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European Journal of Plant Pathology

, Volume 136, Issue 3, pp 523–533 | Cite as

Combination of a simple differential medium and toxA-specific PCR for isolation and identification of phytopathogenic Burkholderia gladioli

  • Yung-An Lee
  • Chin-Sheng Chao
  • Chi-Hung Jung
Article

Abstract

The NGM medium developed in a previous study was used for differential isolation of Pectobacterium chrysanthemi, Burkholderia gladioli, and B. glumae. P. chrysanthemi developed blue colonies, and all B. gladioli and B. glumae strains tested produced diffusible yellow pigments on the NGM medium, easily distinguishable from other Burkholderia spp. and plant pathogenic bacteria. The produced yellow pigments contained a toxoflavin determined by the TLC and orchid leaf chlorosis tests. A specific oligonucleotide primer pair was designed for the detection of toxA, which is involved in toxoflavin biosynthesis. All B. gladioli and B. glumae strains tested contained toxA as determined by PCR amplification. No amplification was observed with other plant pathogenic bacteria. In addition, the toxA-based species-specific PCR assays, based on the nucleotide sequence differences in the promoter region of toxA, were developed for identification of B. gladioli and B. glumae, respectively. The NGM medium and the toxA-based PCR assays were used to determine the causal agents of leaf rot of Phalaenopsis and Oncidium orchids at three cultivation areas in Taiwan. It was found that both P. chrysanthemi and B. gladioli are important pathogenic bacteria of orchid leaf rot in Taiwan. The results indicate that the combination of NGM medium with toxA-based PCR assays is a newly designed and efficient method for isolation and identification of leaf rot pathogenic bacteria especially from plant hosts on which P. chrysanthemi and B. gladioli (or B. glumae) could cause symptoms.

Keywords

Pectobacterium chrysanthemi Burkholderia glumae Phalaenopsis Oncidium Leaf rot Toxoflavin 

Notes

Acknowledgments

The authors thank Dr. Ting-Fang Hsieh (Floriculture Research Center, Taiwan Agricultural Research Institute) for sampling the diseased orchids with leaf rot, and Dr. Ya-Chun Chang (Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan) for helpful discussions and critical reading of the manuscript. This research was supported by grants from the Council of Agriculture (98AS-9.3.1-BQ-B1 and 99AS-9.3.1-BQ-B1) and National Science Council Project (NSC 97-2317-B-030-001), Taiwan, Republic of China.

References

  1. Balandreau, J., Viallard, V., Cournoyer, B., & Coenye, T. (2001). Burkholderia cepacia genomovar III is a common plant-associated bacterium. Applied and Environmental Microbiology, 67, 982–985.PubMedCrossRefGoogle Scholar
  2. Baxter, I. A., Lambert, P. A., & Simpson, I. N. (1997). Isolation from clinical sources of Burkholderia cepacia possessing characteristics of Burkholderia gladioli. Journal of Antimicrobial Chemotherapy, 39, 169–175.PubMedCrossRefGoogle Scholar
  3. Christenson, J. C., Welch, D., Mukwaya, G., Muszynski, M. J., Weaver, R. E., & Brenner, D. J. (1989). Recovery of Pseudomonas gladioli from respiratory tract specimens of patients with cystic fibrosis. Journal of Clinical Microbiology, 27, 270–273.PubMedGoogle Scholar
  4. Chuenchitt, S., Dhirabhava, W., Karnjanarat, S., Buangsuwon, D., & Uematsu, T. (1983). A new bacterial disease on orchids Dendrobium sp. caused by Pseudomonas gladioli. Kasetsart Journal, 17, 26–36.Google Scholar
  5. Clode, F. E., Kaufmann, M. E., Malnick, H., & Pitt, T. L. (1999). Evaluation of three oligonucleotide primer sets in PCR for the identification of Burkholderia cepacia and their differentiation from Burkholderia gladioli. Journal of Clinical Pathology, 52, 173–176.PubMedCrossRefGoogle Scholar
  6. Coenye, T., & Vandamme, P. (2003). Diversity and significance of Burkholderia species occupying diverse ecological niches. Environmental Microbiology, 5, 719–729.PubMedCrossRefGoogle Scholar
  7. Coenye, T., Gillis, M., & Vandamme, P. (2000). Pseudomonas antimicrobica Attafuah and Bradbury 1990 is a junior synonym of Burkholderia gladioli (Severini 1913) Yabuuchi et al. 1993. International Journal of Systematic and Evolutionary Microbiology, 50, 2135–2139.PubMedCrossRefGoogle Scholar
  8. Furuya, N., Ura, H., Iiyama, K., Matsumoto, M., Takeshita, M., & Takanami, Y. (2002). Specific oligonucleotide primers based on sequences of the 16S–23S rDNA spacer region for the detection of Burkholderia gladioli by PCR. Journal of General Plant Pathology, 68, 220–224.CrossRefGoogle Scholar
  9. Govan, J. R. W., & Deretic, V. (1996). Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aerugionosa and Burkholderia cepacia. Microbiological Reviews, 60, 539–574.Google Scholar
  10. Iiyama, K., Furuya, N., Takanami, Y., & Matsuyama, N. (1995). A role of phytotoxin in virulence of Pseudomonas glumae Kurita et Tabei. Annals of the Phytopathological Society of Japan, 61, 470–476.CrossRefGoogle Scholar
  11. Jeong, Y., Kim, J., Kim, S., Kang, Y., Nagamatsu, T., & Hwang, I. (2003). Toxoflavin produced by Burkholderia glumae causing rice grain rot is responsible for inducing bacterial wilt in many field crops. Plant Disease, 87, 890–895.CrossRefGoogle Scholar
  12. Kawaradani, M., Okada, K., & Kusakari, S. (2000). New selective medium for isolation of Burkholderia glumae from rice seeds. Journal of General Plant Pathology, 66, 234–237.CrossRefGoogle Scholar
  13. Keith, L., Sewake, K. T., & Zee, F. T. (2005). Isolation and characterization of Burkholderia gladioli from orchids in Hawaii. Plant Disease, 89, 1273–1278.CrossRefGoogle Scholar
  14. Lee, Y.-A., & Chan, C.-W. (2007). Molecular typing and presence of genetic markers among strains of banana finger-tip rot pathogen, Burkholderia cenocepacia, in Taiwan. Phytopathology, 97, 195–201.PubMedCrossRefGoogle Scholar
  15. Lee, Y.-A., & Yu, C.-P. (2006). A differential medium for the isolation and rapid identification of a plant soft rot pathogen, Erwinia chrysanthemi. Journal of Microbiological Methods, 64, 200–206.PubMedCrossRefGoogle Scholar
  16. Lee, Y.-A., Fan, S.-C., Chiu, L.-Y., & Hsia, K.-C. (2001). Isolation of an insertion sequence from Ralstonia solanacearum race 1 and its potential use for strain characterization and detection. Applied and Environmental Microbiology, 67, 3943–3950.PubMedCrossRefGoogle Scholar
  17. Lee, Y.-A., Chan, C.-W., & Chao, C.-P. (2006). Physiological and molecular characterizations of banana finger-tip rot and onion decay pathogens in Taiwan. Plant Pathology Bulletin, 15, 117–124.Google Scholar
  18. Mahenthiralingam, E., Baldwin, A., & Vandamme, P. (2002). Burkholderia cepacia complex infection in patients with cystic fibrosis. Journal of Medical Microbiology, 51, 533–538.PubMedGoogle Scholar
  19. Matsuda, I., & Sato, Z. (1988). Relations between pathogenicity and pigment productivity in the causal agent of bacterial grain rot of rice. Annals of the Phytopathological Society of Japan, 54, 378.CrossRefGoogle Scholar
  20. Nandakumar, R., Shahjahan, A. K. M., Yuan, X. L., Dickstein, E. R., Groth, D. E., Clark, C. A., et al. (2009). Burkholderia glumae and B. gladioli cause bacterial panicle blight in rice in the southern United States. Plant Disease, 93, 896–905.CrossRefGoogle Scholar
  21. Ross, J., Holland, S. M., Gill, V. J., DeCarlo, E. S., & Gallin, J. I. (1995). Severe Burkholderia (Pseudomonas) gladioli infection in chronic granulomatous disease: report of two successfully treated cases. Clinical Infectious Diseases, 21, 1291–1293.PubMedCrossRefGoogle Scholar
  22. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory.Google Scholar
  23. Sato, Z., Koiso, Y., Iwasaki, S., Matsuda, I., & Shirata, A. (1989). Toxins produced by Pseudomonas glumae. Annals of the Phytopathological Society of Japan, 55, 353–356.CrossRefGoogle Scholar
  24. Simpson, I. N., Finlay, J., Winstanley, D. J., Dewhurst, N., Nelson, J. W., Butler, S. L., et al. (1994). Multi-resistance isolates possessing characteristics of both Burkholderia (Pseudomonas) cepacia and Burkholderia gladioli from patients with cystic fibrosis. Journal of Antimicrobial Chemotherapy, 34, 353–361.PubMedCrossRefGoogle Scholar
  25. Suzuki, F., Sawada, H., Azegami, K., & Tsuchiya, K. (2004). Molecular characterization of the tox operon involved in toxoflavin biosynthesis of Burkholderia glumae. Journal of General Plant Pathology, 70, 97–107.CrossRefGoogle Scholar
  26. Takeuchi, T., Sawada, H., Suzuki, F., & Matsuda, I. (1997). Specific detection of Burkholderia plantarii and B. glumae by using primers selected from the 16S–23S rDNA spacer regions. Annals of the Phytopathological Society of Japan, 63, 455–462.CrossRefGoogle Scholar
  27. Tsushima, S., Wakimoto, T., & Mogi, S. (1986). Selective medium for detecting Pseudomonas glumae Kurita et Tabei, the causal bacterium of grain rot of rice. Annals of the Phytopathological Society of Japan, 52, 253–259.CrossRefGoogle Scholar
  28. Whitby, P. W., Pope, L. C., Carter, K. B., Lipuma, J. J., & Stull, T. L. (2000). Species-specific PCR as a tool for the identification of Burkholderia gladioli. Journal of Clinical Microbiology, 38, 282–285.PubMedGoogle Scholar

Copyright information

© KNPV 2013

Authors and Affiliations

  1. 1.Department of Life ScienceFu Jen Catholic UniversityNew Taipei CityRepublic of China
  2. 2.Graduate Institute of Applied Science and EngineeringFu Jen Catholic UniversityNew Taipei CityRepublic of China

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