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Conjugal plasmid transfer was examined on the phylloplane of bean (Phaseolus vulgaris) and related to the spatial distribution pattern and metabolic activity of the bacteria. The donor (Pseudomonas putida KT2442) harbored a derivative of the TOL plasmid, which conferred kanamycin resistance and had thegfp gene inserted downstream of a lac promoter. A chromosomal insertion of lacI q prevented expression of the gfp gene. The recipient (P. putida KT2440) had a chromosomal tetracycline resistance marker. Thus, transconjugants could be enumerated by plating and visualized in situ as green fluorescent cells. Sterile bean seedlings were inoculated with donors and recipients at densities of approximately 105 cells per cm2. To manipulate the density and metabolic activity (measured by incorporation of [3H]leucine) of the inoculated bacteria, plants were grown at various relative humidities (RH). At 100% RH, the transconjugants reached a density of 3 � 103CFU/cm2, corresponding to about one-third of the recipient population. At 25% RH, numbers of transconjugants were below the detection limit. Immediately after inoculation onto the leaves, the per-cell metabolic activity of the inocula increased by up to eight times (100% RH), followed by a decrease to the initial level after 96 h. The metabolic activity of the bacteria was not rate limiting for conjugation, and no correlation between the two parameters was observed. Apparently, leaf exudates insured that the activity of the bacteria was above a threshold value for transfer to occur. Transconjugants were primarily observed in junctures between epidermal cells and in substomatal cavities. The distribution of the transconjugants was similar to the distribution of indigenous bacteria on nonsterile leaves. Compared to polycarbonate filters, with cell densities equal to the overall density on the leaves, transfer ratios on leaves were up to 30 times higher. Thus, aggregation of the bacteria into microhabitats on the phylloplane had a great stimulatory effect on transfer.

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.The use of antagonistic bacteria for the protection of crops against soilborne pathogens provides a promising environment-friendly alternative to chemical pesticides. However, the root colonization efficiency of introduced biocontrol strains is often limited, potentially reducing the effectiveness of protection (8,25,32). The selection and use of biocontrol strains therefore depend heavily on our knowledge of survival of the inoculant and its potential activity in the rhizosphere ecosystem of a particular plant and soil. Consequently, successful biological control with inoculated strains requires an understanding of the dynamics and composition of the bacterial communities colonizing the rhizosphere.Previous studies, employing cultivation-based, laboratory methods or microscopy, have shown that different bacterial populations are present or active at different stages of root development and that rhizosphere communities are distinct from those found in bulk soil (1,14,17,19,21,27,30). However, recent molecular studies involving PCR amplification of 16S rRNA genes (rDNA), question some of these results. Duineveld et al. (9) reported that the bacterial communities of the Chrysanthemum rhizosphere, as measured by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA PCR products, changed very little with plant age and were similar to those of bulk soil. In contrast, different bacterial communities were identified in soil and in the root tissue of white clover and ryegrass by cluster analysis of a 16S rDNA clone library (16). Furthermore, DGG...

The gfp-tagged Pseudomonas fluorescens biocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3-to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.

Access to the published version may require journal subscription. Published with permission from: Elsevier.Standard set statement from the publisher: NOTICE: this is the author's version of a work that was accepted for publication in Ecological Indicators. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in ECOLOGICAL INDICATORS, 13, 1 (February AbstractIn 2002, world leaders made a commitment through the Convention on Biological Diversity (CBD), to achieve a significant reduction in the rate of biodiversity loss by 2010. At the Conference of the Parties of the CBD in Nagoya, Japan in 2010, the target was renewed for 2020. We have developed a Biodiversity Change Index (BCI) to help measure progress towards this target. The BCI is constructed with a two-dimensional resolution, allowing for a direct evaluation of the relative importance of changes in quantity and quality, respectively, to the overall change in biodiversity. Quantity is measured as the area of a specified habitat type and quality as the abundance of indicator species and other habitat quality parameters, such as the proportion of old trees or dead wood in forests. The BCI enables easy comparison of changes in biodiversity between different habitat types and between different regions and nations. We illustrate the use of BCI by calculating the index for the Nordic countries for two common habitat types, farmland and forest, and one habitat type of similar importance in the northern hemisphere; mires. In the period 1990-2005 declines in biodiversity of similar magnitudes are seen for farmland and mires across the Nordic countries, while for forest, trends vary considerably. Our results show that the BCI framework can be a useful tool to communicate the complex issue of biodiversity change in a simple manner. However, in accordance with other studies of biodiversity change we conclude that existing monitoring data are too scarce to consistently calculate BCI for all habitat types in all Nordic countries. In order to reasonably evaluate changes in biodiversity, further efforts towards monitoring programmes to obtain reliable and quality assured data on biodiversity at acceptable spatial and temporal resolutions are needed. Moreover, common methods to apply and harmonise data from different monitoring schemes should be developed.

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A single-cell approach for studying the growth potential and the establishment of bacteria in the barley phytosphere is presented, using Pseudomonas fluorescens strain with the capability for biological control. The incidence of growth of one to four bacterial cells dispersed to the young rhizosphere approximated to 100%, and specific growth rate averaged 0.05. Net growth occurred for cells added to the rhizosphere at densities between 1 and 100,000 cells, while at higher densities population sizes declined, but always approached 10(5)-10(6) cells per rhizosphere. No net growth was observed in bulk soil, and cells died in the phyllosphere. Our results showed that bacterial establishment was more related to the availability of microhabitats supporting growth, than related to the number of bacteria released.