No abstract

Vertical soil microcosms flushed with groundwater were used to study the influence of water movement on survival and transport of a genetically engineered Pseudomonas fluorescens C5t strain through a loamy sand and a loam soil. Transport of cells introduced into the top 1 cm of the vertical soil microcosms was dependent on the flow rate of water and the number of times microcosms were flushed with groundwater. The presence of wheat roots growing downward in the microcosms contributed only slightly to the movement of P. fluorescens C5t cells to lower soil regions of the loamy sand microcosms, but enhanced downward transport in the loam microcosms. Furthermore, the introduced P. fluorescens C5t cells were detected in the effluent water samples even after three flushes of groundwater and 10 days of incubation. As evidenced by a comparison of counts from immunofluorescence and selective plating, nonculturable C5t cells occurred in day 10 soil and percolated water samples, primarily of the loamy sand microcosms. Vertical soil microcosms that use water movement may be useful in studying the survival and transport of genetically engineered bacteria in soil under a variety of conditions prior to field testing.

Survival of Pseudomonas fluorescens R2f containing either the conjugative plasmid RP4 or the nonconjugative plasmid pRK2501, and stability of the plasmids were studied in two soils, Ede loamy sand and Guelph loam, and in extracts prepared from these soils. In sterile soils, the introduced bacterial populations initially increased and then remained stable over a 47-day period. The presence of wheat roots did not significantly influence bacterial numbers in Guelph loam, whereas a slight increase occurred in Ede loamy sand. In Guelph loam, both plasmids were stably maintained in the introduced populations, but in Ede loamy sand substantial plasmid loss was observed. The presence of added phosphate in Ede loamy sand enhanced plasmid maintenance in the introduced R2f population. In nonsterile Guelph loam, a slow decline in the introduced populations was noted, regardless of plasmid type, whereas in Ede loamy sand the decline was more rapid. There was no detectable effect of plasmid type on host survival. Both plasmids RP4 and pRK2501 remained present in the R2f populations in these soils. The results obtained with sterile soil extracts substantiated the data on plasmid loss in both soils; both plasmids were rather unstable during starvation in minimal medium. The results indicated the absence of an effect of plasmid type on host survival. Soil type significantly affected host survival and plasmid maintenance, and higher survival and stability were observed in the heavier-textured Guelph loam.Key words: survival, plasmid stability, Pseudomonas spp., soil, microcosms.

The effect of complexation and pH on heavy metal (Cu, Zn, Pb) toxicity to a freshwater green alga, Scenedesmus quadricauda, was investigated. Extracellular ligands produced by S. quadricauda were capable of binding heavy metals and reducing their single and combined toxicities. Apparent complexing capacities and the ability of the sediment humics and artificial complexing agents such as ethylenediaminetetraacetic acid (EDTA), citric acid, and glycolic acid to ameliorate Cu, Zn, or Pb toxicity were also assessed. The toxicity of metals to algal growth was enhanced at acidic pH. Combined toxicity of these metals was significantly greater at pH 4.5 than at pH 8.5 or pH 6.5. Synergistic effects (between Cu, Zn, and Pb) towards algal growth increased at low pH. Specific heavy metals, their respective concentrations, the presence of complexing ligands, and pH influence both individual and combined heavy metal toxicities.

One year after introduction into agricultural drainage waterPseudomonas fluorescens R2f (RP4),Pseudomonas putida CYM318 (pRK2501), andKlebsiella aerogenes NCTC418 (pBR322) could be recovered on agar media. Survival of the introduced strains depended on competition with the indigenous microflora, the presence of nutrients, and the availability of air.In contrast toK. aerogenes NCTC418 (pBR322), bothPseudomonas species lost their plasmids, as indicated by the consistently lower colony counts on selective medium compared with the counts on nonselective medium. The plasmid loss did not depend on nutrient status and oxygen supply. P. fluorescens R2f cells could be detected with the immunofluorescence (IF) technique. Total cell counts determined by IF were consistently higher than corresponding colony counts. Even in samples where no colonies were recovered, R2f cells could be detected by IF. This indicated the occurrence of nonculturable R2f cells in drainage water. Homology with(32)P-labelled plasmid RP4 DNA was found in several drainage water samples that originally receivedP. fluorescens R2f (RP4), by using the cell suspension filter hybridization technique. P. putida CYM318 andK. aerogenes NCTC418 cells could also be detected in sterile drainage water samples, after nonspecific staining with fluorescein isothiocyanate. Cell counts of both strains were consistently higher than corresponding plate counts.