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Clinical and nonclinical isolates of the lactose-positive Vibrio vulnificus were compared with Vibrio strains isolated from lesions on eels (Anguilla japonica) cultured commercially in Japan. Strains were compared phenotypically and antigenically, for pathogenicity to mice and eels, and for genetic relatedness. The strains isolated from diseased eels differed phenotypically from the original species description of V. vulnificus in that they were negative for indole production, ornithine decarboxylase activity, growth at 42°C, and acid production from mannitol and sorbitol. No relationship between the surface antigens of V. vulnificus strains from environmental and clinical sources and the strains from diseased eels was observed. Typical V. vulnificus strains and the eel isolates were pathogenic to mice; however, only those strains originally isolated from diseased eels were found to be pathogenic to eels. Results of DNA-DNA competition experiments revealed that there was greater than 90% relative reassociation between clinical and nonclinical V. vulnificus and strains from diseased eels. Based on the results of the DNA-DNA competition experiments, we conclude that the strains isolated from diseased eels were V. vulnificus; however, the differences in phenotypic characteristics and eel pathogenicity indicated that these strains represent a different biogroup. Therefore, we propose that strains phenotypically similar to the type strain of the species (ATCC 27562) be classified as V. vulnificus biogroup 1 and the strains phenotypically similar to those isolated from diseased eels be classified as V. vulnificus biogroup 2 represented by the reference strain ATCC 33148.

Transgenic plants that produce pesticidal proteins will release these proteins into the soil when these plants are incorporated into the soil by tillage or as leaf litter. Little is known about the fate and persistence of transgenic plant pesticidal products in the soil. We used a model system of transgenic cotton that produces Bacillus thuringlensis var. kurstaki δ-endotoxin (Btk toxin) to evaluate the persistence of transgenic pesticides in soil. Purified Btk toxin or transgenic cotton leaves containing Btk toxin were added to soil in five different microcosm experiments in concentrations ranging from 1 to 1600 ng Btk toxin/g soil. The concentration of the extractable Btk toxin was measured for up to 140 days. An initial rapid decline in extractable toxin concentration in the first 14 days, followed by a slower decline, was observed in four of the five experiments. At the end of the experiments, Btk toxin from transgenic plant tissue was undetectable (less than 0.1% of starting concentration) in two of the microcosm experiments and at 3, 16, and 35% of the original amounts in the other experiments. In addition, experiments using γ-irradiated sterilized soil indicated that the observed decline in extractable toxin concentration was due largely to biotic degradation rather than to physical adsorption by the soil.Key words: transgenic plants, Bacillus thuringlensis toxin, risk assessment.

Genetic engineering offers the opportunity to generate plants with useful new traits conferred by genes originating from a variety of organisms. The objectives of this study were to establish methods for investigating persistence of recombinant plant marker DNA after introduction into soil and to collect data from controlled laboratory test systems. As a model system, we studied the stability of DNA encoding recombinant neomycin phosphotransferase II (rNPT‐II), a neomycin/kanamycin resistance marker, used in plant genetic engineering. The recombinant nature of the target (i.e. fusion of nopaline synthase promoter and NPT‐II coding region) allowed us to design a rNPT‐II‐specific PCR primer pair. DNA preparation and quantitative PCR protocols were established. Effects of temperature and moisture, on DNA persistence in soil were determined in two laboratory test systems. In the first system, purified plasmid DNA was added to soil and incubated under controlled conditions. Up to 0.08% of the rNPT‐II target sequences were detectable after 40 days. In the second system, fresh leaf tissue of transgenic tobacco was ground, added to soil, and incubated under controlled conditions. After 120 days, up to 0.14% of leaf tissue‐derived genomic rNPT‐II sequences were detectable. Under most experimental conditions, leaf tissue‐derived and plasmid DNA were initially degraded at a high rate. A small proportion of the added DNA resisted degradation and was detectable for several months. We hypothesize that this DNA may have been adsorbed to soil particles and was protected from complete degradation.

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection ofPseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection ofPseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas16S rRNA 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 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target 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, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool forPseudomonas population structure analyses and taxonomic confirmations.