1997 Volume 63 Issue 6 Pages 455-462
Specific polymerase chain reaction (PCR) primers targeting genomic DNA were selected for rapid, sensitive and specific detection of Burkholderia plantarii and B. glumae, the causal agents of bacterial seedling blight of rice and bacterial seedling rot of rice, respectively. The complete sequences of the spacer region between the 16S and 23S rRNA genes of B. plantarii, B. glumae, B. gladioli, B. cepacia, B. caryophylli, B. andropogonis, B. solanacearum and Pseudomonas corrugata, were determined. Among strains of B. plantarii from diverse geographical regions in Japan, the degree of sequence similarity was more than 93%. All strains of B. glumae isolated in diverse geographical regions in Japan had the same sequence. The degree of similarity among the strains of Burkhorderia spp. ranged from 60% to 90%, but less than 59% between strains of Burkholderia spp. and strains of P. corrugata, P. fluorescens and Escherichia coli. These results suggest that the sequences are conserved within species, but are variable between species. Since strains of B. plantarii, B. glumae and B. gladioli exhibited a relatively high degree of sequence similarity (81-90%) to each other, we designed species-specific primers from the sequence of the regions that were conserved within species, but not between species. In PCR with PL-12f (5'-AGCCAGTCAGAGGATAAGTC-3') and PL-11r (5'-CAATTGAGCCGAACATTTAAG-3') primers, an approximately 180-bp fragment was amplified in all 45 strains of B. plantarii. No PCR products were obtained from other bacteria tested. Primers GL-13f (5'-ACACGGAACACCTGGGTA-3') and GL-14r (5'-TCGCTCTCCCGAAGAGAT-3') amplified an approximately 400-bp fragment in all 20 isolates of B. glumae, whereas no PCR products were obtained from other species of bacteria. Using the specific primers designed in this study, the PCR method can detect and identify B. glumae and B. plantarii in rice samples within 6 hr.