A HIGHLY SELECTIVE PCR PROTOCOL FOR DETECTING 16S RIBOSOMAL-RNA GENESOF THE GENUS PSEUDOMONAS (SENSU-STRICTO) IN ENVIRONMENTAL-SAMPLES

Pseudomonas species are plant, animal, and human pathogens; exhibit pl ant pathogen-suppressing properties useful in biological control; or e xpress metabolic versatilities valued in biotechnology and bioremediat ion, Specific detection of Pseudomonas species in the environment may help us gain a more complete understanding of the ecological significa nce of these microorganisms. The objective of this study was to develo p a PCR protocol for selective detection of Pseudomonas (sensu stricto ) in environmental samples. Extensive database searches identified a h ighly selective PCR primer pair for amplification of Pseudomonas 16S r RNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 t arget and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences fro m purified bulk soil DNA followed by cloning of PCR products, Restrict ion analysis with HaeIII revealed eight different fragmentation patter ns among 36 clones. Sequencing and phylogenetic analysis of 8 represen tative clones indicated that 91.7% of the products were derived from t arget organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clust ered with sequences of confirmed Pseudomonas species, whereas two patt erns, representing 30.6% of the clones, formed a novel phylogenetic cl uster closely associated with Pseudomonas species. The results indicat ed that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool for Pseudomonas population structure an alyses and taxonomic confirmations.