Combination of a simple differential medium and toxA-specific PCR for isolation and identification of phytopathogenic Burkholderia gladioli
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.
KeywordsPectobacterium chrysanthemi Burkholderia glumae Phalaenopsis Oncidium Leaf rot Toxoflavin
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.
- 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
- 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
- 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
- Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory.Google Scholar
- 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