Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

The use of subtractive hybridization to obtain a DNA probe specific for Pseudomonas solanacearum race 3


Pseudomonas solanacearum, the causal agent of bacterial wilt, has been classified into three races based on host range and into five biovars based on physiological properties. Strains of race 3 belong exclusively to biovar 2 and primarily affect potatoes. Although this race is thought to have originated in the Andean highlands, it has unusual physiological properties that make it a potential threat to potatoes grown at the cooler latitudes worldwide. Consequently, there is need for a rapid and sensitive method for detection of race 3 strains. We have used subtractive hybridization to enrich for race 3-specific DNA sequences in total race 3 genomic DNA, and thereby obtained a 2 kb clone homologous to DNA from all 28 race 3 strains tested, but with only five of 90 non-race 3 strains. In addition, two larger regions of the genome, containing a minimum of 23 kb of DNA, are also specific for race 3. Deletion of this DNA did not affect virulence. This race 3-specific DNA is a potentially useful diagnostic tool for the detection of race 3 strains.

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


  1. Amasino RM (1986) Acceleration of nucleic acid hybridization rate by polyethylene glycol. Anal Biochem 152:304–345

  2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1988) Current Protocols in Molecular Biology. John Wiley and Sons, New York

  3. Barker RH, Suebsaeng L, Rooney W, Alicrim GC, Dourado HV, Wirth DF (1986) Specific DNA probe for the diagnosis of Plasmodium falciparum malaria. Science 231:1434–1436

  4. Boucher CA, Barberis PA, Arlat M (1988) Acridine orange selects for deletion of hrp genes in all races of Pseudomonas solanacearum. Mol Plant-Microbe Interact 1:282–288

  5. Buddenhagen I, Sequeira L, Kelman A (1962) Designation of races in Pseudomonas solanacearum. Phytopathology 52:726

  6. Buddenhagen I (1985) Bacterial wilt revisited. In: Persley GJ (ed) Bacterial Wilt Disease in Asian and the South Pacific, ACIAR Proceedings No 13. ACIAR, Canberra, pp 126–143

  7. Ciampi L, Sequeira L (1980) Influence of temperature on virulence of race 3 strains of Pseudomonas solanacearum. Am Potato J 57:307–317

  8. Cook D, Barlow L, Sequeira L (1989) Genetic diversity of Pseudomonas solanacearum: detection of restriction fragment length polymorphisms with DNA probes that specify virulence and the hypersensitive response. Mol Plant-Microbe Interact 2:113–121

  9. Coplin DL, Cook D (1990) Molecular genetics of extracellular polysaccharide biosynthesis in vascular phytopathogenic bacteria. Mol Plant-Microbe Interact, in press

  10. Harris D (1972) Intra-specific variation in Pseudomonas solanacearum. In: Proceedings of the third international conference of plant pathogenic bacteria, Wageningen pp 289–292

  11. Hayward A (1964) Characteristics of Pseudomonas solanacearum. J Appl Bact 27:265–277

  12. Hendrick CA, Sequeira L (1984) Lipopolysaccharide-defective mutants of the wilt pathogen Pseudomonas solanacearum. Appl Environ Microbiol 48:389–395

  13. Hyman HC, Yogev D, Razin S (1987) DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium. J Clin Microbiol 25:726–728

  14. Johansen IE, Rasmussen OF, Heide M (1989) Specific identification of Clavibacter michiganense subsp. sepedonicum by DNA-hybridization probes. Phytopathology 79:1019–1023

  15. Kelman A (1954) The relationship of pathogenicity of Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693–695

  16. Kelman A, Hruschka J (1973) The role of motility and aerotaxis in the selective increase of avirulent bacteria in still broth cultures of Pseudomonas solanacearum. J Gen Microbiol 76:177–188

  17. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  18. Martin C, French ER, Nydegger U (1981) Bacterial wilt of potatoes in the Amazon basin. Plant Disease 65:246–248

  19. Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  20. Morton D, Dukes P, Jenkins S (1966) Serological relationships of races 1, 2 and 3 of Pseudomonas solanacearum. Plant Disease Reporter 50:275–277

  21. Olson K (1976) Overwintering of Pseudomonas solanacearum in Sweden. In: Sequeira L, Kelman A (eds) Planning Conference and Workship on the Ecology and Control of Bacterial Wilt Caused by Pseudomonas solanacearum. North Carolina State University, Raleigh, pp 105–109

  22. Seneviratne A (1969) On the occurrence of Pseudomonas solanacearum in the hill country of Ceylon. J Hort Sci 44:393–402

  23. Thurston HD (1963) Bacterial wilt of potatoes in Colombia. Am Potato J 40:381–390

  24. Wood PK, Morrie JG, Small PLC, Sethabutr O, Regina M, Trabulsi L, Kaper JB (1986) Comparison of DNA probes and the Sereny test for identification of invasive Shigella and Escherichia coli strains. J Clin Microbiol 24:498–500

Download references

Author information

Correspondence to Douglas Cook.

Additional information

Communicated by J. Schell

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Cook, D., Sequeira, L. The use of subtractive hybridization to obtain a DNA probe specific for Pseudomonas solanacearum race 3. Molec. Gen. Genet. 227, 401–410 (1991). https://doi.org/10.1007/BF00273930

Download citation

Key words

  • Bacterial wilt
  • Pseudomonas solanacearum
  • DNA probe
  • Potato
  • Subtractive hybridization