Predicting survival of a genetically engineered microorganism,Pseudomonas chlororaphis 3732RN-L11, in soil and wheat rhizosphere across Canada with linear multiple regression models

Pseudomonas chlororaphis 3732RN-L11 survival rates in soil and wheat rhizosphere were measured using intact soil core microcosms representing 23 sites across Canada. Linear multiple regression (LMR) models were developed to predict the survival rate of this genetically engineered microorganism (GEM) as a function of soil parameters measured at the time of microcosm inoculation. LMR models were tested by comparing their predicted survival rates with observed survival rates from environmental introductions of the GEM by Gagliardi et al. (2001) at five field sites across Canada over two years. No soil parameter (e.g., % clay) was highly correlated with GEM survival rates in soil or wheat rhizosphere. Total fungal colony-forming units (CFUs), % soil titanium (positive correlations), and % soil magnesium (negative correlation) were found to be the best LMR predictors of GEM survival rates in soil over two years. Total soil bacterial CFUs, nitrate, % soil potassium (positive correlations), and exchangeable magnesium (negative correlation) were found to be the best LMR predictors of GEM survival rate in wheat rhizosphere over two years. While LMR models were statistically significant, they were unable to reliably predict the survival rate of the GEM in field trial introductions. The results indicate that there can be considerable uncertainty associated with predicting GEM survival for multi-site environmental introductions.     

An intact soil-core microcosm method to evaluate the survival and vertical dispersal of Trichoderma atroviride SC1

Aim:  To develop an intact soil-core microcosm method to study the survival and vertical dispersal of an experimental biocontrol agent ( Trichoderma atroviride SC1) applied to the soil surface.
Methods and Results:  The soil for the microcosms was collected using iron pipes with perforations corresponding to different soil layers. The tool was inserted into the soil and gently removed with the soil core inside. Trichoderma atroviride SC1 was mixed with the top layer of soil in the pipe. The experiment was performed in 2006 and 2007, and data from the microcosms were compared with results obtained under field conditions in the locations in which, the microcosms were collected, in the same periods. The concentrations of T. atroviride SC1 in the soil were estimated immediately after treatment, and 1, 5, 9 and 18 weeks after treatment at both the soil surface and the above-mentioned depths. The development of T. atroviride SC1 populations in the microcosms during the 18 weeks of monitoring was similar to that observed under field conditions. The dispersal of conidia was affected by the application of water to the soil.
Conclusions:  Results demonstrate that this microcosm prototype can be used to model the behaviour of T. atroviride SC1 in soil.
Significance and Impact of the Study:  The intact soil-core microcosm is a reliable, easy-to-use, fast and cheap method that could also be used in studies of similar filamentous fungi to study their probable fate in the soil prior to their being introduced into the environment.     

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.     

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere.

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.     

Selection of rhizosphere-competent Pseudomonas strains as biocontrol agents in tropical soils

Pseudomonas strains were isolated from the rhizosphere of maize grown in yellow-red latosol from Rio de Janeiro, Brazil, to serve as a delivery system for heterologous genes and for risk assessment studies in tropical soils. Selected strains were modified by insertion of the cryIVB gene from Bacillus thuringiensis and tested for pathogenicity gene expression against larvae of a susceptible model species, Anopheles aquasalis. Modified strains Br8 and Br12 showed similar survival performance to their parental strains, and presented a viable density of 10⁷ c.f.u./g dry soil 30 days after release. A strain of P. fluorescens (Br12) that presented positive results for gene expression and the best survival performance, was selected for risk assessment studies in soil microcosms.     

Spatial variation of active microbiota in the rice rhizosphere revealed by in situ stable isotope probing of phospholipid fatty acids

This report is part of a serial study applying stable isotope labelling to rice microcosms to track the utilization of recently photosynthesized carbon by active microbiota in the rhizosphere. The objective of the present study was to apply phospholipid fatty acid-based stable isotope probing (PLFA-SIP) to detect the spatial variation of active microorganisms associated with rhizosphere carbon flow. In total, 49 pulses of 13CO2 were applied to rice plants in a microcosm over a period of 7 days. Rhizosphere soil was separated from bulk soil by a root bag. Soil samples were taken from rhizosphere and bulk soil, and the bulk soil samples were further partitioned both vertically (up layer and down layer) and horizontally with increasing distance to the root bag. Incorporation of 13C into PLFAs sharply decreased with distance to the roots. The labelling of 16:1omega9, 18:1omega7, 18:1omega9, 18:2omega6,9 and i14:0 PLFAs was relatively stronger in the rhizosphere while that of i15:0 and i17:0 increased in the bulk soil. The microorganisms associated with 16:1omega9 were active in both up- and down-layer soils. The microorganisms represented by i14:0, 18:1omega7 and 18:2omega6,9 exhibited a relatively higher activity in up-layer soil, whereas those represented by i15:0 and i17:0 were more active in down-layer soil. These results suggest that in the rhizosphere Gram-negative and eukaryotic microorganisms were most actively assimilating root-derived C, whereas Gram-positive microorganisms became relatively more important in the bulk soil. The active populations apparently differed between up- and down-layer soil and in particular changed with distance to the roots, demonstrating systematic changes in the activity of the soil microbiota surrounding roots.     

Impact of flooding on soil bacterial communities associated with poplar (Populus sp.) trees

Soil bacterial communities were analyzed in different habitats (bulk soil, rhizosphere, rhizoplane) of poplar tree microcosms (Populus tremulaxP. alba) using cultivation-independent methods. The roots of poplar trees regularly experience flooded and anoxic conditions. Therefore, we also determined the effect of flooding on microbial communities in microcosm experiments. Total community DNA was extracted and bacterial 16S rRNA genes were amplified by PCR and analyzed by terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and sequencing. Clone libraries were created from all three habitats under both unflooded and flooded conditions resulting in a total of 281 sequences. Numbers of different sequences (<97% similarity) in the different habitats represented 16-55% of total bacterial species richness determined from the nonparametric richness estimator Chao1. According to the number of different terminal restriction fragments (T-RFs), all of the different habitats contained approximately 20 different operational taxonomic units (OTUs), except the flooded rhizoplane habitat whose community contained less OTUs. Results of cloning and T-RFLP analysis generally supported each other. Correspondence analysis of T-RFLP patterns showed that the bacterial communities were different in bulk soil, rhizosphere and rhizoplane and changed upon flooding. For example OTUs representing Bacillus sp. were highest in the unflooded bulk soil and rhizosphere. Sequences related to Aquaspirillum, in contrast, were predominant on the poplar roots and in the rhizosphere of flooded microcosms but were rarely found in the other habitats.     

Validation of microbial community structure and ecological functional parameters in an aquatic microcosm designed for testing genetically engineered microorganisms

Microcosms were designed to facilitate studies of the fate, functioning, and ecological effects of microorganisms released into the aquatic environment. The microcosms were three-phase systems (sediment/water/air) with three compartments (a primary producer component, a herbivore grazer component, and intact sediment cores). The microcosms were validated by comparing gross ecological parameters and microbial community structure between the microcosms and the eutrophic Lake Bagsværd, which was simulated in the model. The photosynthetic potential and chlorophyll a concentrations were significantly lower in the microcosms than in the lake, which apparently was due to inorganic nutrient limitation. In the microcosms, total bacterial numbers and metabolic activity by [³H]thymidine incorporation were unaffected by the reduced algal biomass and primary production, simulating field conditions closely, with a strong dependence on temperature. Two days after filling the microcosms, the percentage of similarity of the microbial communities in the microcosm and Lake Bagsværd was 40%, measured by hybridizations of total microbial DNA. The similarity increased during the 10-day experimental period to 63–76%. In two experiments, Alcaligenes eutrophus AEO106(pRO101) was released into the microcosms. The release reduced the similarity between microcosms and lake to 2% and 27%, depending on the number of introduced cells. Concomitant to a decline in the A. eutrophus AEO106(pRO101) population, the similarity gradually recovered. It is concluded that the microcosms can simulate a freshwater lake ecosystem, but care has to be taken when extrapolating microcosm results to the source ecosystem because of the possible different selective conditions in the microcosm.     

Intact soil-core microcosms compared with multi-site field releases for pre-release testing of microbes in diverse soils and climates

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.     

A microcosm for measuring survival and conjugation of genetically engineered bacteria in rhizosphere environments

A microcosm is described to evaluate and measure bacterial conjugation in the rhizosphere of barley and radish with strains ofPseudomonas cepacia. The purpose was to describe a standard method useful for evaluating the propensity of genetically engineered microorganisms (GEMs) to transfer DNA to recipient bacteria. Results demonstrated the formation of transconjugants from the rhizosphere of each plant 24 h after inoculation. Transconjugant populations peaked at 1.8 × 10² colony forming units (CFU)/g root and associated soil in barley and 2.0×10² CFU/g root and associated soil in radish; they then declined over the next five days of the experiment. No significant differences were found in the survival of transconjugant populations monitored from the two plant species. The microcosm was also used to document the formation of false positive transconjugants, which resulted from donor and recipientP. cepacia mating on the surface of selective agar plates instead of in microcosms. Transconjugants resulting from such plate mating occurred in substantial numbers during the first 5 days of the experiment but declined to undetectable numbers by day 7. The use of nalidixic acid was investigated to determine the magnitude of plate mating. The number of transconjugants detected from radish rhizosphere was reduced by two orders of magnitude by including nalidixic acid in the plating medium; this indicated that 99% of the transconjugants were a result of plate mating.     

Field calibration of soil-core microcosms: Ecosystem structural and functional comparisons

Microcosms containing intact soil-cores are a potential biotechnology risk assessment tool for assessing the ecological effects of genetically engineered microorganisms before they are released to the field; however, microcosms must first be calibrated to ensure that they adequately simulate key field parameters. Soil-core microcosms were compared with the field in terms of ecological response to the introduction of a large inoculum of a rifampicin-resistant rhizobacterium,Pseudomonas sp. RC1. RC1 was inoculated into intact soil-core microcosms incubated in the laboratory at ambient temperature (22°C) and in a growth chamber with temperature fluctuations that mimicked a verage field values, as well as into field lysimeters and plots. The effect of the introduced bacterium on ecosystem structure, including wheat rhizoplane populations of total and fluorescent pseudomonads, total heterotrophic bacteria, and the diversity of total heterotrophic bacteria, was determined. Fluorescent pseudomonads were present on the rhizoplane in significantly lower numbers in soil inoculated with RC1, in both microcosms and the field. Conditions for microbial growth appeared to be most favorable in the growth chamber microcosm, as evidenced by higher populations of heterotrophs and a greater species diversity on the rhizoplane at the three-leaf stage of wheat growth. Ecosystem functional parameters, as determined by soil dehydrogenase activity, plant biomass production, and¹⁵N-fertilizer uptake by wheat, were different in the four systems. The stimulation of soil dehydrogenase activity by the addition of alfalfa was greater in the microcosms than in the field. In general, growth chamber microcosms, which simulated average field temperatures, were better predictors of field behavior than microcosms incubated continuously at 22°C.     

The fate of a genetically modified Pseudomonas strain and its transgene during the composting of poultry manure

The fate of the genetically modified (GM) Pseudomonas chlororaphis strain 3732 RN-L11 and its transgene (lacZ insert) during composting of chicken manure was studied using plate count and nested polymerase chain reaction (PCR) methods. The detection sensitivity of the nested PCR method was 165 copies of the modified gene per gram of moist compost or soil. Compost microcosms consisted of a 100-g mixture of chicken manure and peat, whereas soil microcosms were 100-g samples of sandy clay loam. Each microcosm was inoculated with 4 x 1010 CFU of P. chlororaphis RN-L11. In controlled temperature studies, neither P. chlororaphis RN-L11 nor its transgene could be detected in compost microcosms after incubation temperature was elevated to 45 degrees C or above for one or more days. In contrast, in the compost microcosms incubated at 23 degrees C, the target organism was not detected by the plate count method after 6 days, but its transgene was detectable for at least 45 days. In compost bins, the target organism was not recovered from compost microcosms or soil microcosms at different levels in the bins for 29 days. However, the transgene was detected in 8 of the 9 soil microcosms and in only 1 of the 9 compost microcosms. The compost microcosm in which transgene was detected was at the lower level of the bin where temperatures remained below 45 degrees C. The findings indicated that composting of organic wastes could be used to reduce or degrade heat sensitive GM microorganisms and their transgenes.     

Survival of Escherichia coli O157:H7 in soil and on lettuce after soil fumigation

Long-term survival of Escherichia coli O157:H7 in soil and in the rhizosphere of many crops after fumigation is relatively unknown. One of the critical concerns with food safety is the transfer of pathogens from contaminated soil to the edible portion of the plants. Multiplex fluorogenic polymerase chain reaction was used in conjunction with plate counts to quantify the survival of E. coli O157:H7 in soil after fumigation with methyl bromide and methyl iodide in growth chamber and microcosm laboratory experiments. Plants were grown at 20 degrees C in growth chambers during the first experiment and soils were irrigated with water contaminated with E. coli O157:H7. For the second experiment, soil microcosms were used in the laboratory without plants and were inoculated with E. coli O157:H7 and spiked with the two fumigants. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7. Both fumigants were effective in reducing pathogen concentrations in soil, and when fumigated soils were compared with nonfumigated soils, pathogen concentrations were significantly higher in the nonfumigated soils throughout the study. This resulted in a longer survival of the pathogen on the leaf surface especially in sandy soil than observed in fumigated soils. Therefore, application of fumigant may play some roles in reducing the transfer of E. coli O157:H7 from soil to leaf. Regression models showed that survival of the pathogen in the growth chamber study followed a linear model while that of the microcosm followed a curvilinear model, suggesting long-term survival of the pathogen in soil. Both experiments showed that E. coli O157:H7 can survive in the environment for a long period of time, even under harsh conditions, and the pathogen can survive in soil for more than 90 days. This provides a very significant pathway for pathogen recontamination in the environment.     

Survival of native <Emphasis Type="Italic">Pseudomonas</Emphasis> in soil and wheat rhizosphere and antagonist activity against plant pathogenic fungi

Survival of Pseudomonas sp. SF4c and Pseudomonas sp. SF10b (two plant-growth-promoting bacteria isolated from wheat rhizosphere) was investigated in microcosms. Spontaneous rifampicin-resistant mutants derived from these strains (showing both growth rate and viability comparable to the wild-strains) were used to monitor the strains in bulk soil and wheat rhizosphere. Studies were carried out for 60 days in pots containing non-sterile fertilized or non-fertilized soil. The number of viable cells of both mutant strains declined during the first days but then became established in the wheat rhizosphere at an appropriate cell density in both kinds of soil. Survival of the strains was better in the rhizosphere than in the bulk soil. Finally, the antagonism of Pseudomonas spp. against phytopatogenic fungi was evaluated in vitro. Both strains inhibited the mycelial growth (or the resistance structures) of some of the phytopathogenic fungi tested, though variation in this antagonism was observed in different media. This inhibition could be due to the production of extracellular enzymes, hydrogen cyanide or siderophores, signifying that these microorganisms might be applied in agriculture to minimize the utilization of chemical pesticides and fertilizers.     

Fate of the biological control agent Pseudomonas aureofaciens TX-1 after application to turfgrass

The fate and impact of Pseudomonas aureofaciens TX-1 following application as a biocontrol agent for fungi in turfgrass were studied. The organism was applied with a modified irrigation system by using a preparation containing 1 x 10(6) P. aureofaciens TX-1 CFU ml(-1) about 100 times between May and August. We examined the impact of this repeated introduction of P. aureofaciens TX-1 (which is known to produce the antimicrobial compound phenazine-1-carboxylic acid) on the indigenous microbial community of the turfgrass system and on establishment of introduced bacteria in the soil system. A PCR primer-DNA hybridization probe combination was developed to accurately monitor the fate of P. aureofaciens TX-1 following application in irrigation water. To assess the impact of frequent P. aureofaciens TX-1 applications on the indigenous bacterial community, turfgrass canopy, thatch, and rhizosphere samples were obtained during the growing season from control and treated plots and subjected to DNA extraction procedures and denaturing gradient gel electrophoresis (DGGE). PCR amplification and hybridization of extracted DNA with the P. aureofaciens TX-1-specific primer-probe combination revealed that P. aureofaciens TX-1 not only became established in the rhizosphere and thatch but also was capable of overwintering. Separation of PCR-amplified partial 16S rRNA genes by DGGE showed that the repeated application of P. aureofaciens TX-1 in irrigation water resulted in transient displacement of a leaf surface bacterial community member. There was no obvious alteration of any dominant members of the thatch and rhizosphere microbial communities.     

Pelagic Diatom Populations in Lentic Freshwater Microcosms

Three experiments, involving simultaneous monitoring of selected biological and chemical parameters in 50 l laboratory microcosms and the epilimnia of their parent reservoirs, were carried out from the autumn of 1978 to the winter of 1980. Experiments lasted 8-13 weeks, their aim being to ascertain degree of similarity between laboratory and field systems. Microcosm dynamics, specifically diatom dynamics, most closely paralleled that found in reservoirs during late spring and early summer, a time of thermal stratification. During winter months when thermal stratification was absent or less pronounced, microcosm diatom populations diverged significantly from reservoir populations within 24 days. It is inferred that microcosm design and operating conditions have a major bearing on microcosm usefulness for environmental assessment.     

Effects of three soil-applied herbicides on populations of plant disease suppressing bacteria in the cotton rhizosphere

The potential impact of three widely used herbicides, pendimethalin, prometryn, and trifluralin, on populations of five plant disease suppressing bacterial isolates (three isolates of Pseudomonas fluorescens and two isolates of Burkholderia cepacia) in the rhizosphere of cotton seedlings was investigated. All isolates are efficient cotton root colonizers and each is capable of suppressing a plant disease. In microcosm experiments, application of each of the test herbicides at the rates of 1, 2, and 4 µg active ingredient (a.i.) g^-1 soil caused significant (p<0.05) reductions in populations of most of the isolates in the rhizosphere, 14 days after the release of bacteria into the soil by seed coating. The responses of the isolates to the herbicides varied depending on the isolate and the type and concentration of the herbicides. In microcosm experiments the impact of pendimethalin, prometryn, and trifluralin at the respective concentrations of 2.4, 3.6, and 1.8 g a.i. g^-1 soil on the population of isolate D1 in the cotton rhizosphere declined with time during a four week period of monitoring following the release of the isolate into the soil by seed coating. The impact of soil applied test herbicides on the population sizes of D1 in cotton rhizosphere was also studied in two field experiments (Safford and Tucson, Arizona) where the bacteria were added as a soil drench. In the Safford experiment pendimethalin and prometryn, but not trifluralin, caused significant (p <0.05) reductions in the population of the bacterium 15 days after sowing. In the Tucson experiment a significant (p < 0.05) reduction in the population of the bacterium was observed 15 and 25 days after sowing in soils treated with pendimethalin and prometryn and 25 days after sowing in soils treated with trifluralin.     

Soil Phosphorus Uptake by Continuously Cropped Lupinus albus: A New Microcosm Design

When grown in soils with sparingly available phosphorus (P), white lupin (Lupinus albus L.) forms special root structures, called cluster roots, which secrete large amounts of organic acids and concomitantly acidify the rhizosphere. Many studies dealing with the understanding of this P acquisition strategy have been performed in short time experiments either in hydroponic cultures or in small microcosm designs with sand or sand:soil mixtures. In the present study, we applied an experimental design which came nearer to the natural field conditions: we performed a one-year experiment on large microcosms containing 7 kg of soil and allowing separation of rhizosphere soil and bulk soil. We planted six successive generations of lupins and analysed P uptake, organic P desorption, phosphatase activities and organic acid concentrations in different soil samples along a spatio-temporal gradient. We compared the rhizosphere soil samples of cluster (RSC) and non-cluster roots (RSNC) as well as the bulk soil (BS) samples. A total shoot biomass of 55.69 ± 1.51 g (d.w.) y⁻¹ was produced and P uptake reached 220.59 ± 5.99 mg y⁻¹. More P was desorbed from RSC than from RSNC or BS (P < 0.05). RSC and RSNC showed a higher activity of acid and alkaline phosphatases than BS samples and a higher acid phosphatase activity was observed in RSC than in RSNC throughout the one-year experiment. Fumarate was the most abundant organic acid in all rhizosphere soil samples. Citrate was only present in detectable amounts in RSC while malate and fumarate were recovered from both RSC and RSNC. Almost no organic acids could be detected in the BS samples. Our results demonstrated that over a one-year cultivation period in the absence of an external P supply, white lupin was able to acquire phosphate from the soil and that the processes leading to this P uptake took place preferentially in the rhizosphere of cluster roots.     

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract: Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.