Ian P. Thompson

Site directed chromosomal marking of a fluorescent pseudomonad isolated from the phytosphere of sugar beet; stability and potential for marker gene transfer.

A plasmid‐free, non‐pathogenic, ribosomal RNA group 1 fluorescent pseudomonad, Pseudomonas fluorescens SBW25, was selected from the microflora of sugar beet (Beta vulgaris) and modified to contain constitutively expressed marker genes. By site directed homologous recombination a KX cassette [kanamycin resistance (kanτ) and catechol 2,3 dioxygenase (xylE)] and a ZY cassette [lactose utilization (lacZY, β‐galactosidase, lactose permease)] were introduced at least 1 Mbp apart on the 6.6 Mbp bacterial chromosome. Separate sites were selected to provide sensitive detection methods and allow assessments of marker gene stability of the genetically modified micro‐organism (GMM), SBW25EeZY6KX, when it colonized the leaves and roots of sugar beet plants following seed inoculation.

Response of soil microbial communities to single and multiple doses of an organic pollutant

The effect of 100 μg 1,2-dichlorobenzene (1,2-DCB) g−1 dry weight (dw) of soil introduced either as a single dose or multiple (10 fortnightly) doses of 10 μg g−1 dw, on the microbial biomass, diversity of culturable bacterial community and the rate of 1,2-DCB mineralisation, were compared. After 22 weeks exposure both application regimes significantly reduced total bacterial counts and viable fungal hyphal length. The single dose had the greatest overall inhibitory effect, although the extent of inhibition varied throughout the study. Total culturable bacterial counts, determined after 22 weeks exposure showed little response to 1,2-DCB, but pseudomonad counts in single and multiple treatments were reduced to 9.7 and 0.147%, respectively, of the numbers detected in the control soil. The effect of 1,2-DCB application on the taxonomic composition of the culturable bacteria community was determined by fatty acid methyl ester (FAME) analysis. Compared to control soils, the single dose treatment had a lower percentage of Arthrobacter and Micrococcus. Multiple applications had a significant effect upon pseudomonad abundance, which represented only 2% of the identified community, compared to 45.6% in the control. The multi-dosed soils contained a high percentage of bacilli (>25%). The effects of 1,2-DCB applications on the metabolic potential of the soil microbial community was determined by BIOLOG profiling. The number of carbon compounds utilised by the community in the multi-dosed soils (49 positives) was significantly less (P<0.05) than detected in the single dose treatment (76) and control (66). The rate of 1,2-DCB mineralisation, determined by 14CO2 production from radiolabelled [UL–14C] 1,2-DCB, declined throughout the study, and after 22 weeks was slightly but significantly (P<0.05) lower in the multiply- than the singly-dosed soils. The differential response to 1,2-DCB treatments was attributed to its reduced bioavailability in soils after a single exposure, compared to multiple applications.

Monitoring temporal and spatial variation in rhizosphere bacterial population diversity: A community approach for the improved selection of rhizosphere competent bacteria

The potential for developing a reliable strategy for selecting rhizosphere competent bacteria, based on an improved understanding of the community diversity and population dynamics of fluorescent pseudomonads, was investigated. Isolates from a collection of over 690 fluorescent pseudomonads, obtained from sugar beet and wheat plants grown in field soils in laboratory microcosms, were genotypically and phenotypically characterised. RFLP rRNA analysis (ribotyping) revealed that the sampled population was composed of 385 related but distinct ribotypes. Most ribotypes were isolated only once and represented a transient colonising population. However, representatives of 26 ribotypes were detected more often, of which five were isolated from rhizosphere soils sampled 7 months after the first sampling. Comparative phenotypic analysis of isolates (motility, antibiotic resistance and production, adherence, fatty acid composition, substrate utilisation patterns) demonstrated that the ability to utilise organic acids as carbon sources correlated with rhizosphere competence. Single inoculum and competitive colonisation studies in planted microcosms confirmed rhizosphere competence, but also demonstrated synergistic interactions. The colonisation ability and population densities of transient strains were significantly increased when co-inoculated with rhizosphere competent isolates. These data demonstrate potential cross-feeding and combined niche exploitation, rather than direct competition, confirming the multi-factorial nature of rhizosphere competence in diverse fluorescent pseudomonad communities. They also highlight the need to consider the use of mixed inocula for plant growth promotion and the systematic selection of strains for effective biotechnological exploitation.

Assessment of the Potential for Gene Transfer in the Phytosphere of Sugar Beet

A comprehensive understanding of the microbial ecology of the phytosphere of sugar beet is required to make valid assessments concerning the fate of genetically modified microorganisms (GMOs) and their DNA in this habitat. Descriptions of resident populations are considered necessary as indigenous communities are potential mediators of in situ gene transfer events to and from introduced GMOs. In order to model impact analysis a survey, over an entire growing season, of the bacteria, yeast and filamentous fungi present on the aerial surface of field grown sugar beet (Beta vulgaris var. amethyst, Germians seeds, U.K), was undertaken. Bacteria were isolated on Tryptic Soy Agar (Difco) and identified to the species level by gas chromatography (Hewlett Packard 5096A) of component cellular fatty acid methyl esters using a commercially available database (MIDI-MIS, Delaware, USA). Figures 1 and 2 show the temporal and spatial distribution of the natural background bacterial populations. Members of the Pseudomonadaceae and Enterobacteriaceae were the most abundant colonizers isolated and within these groups, subtle fluctuations in the distribution of component species were recorded.