
  3. Becton Dickenson, 1985. Microbiologische Handbuch. Becton Dickenson Deutschland, Heidelberg: 40.Google Scholar
  4. Commission of the European Communities, 1987. Scheme for the detection and diagnosis of the ring rot bacteriumCorynebacterium sepedonicum in batches of potato tubers. Published by the Commission of the European Communities EUR 11288, Luxembourg: 1–21.Google Scholar
  5. De Boer, S.H. & Copeman, R. J., 1980. Bacterial ring rot testing with the indirect fluorescent antibody staining procedure. American Potato Journal 57: 457–465.Google Scholar
  6. De Bruyne, E., 1985. Fenotypische karakterisatie vanCorynebacterium sepedonicum, verwekker van ringrot bij aardappelen. Faculteit van de Wetenschappen, Laboratorium voor Microbiologie en Microbiële Genetica, Gent: 1–89.Google Scholar
  7. Fatmi, M. & Schaad, N., 1988. Semiselective agar medium for isolation ofClavibacter michiganense subsp.michiganense from tomato seed. Phytopathology 78: 121–126.Google Scholar
  8. Janse, J.D. & Van Vaerenbergh, J., 1987. Interpretation of the EC method for the detection of latentCorynebacterium sepedonicum infections in potato. EPPO Bulletin 17: 1–10.Google Scholar
  9. Lelliott, R.A. & Stead, D.E., 1987. Methods for the diagnosis of bacterial diseases of plants. Blackwell Scientific Publications, Oxford: 1–216.Google Scholar
  10. Lachance, R.A., 1962. The amino-acid requirements ofCorynebacterium sepedonicum (Spieck. & Kott.) Skapt. & Burkh. Canadian Journal of Microbiology 8: 321–325.PubMedGoogle Scholar
  11. Miller, H.J., 1984. Cross-reactions ofCorynebacterium sepedonicum antisera with soil bacteria associated with potato tubers. Netherlands Journal of Plant Pathology 90: 23–28.Google Scholar
  12. Paquin, R. & Lachance, R.A., 1970. Sur la nutrition aminée deCorynebacterium sepedonicum (Spieck. et Kott.) Skapt. et Burkh. et la résistence de la pomme de terre au flétrissement bactérien. Canadian Journal of Microbiology 16: 719–726.PubMedGoogle Scholar
  13. Paquin, R. & Pelletier, G., 1966. Etude de pH de millieux de culture synthéthiques et naturels en fonction de la résistence des pommes de terre au flétrissement bactérien. Phytoprotection 47: 95–103.Google Scholar
  14. Paquin, R. & Pelletier, G., 1969. Etude du rôle des sucres dans la résistence des pommes de terre au flétrissement bactérien. Canadian Journal of Microbiology 15: 907–916.PubMedGoogle Scholar
  15. Pratt, W.B. & Fekety, R., 1986. The antimicrobial drugs. Oxford Univ. Press, New York: 1–501.Google Scholar
  16. Snieszko, S.F. & Bonde, R., 1943. Studies on the morphology, physiology, serology, longevity, and pathogenicity ofCorynebacterium sepedonicum. Phytopathology 33: 1032–1044.Google Scholar
  17. Underberg, H.A. & Sander, E., 1991. Detection ofCorynebacterium sepedonicum with antibodies raised in chicken egg yolks. Journal of Plant Diseases and Protection 98: 188–196.Google Scholar
  18. Van Vuurde, J.W.L. & Roozen, N.J.M., 1990. Comparison of immunofluorescence colonystaining in media, selective isolation on pectate medium, ELISA and immunofluorescene cell staining for detection ofErwinia carotovora subsp.atroseptica andE. chrysanthemi in cattle manure slurry. Netherlands Journal of Plant Pathology 96: 75–89.Google Scholar
  19. Vruggink, H. & Maas Gesteranus, H.P., 1975. Serological recognition ofErwinia carotovora var.atroseptica, the causal organism of potato blackleg. Potato Research 18: 546–555.Google Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 1991

Authors and Affiliations

  • N. J. M. Roozen
    • 1
  • J. W. L. Van Vuurde
    • 1
  1. 1.Research Institute for Plant Protection (IPO-DLO)Wageningenthe Netherlands

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