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1 Laboratory of Plant Pathology and Biotechnology, Kochi University, 200 Monobe, Nankoku, Kochi 783-8502, Japan
2 National Agricultural Research Center for Tohoku Region, Fukushima 960-2156, Japan
3 Research Institute of Molecular Genetics, Kochi University, 200 Monobe, Nankoku, Kochi 783-8502, Japan
4 Laboratory of Plant Pathology, Kyushu University, Fukuoka 812-8581, Japan
5 Department of Microbiology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan
6 Laboratorium voor Microbiologie, Universiteit Gent, B-9000 Gent, Belgium
7 National Institute for Agro-Environmental Science, Tsukuba, Ibaraki 305-8604, Japan
Correspondence
Yasufumi Hikichi
yhikichi{at}cc.kochi-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the gyrB and rpoD gene sequences determined in this study are given in Table 1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and seedling blight (Azegami et al., 1987) caused by Burkholderia glumae and Burkholderia plantarii, respectively, are devastating diseases of rice seedling cultivation in nursery boxes. During germination, these pathogens infect seeds and invade plumules through stomata and wounds and proliferate in the intercellular spaces of the parenchyma (Azegami et al., 1988; Hikichi et al., 1995). Proliferation of B. glumae and B. plantarii in the plumules leads to production of the toxic materials toxoflavin (Sato et al., 1989; Yoneyama et al., 1998) and tropolone (Azegami et al., 1985), resulting in rot and blight in rice seedlings, respectively.
Within Burkholderia gladioli, three pathovars, all of which are recognized as phytopathogenic bacteria, have been delineated: B. gladioli pv. gladioli, which causes gladiolus rot (Hildebrand et al., 1973); B. gladioli pv. alliicola, which causes onion bulb rot (Young et al., 1978); and B. gladioli pv. agaricicola, which causes rapid soft rot of cultivated mushrooms (Lincoln et al., 1991). B. gladioli has also been isolated from rice seedlings showing bleaching symptoms, suggesting that this bacterium also infects rice seedlings (Kato et al., 1992). On the other hand, proliferation of B. glumae and B. plantarii is suppressed in rice seeds infected with B. gladioli (Miyagawa, 2000). Therefore, ecological studies on B. gladioli in rice plants are important to understand disease development caused not only by B. gladioli but also by B. glumae and B. plantarii. Such ecological studies require efficient detection and identification of these three rice-pathogenic Burkholderia species. Although the diversity of Burkholderia species has been analysed using 16S rRNA gene sequences (Hu et al., 2001; Salles et al., 2002), the discriminatory power of this gene is too restricted to reveal the detailed phylogenetic relationships among B. plantarii, B. glumae and B. gladioli. The 16S rRNA gene has been widely used for designing taxonomically meaningful, highly specific PCR primers, providing enough sequence information to allow the analysis of both close and distant phylogenic relationships among micro-organisms (Stackebrandt & Goebel, 1994). However, the degree of resolution obtained with 16S rRNA gene sequence analysis is not sufficiently discriminatory to permit resolution of intrageneric relationships among closely related micro-organisms, because of the extremely slow rate of evolution of the 16S rRNA gene (Yamamoto & Harayama, 1998). The genes encoding the 
-subunit polypeptide of DNA gyrase (gyrB) and 
70 factor (rpoD) are estimated to evolve much faster than the 16S rRNA gene (Yamamoto & Harayama, 1998).
In the present study, we analysed the phylogenetic diversity in the gyrB and rpoD gene sequences of these three rice-pathogenic Burkholderia species, in order to develop a specific and sensitive detection method.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Each bacterial isolate was grown aerobically in nutrient broth at 30 °C. Chromosomal DNA from the bacteria used as the PCR template was prepared using an AquaPure Genomic DNA Isolation kit (Bio-Rad), according to the supplier's instructions.
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. PCR was performed with a thermal cycler (TaKaRa) using PCR buffer (TaKaRa) containing 200 µM of each of the dNTPs, 0.5 µM of each primer, 0.2 µg template DNA and 2.5 U Ex-Taq polymerase (TaKaRa), in a total volume of 40 µl. A total of 35 amplification cycles were performed with template DNA denaturation at 94 °C for 1 min, primer annealing at 58 °C for 1 min and primer extension at 72 °C for 1 min. PCR products were electrophoresed on 1.0 % agarose gels and purified using Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction spin columns (Bio-Rad), following the manufacturer's instructions. The nucleotide sequences of gyrB and rpoD were determined directly from the PCR fragments using primers M13R (5'-CAGGAAACAGCTATGACC-3') and M13(–21) (5'-TGTAAAACGACGGCCAGT-3'), and 70Fs (5'-ACGACTGACCCGGTACGCATGTA-3') and 70Rs (5'-ATAGAAATAACCAGACGTAAGTT-3'), respectively. Sequencing was carried out using an ABI Automated DNA Sequencer model 373 (Applied Biosystems) and analysed using DNASIS-Mac software (Hitachi Software Engineering).
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), using genetic distances computed with Kimura's two-parameter model (Kimura, 1980). The neighbour-joining phylogenetic tree was drawn using TreeView. The nucleotide sequences of gyrB and rpoD from Escherichia coli K-12 were used as the outgroup for phylogenetic tree reconstructions.
Analysis of the phylogenetic diversity of B. plantarii strains.
Aliquots (40 µl) of each PCR product from the genomic DNA of B. plantarii strains obtained using primers UP-1E and AprU were ethanol-precipitated and the pellets were dissolved in 10 µl distilled water. The DNA in the solutions was digested with SacI and HaeII (both from TaKaRa) at 37 °C for 6 h, loaded onto horizontal 1.2 % TAE agarose gels and stained with ethidium bromide after electrophoresis for detection of specific DNA fragments corresponding to the gyrB nucleotide sequences of the strains.
Multiplex PCR.
To detect and discriminate B. plantarii, B. glumae and B. gladioli using single-step PCR, primers (Table 2) developed for the gyrB gene of the bacteria were used for development of a multiplex-PCR protocol. PCR was performed with one cycle of 94 °C for 2 min and 35 cycles of 94 °C for 1 min, 63 °C for 1 min and 72 °C for 1 min. Aliquots (9 µl) of each PCR product were loaded onto horizontal 1.5 % TAE agarose gels and stained with ethidium bromide after electrophoresis for detection of 597, 529 and 479 bp DNA fragments corresponding to the gyrB nucleotide sequences of B. plantarii, B. glumae and B. gladioli, respectively.
Analysis of rice seeds infected with B. plantarii, B. glumae and B. gladioli.
To test whether infection of rice seeds with B. plantarii, B. glumae and B. gladioli could be detected using multiplex PCR, rice seeds naturally infected with B. plantarii and B. glumae were obtained from Dr H. Miyagawa, National Agricultural Research Center for Western Region, Japan. Flowering spikelets of rice plants cultivated in pots were inoculated with a suspension of B. plantarii MAFF 302391, MAFF 302907, MAFF 302924, MAFF 311030 or MAFF 301723T, B. glumae MAFF 301169T or B. gladioli MAFF 302386, MAFF 302543, MAFF 302918, MAFF 302919 or T-1 at 1.0x108 c.f.u. ml–1 by spray application at 10 ml per plant, and the resultant rice seeds infected with each strain were used in this study. Non-infected rice seeds were used as a control. One gram of rice seeds was ground in a mortar with 1.0 ml distilled water and the suspension was then placed in a microtube and centrifuged at 1000 g for 1 min. The supernatant was held at 100 °C for 8 min and then centrifuged at 12 000 g for 3 min. Twenty-five microlitres of the supernatant was used as a template for the multiplex PCR.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The combined gyrB and rpoD nucleotide sequences showed high similarity and the sequence similarity between B. glumae MAFF 301169T and B. plantarii MAFF 301723T was 96.2 %, indicating a close phylogenetic relationship between the two species. Furthermore, the B. plantarii, B. glumae and B. gladioli clusters were supported by high bootstrap probabilities, indicating that they each form a tight monophylogenetic branch (Fig. 1).
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). The remaining strain, LMG 16020, is the type strain of B. vandii that is now classified as a strain of B. plantarii (Coenye et al., 1999). Whereas the Vanda strain LMG 16020 occupied a distinct position in the tree, the 23 rice strains were divided into three subgroups (Fig. 1). Nucleotide sequences of the gyrB PCR products from strains in subgroup I showed one restriction site for both HaeII and SacI (data not shown). One restriction site for HaeII was located in the gyrB PCR products from strains in subgroup II. No HaeII and SacI restriction sites were present in the gyrB PCR products from strains in subgroup III. SacI and HaeII digestion of the gyrB PCR products allowed discrimination of strains among subgroups I, II and III (Fig. 2).
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Phylogenetic relationships among B. gladioli strains
B. gladioli R-20202, which was obtained from a specimen from a cystic fibrosis patient in France, occupied a unique position among the 37 B. gladioli strains examined (Fig. 1). All other isolates formed a rather loose assemblage without subdivision supported by high bootstrap values. The combined nucleotide sequences of gyrB and rpoD of two rice strains, MAFF 302544 and MAFF 302914, were identical to those of the mung bean strain MAFF 302409 and the Cymbidium strain MAFF 302424. Furthermore, those of strain T-1 were identical to those of the Tulip strain MAFF 302515. Moreover, the combined translated amino acid sequences of gyrB and rpoD of the rice strains MAFF 302543, E-14 and H-1 were identical not only to those of the corn flour strain GTC 1088, B. gladioli pv. agaricicola GTC 1730, the gladiolus strain ATCC 10248T and soil strains MAFF 302533 and MAFF 302534, but also to the cystic fibrosis patient strains LMG 18157 (USA) and R-15279 (Germany). These results indicate that the pathovar, host plant and geographical origin of the strains correlate poorly with the phylogenetic diversity among the B. gladioli strains.
Detection of B. gladioli, B. glumae and B. plantarii in infected rice seeds by multiplex PCR
A multiplex PCR-based detection method for B. gladioli, B. glumae and B. plantarii was developed using the gyrB nucleotide sequences (Table 2). A 479 bp fragment specific for B. gladioli was amplified from the genomic DNA of all B. gladioli strains used in this study (Fig. 3). From the genomic DNA of all B. glumae strains tested, a 529 bp DNA fragment was specifically amplified in the multiplex PCR. A 597 bp DNA fragment was specifically amplified from the genomic DNA of all B. plantarii strains tested, including LMG 16020 from Vanda sp. No DNA fragments were amplified from the genomic DNA of B. cepacia ATCC 25416T. Sequencing of the PCR products confirmed the specificity of the multiplex PCR (data not shown).
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). One fragment of 529 bp in length was amplified from DNA of rice seeds from plants inoculated with B. glumae MAFF 301169T. A 597 bp DNA fragment was amplified from DNA of rice seeds from plants inoculated with B. plantarii MAFF 302391, MAFF 302907, MAFF 302924, MAFF 311030 and MAFF 301723T. Furthermore, two fragments of 529 and 597 bp in length were amplified from DNA of rice seeds naturally infected with B. glumae and B. plantarii, respectively (Fig. 4b). Sequencing of the PCR products confirmed the specificity obtained by PCR (data not shown). No products were obtained from DNA from non-infected rice seeds (Fig. 4b). These results show that the multiplex-PCR protocol facilitates specific detection and discrimination of rice seeds infected with B. plantarii, B. glumae and B. gladioli.
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) shows that an understanding of the ecology of B. gladioli on rice plants would facilitate not only the development of control systems for B. gladioli-induced disease, but also an understanding of the ecology of B. glumae and B. plantarii on rice plants. In the present study, we have designed a multiplex PCR that can be used for the simultaneous detection of B. plantarii, B. glumae and B. gladioli in rice seeds infected with these Burkholderia species. Therefore, this assay might facilitate an understanding of the ecological significance of Burkholderia species, especially the interactions that control their survival fitness on rice plants, thus leading to the development of relevant control systems.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Azegami, K., Nishiyama, K., Watanabe, Y., Kadota, I., Ohuchi, A. & Fukazawa, C. (1987). Pseudomonas plantarii sp. nov., the causal agent of rice seedling blight. Int J Syst Bacteriol 37, 144–152.
Azegami, K., Nishiyama, K. & Tabei, H. (1988). Infection counts of rice seedlings with Pseudomonas plantarii and Pseudomonas glumae. Ann Phytopathol Soc Jpn 54, 337–341.
Coenye, T., Holmes, B., Kersters, K., Govan, J. R. W. & Vandamme, P. (1999). Burkholderia cocovenenans (van Damme et al. 1960) Gillis et al. 1995 and Burkholderia vandii Urakami et al. 1994 are junior synonyms of Burkholderia gladioli (Severini 1913) Yabuuchi et al. 1993 and Burkholderia plantarii (Azegami et al. 1987) Urakami et al. 1994, respectively. Int J Syst Bacteriol 49, 37–42.
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Hu, F.-P., Young, J. M., Triggs, C. M., Park, D.-C. & Saul, D. J. (2001). Relationships within the Proteobacteria of plant pathogenic Acidovorax species and subspecies, Burkholderia species, and Herbaspirillum rubrisubalbicans by sequence analysis of 16S rDNA, numerical analysis and determinative tests. Antonie van Leeuwenhoek 80, 201–213.[Medline]
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Salles, J. F., De Souza, F. A. & van Elsas, J. D. (2002). Molecular method to assess the diversity of Burkholderia species in environmental samples. Appl Environ Microbiol 68, 1595–1603.
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