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PubMed2002

Survival of faecal indicator bacteria in tropical estuarine waters (Bangpakong River, Thailand).

AIMS: To investigate the survival of cultivable bacteria in the tropical Bangpakong estuary (Eastern Thailand) under different salinities and light conditions. METHODS AND RESULTS: Dark and light microcosm experiments using membrane diffusion chambers were carried out under three different experimental conditions, namely (i) low salinity, (ii) progressive mixing with brackish water and (iii) fast mixing with high salinity water spiked with raw urban sewage. Faecal coliforms declined faster than faecal enterococci, as shown by survival T90 values ranging from 82.2 +/- 4.2 to 14.5 +/- 0.8 h and 97.5 +/- 0.4-20.6 +/- 1.2 h, respectively. The survival of freshwater heterotrophic bacteria was higher but variable (121.2 +/- 5.0-30.1 +/- 14.3 h), whereas that of heterotrophic marine bacteria was rather stable (81.5 +/- 4.2-44.6 +/- 2.5 h). CONCLUSIONS: Overall survival was higher in low salinities. Light had a further deleterious effect, since it accelerated the decay of faecal indicators, particularly in high salinities. Faecal enterococci had a higher resistance to environmental conditions compared with faecal coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is relevant to the understanding of the behaviour of different faecal indicator bacteria and the optimization of sewage treatment plants aimed at the reduction and/or elimination of faecal load discharged into estuarine waters submitted to salinity variations.

Bacteria↗
PubMed1998

Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR.

The aim of the present work was to study colonization patterns in roots by different arbuscular mycorrhizal fungi developing from a mixed community in soil. As different fungi cannot be distinguished with certainty in planta on the basis of fungal structures, taxon-discriminating molecular probes were developed. The 5' end of the large ribosomal subunit containing the variable domains D1 and D2 was amplified by PCR from Glomus mosseae (BEG12), G. intraradices (LPA8), Gigaspora rosea (BEG9) and Scutellospora castanea (BEG1) using newly designed eukaryote-specific primers. Sequences of the amplification products showed high interspecies variability and PCR taxon-discriminating primers were designed to distinguish between each of these four fungi. A nested PCR, using universal eukaryotic primers for the first amplification and taxon-discriminating primers for the second, was performed on individual trypan blue-stained mycorrhizal root fragments of onion and leek, and root colonization by four fungi inoculated together in a microcosm experiment was estimated. More than one fungus was detected in the majority of root fragments and all four fungi frequently co-existed within the same root fragment. Root colonization by G. mosseae and G. intraradices was similar from individual mixed inoculum, whilst the frequency of S. castanea and Gig. rosea increased in the presence of the two Glomus species, suggesting that synergistic interactions may exist between some arbuscular mycorrhizal fungi.

Allium↗
PubMed1999

Natural (13)C abundance reveals trophic status of fungi and host-origin of carbon in mycorrhizal fungi in mixed forests.

Fungi play crucial roles in the biogeochemistry of terrestrial ecosystems, most notably as saprophytes decomposing organic matter and as mycorrhizal fungi enhancing plant nutrient uptake. However, a recurrent problem in fungal ecology is to establish the trophic status of species in the field. Our interpretations and conclusions are too often based on extrapolations from laboratory microcosm experiments or on anecdotal field evidence. Here, we used natural variations in stable carbon isotope ratios (delta(13)C) as an approach to distinguish between fungal decomposers and symbiotic mycorrhizal fungal species in the rich sporocarp flora (our sample contains 135 species) of temperate forests. We also demonstrated that host-specific mycorrhizal fungi that receive C from overstorey or understorey tree species differ in their delta(13)C. The many promiscuous mycorrhizal fungi, associated with and connecting several tree hosts, were calculated to receive 57-100% of their C from overstorey trees. Thus, overstorey trees also support, partly or wholly, the nutrient-absorbing mycelia of their alleged competitors, the understorey trees.

Biodegradation, Environmental↗
PubMed2003

Fundamental predictability in multispecies competition: the influence of large disturbance.

Empirical evidence of chaos, or complex behavior, in ecosystems is scarce, presumably due to high system-level noise and/or the rarity of conditions necessary for complex behavior to arise. An alternative explanation might be that complex behavior is fragile and readily suppressed by disturbances that are common in many ecosystems. Here we investigated the role of disturbance frequency and magnitude on complex behavior and focused on population succession trajectories in a plankton system. Because of its prominence in aquatic ecology, we used hydraulic flushing and nutrient loading as disturbances. Our findings from numerical modeling exercises and laboratory microcosm experiments using natural plankton assemblages indicated that one aspect of complex behavior, divergence of nearby trajectories, was suppressed when the magnitude and periodicity of hydraulic flushing and nutrient loading were large. In other words, complex succession became determinable. Divergence of nearby trajectories was relatively robust, however, because pulses of not less than 85% of the total inflow were required to suppress this behavior. Our numerical findings also revealed that large hydraulic disturbances could introduce to the system another aspect of complex behavior, aperiodic succession.

Ecosystem↗
PubMed2003

Determinism in a transient assemblage: the roles of dispersal and local competition.

Both dispersal and local competitive ability may determine the outcome of competition among species that cannot coexist locally. I develop a spatially implicit model of two-species competition at a small spatial scale. The model predicts the relative fitness of two competitors based on local reproductive rates and regional dispersal rates in the context of the number, size, and extinction probability of habitat patches in the landscape. I test the predictions of this model experimentally using two genotypes of the bacteriophagous soil nematode Caenorhabditis elegans in patchy microcosms. One genotype has higher fecundity while the other is a better disperser. With such a fecundity-dispersal trade-off between competitors, the model predicts that relative fitness will be affected most by local population size when patches do not go extinct and by the number of patches when there is a high probability of patch extinction. The microcosm experiments support the model predictions. Both approaches suggest that competitive dominance in a patchily distributed transient assemblage will depend upon the architecture and predictability of the environment. These mechanisms, operating at a small scale with high spatial admixture, may be embedded in a larger metacommunity process.

Animals↗
PubMed1999

Predicting variability in biological control of a plant-pathogen system using stochastic models.

A stochastic model for the dynamics of a plant-pathogen interaction is developed and fitted to observations of the fungal pathogen Rhizoctonia solani (Kühn) in radish (Raphanus sativus L.), in both the presence and absence of the antagonistic fungus Trichoderma viride (Pers ex Gray). The model incorporates parameters for primary and secondary infection mechanisms and for characterizing the time-varying susceptibility of the host population. A parameter likelihood is developed and used to fit the model to data from microcosm experiments. It is shown that the stochastic model accounts well for observed variability both within and between treatments. Moreover, it enables us to describe the time evolution of the probability distribution for the variability among replicate epidemics in terms of the underlying epidemiological parameters for primary and secondary infection and decay in susceptibility. Consideration of profile likelihoods for each parameter provides strong evidence that T. viride mainly affects primary infection. By using the stochastic model to study the dependence of the probability distribution of disease levels on the primary infection rate we are therefore able to predict the effectiveness of a widely used biological control agent.

Models, Biological↗
PubMed2003

Specialized cheating of the ectomycorrhizal symbiosis by an epiparasitic liverwort.

Many non-photosynthetic vascular plants in 10 diverse families obtain all of their carbon from fungi, but in most cases the fungi and the ultimate sources of carbon are unknown. In a few cases, such plants have been shown to be epiparasitic because they obtain carbon from neighbouring green plants through shared mycorrhizal fungi. In all such cases, the epiparasitic plants have been found to specialize upon narrow lineages of ecto- or arbuscular mycorrhizal fungi. Here we show that a non-vascular plant, the non-photosynthetic liverwort Cryptothallus mirabilis, is epiparasitic and is specialized on Tulasnella species that form ectomycorrhizae with surrounding trees at four locations in England, France and Portugal. By using microcosm experiments we show that the interaction with Tulasnella is necessary for growth of Cryptothallus, and by using labelling experiments we show that (14)CO(2) provided to birch seedlings is transferred to Cryptothallus by Tulasnella. This is one of the first documented cases of epiparasitism by a non-vascular plant and of ectomycorrhizal formation by Tulasnella. These results broaden the emerging association between epiparasitism and mycorrhizal specialization into a new class of plants and a new order of fungi.

Carbon↗
PubMed1987

Plasmid transfer between strains of Pseudomonas aeruginosa on membrane filters attached to river stones.

A naturally occurring mercury-resistance, conjugative plasmid, designated pQM1, was isolated from a bacterial population on the surface of stones from a river using Pseudomonas aeruginosa as a recipient. This was a narrow-host-range plasmid [IncP-13; 165 MDa; Tra+, Hgr, fluorescein mercuric acetater, merbrominr, Phi(E79), UVr] confined to some Pseudomonas spp. It was used to demonstrate transfer between bacteria on stones in laboratory microcosm experiments and in situ. Transfer occurred (3.3 X 10(-1) to 6.8 X 10(-9) per recipient) at all the temperatures used (6-20 degrees C), although frequencies were lower in the cold. Nutrient status also affected transfer frequency, rich conditions promoting transfer. The presence of competing bacteria in the natural epilithon lowered transfer frequencies, but when unscrubbed stones were heat treated, transfer was enhanced, perhaps because of nutrient release from the heated epilithon.

Fresh Water↗
PubMed2004

Differential response of archaeal and bacterial communities to nitrogen inputs and pH changes in upland pasture rhizosphere soil.

Grassland management regimens influence the structure of archaeal communities in upland pasture soils, which appear to be dominated by as yet uncultivated non-thermophilic Crenarchaeota. In an attempt to determine which grassland management factors select for particular crenarchaeal community structures, soil microcosm experiments were performed examining the effect of increased pH, application of inorganic fertilizer (ammonium nitrate) and sheep urine deposition on both archaeal and bacterial communities in unmanaged grassland soil. As grassland management typically increases pH, a further experiment examined the effect of a reduction in pH, to that typical of unimproved grassland soils, on archaeal and bacterial communities. The RT-PCR amplification of 16S rRNA followed by denaturing gradient gel electrophoresis analysis demonstrated a distinct and reproducible effect on bacterial communities after incubation for 28 or 30 days. In contrast, none of the treatments had a significant effect on the structure of the crenarchaeal community, indicating that these factors are not major drivers of crenarchaeal community structures in grassland soils.

Animals↗
PubMed2005

Soil nutrient heterogeneity interacts with elevated CO2 and nutrient availability to determine species and assemblage responses in a model grassland community.

Interactive effects of atmospheric CO(2) concentration ([CO(2)]), soil nutrient availability and soil nutrient spatial distribution on the structure and function of plant assemblages remain largely unexplored. Here we conducted a microcosm experiment to evaluate these interactions using a grassland assemblage formed by Lolium perenne, Plantago lanceolata, Trifolium repens, Anthoxanthum odoratum and Holcus lanatus. Assemblages exhibited precise root foraging patterns, had higher total and below-ground biomass, and captured more nitrogen when nutrients were supplied heterogeneously. Root foraging responses were modified by nutrient availability, and the patterns of N capture by interactions between nutrient distribution, availability and [CO(2)]. Greater above-ground biomass was observed under elevated CO(2) only under homogeneous conditions of nutrient supply and at the highest availability level. CO(2) interacted with nutrient distribution and availability to determine foliar percentage N and below : above-ground biomass ratios, respectively. Interactions between nutrient distribution and CO(2) determined the relative contribution to above-ground biomass of four of the species. The responses of dominant and subordinate species to [CO(2)] were dependent on the availability and distribution of nutrients. Our results suggest that soil nutrient distribution has the potential to influence the response of plant species and assemblages to changes in [CO(2)] and nutrient availability.

Biomass↗
PubMed1999

Fate of plasmid-bearing, luciferase marker gene tagged bacteria after feeding to the soil microarthropod Onychiurus fimatus (Collembola).

In order to study the potential impact of the soil microarthropod Onychiurus fimatus (Collembola) on the microbial community, we analysed the fate of luciferase marker gene tagged bacterial strains fed to young adult specimens in petri dish microcosm experiments. In faeces collected from O. fimatus, Escherichia coli S17-1/pRP4luc and Sinorhizobium meliloti L33 were only detectable for 2 days after feeding whereas strain HR2/pRP4luc, a close relative of Stenotrophomonas maltophilia, isolated from another collembolan species, could be detected for 16 days. The amount of shed cells of strain HR2 increased during the frequent releases of the cast-off skins (exuvia). In order to analyse whether gut associated bacteria could serve as recipients for mobile genetic elements, plasmid-bearing E. coli donor strains were incubated with faeces in filter mating-like experiments and, in other experiments, directly fed to O. fimatus specimens. Transconjugants were obtained with both the conjugative self-transferable broad host range plasmid pRP4luc and the mobilisable (Mob(+)) broad host range plasmid pSUP104luc, the latter, however, only with a mobilising donor strain. No transfer was detected with the narrow host range plasmids pSUP202luc (Mob(+)), pUC18luc (Mob(-)), or with the broad host range transposon delivery plasmid pUTluxCDABE (Mob(+)). Transconjugants of pRP4luc were detected within one day of the beginning of a feeding experiment and then throughout the incubation period of two weeks, with gaps of no detection after 5, 12 and 14 days, probably caused by moulting. The results of this study indicate that feeding activities of collembola can modify the structure of soil-inhabiting microbial communities and enhance the spread of plasmids from non-indigenous to indigenous soil bacteria.

Journal Article↗
PubMed2006

Physiological traits of bacterial strains isolated from phenol-degrading aerobic granules.

The physiological characteristics of ten bacterial strains isolated from phenol-degrading aerobic granules were evaluated in order to identify competitive traits for dominant growth in aerobic granules. The ten strains showed a wide diversity in specific growth rates and oxygen utilization kinetics, and could be divided into four catabolic types of phenol degradation. While some strains degraded phenol mainly via the meta pathway or the ortho pathway, other strains degraded phenol via both these pathways. The ten strains also exhibited high levels of autoaggregation and coaggregation activity. Within the collection of ten strains, 36.7% of all possible strain pairings displayed a measurable degree of coaggregation. Strain PG-08 possessed the strongest autoaggregation activity and showed significant coaggregation (coaggregation indices of 67% to 74%) with PG-02. The three strains PG-01, PG-02 and PG-08 belonging to dominant groups in the granules possessed different competitive characteristics. Microcosm experiments showed the three strains could not coexist at the high phenol concentration of 250 mg L(-1), but could coexist at lower phenol concentrations in a spatially heterogeneous environment. This study illustrated that the spatial heterogeneity provided by the aerobic granules led to niche differentiation and increased physiological diversity in the resident microbial community.

Aerobiosis↗
PubMedDate not supplied

Extremely alkaline (pH > 12) ground water hosts diverse microbial community.

Chemically unusual ground water can provide an environment for novel communities of bacteria to develop. Here, we describe a diverse microbial community that inhabits extremely alkaline (pH > 12) ground water from the Lake Calumet area of Chicago, Illinois, where historic dumping of steel slag has filled in a wetland. Using microbial 16S ribosomal ribonucleic acid gene sequencing and microcosm experiments, we confirmed the presence and growth of a variety of alkaliphilic beta-Proteobacteria, Bacillus, and Clostridium species at pH up to 13.2. Many of the bacterial sequences most closely matched those of other alkaliphiles found in more moderately alkaline water around the world. Oxidation of dihydrogen produced by reaction of water with steel slag is likely a primary energy source to the community. The widespread occurrence of iron-oxidizing bacteria suggests that reduced iron serves as an additional energy source. These results extend upward the known range of pH tolerance for a microbial community by as much as 2 pH units. The community may provide a source of novel microbes and enzymes that can be exploited under alkaline conditions.

Alkalies↗
PubMed2006

Influences of infaunal burrows on the community structure and activity of ammonia-oxidizing bacteria in intertidal sediments.

Influences of infaunal burrows constructed by the polychaete (Tylorrhynchus heterochaetus) on O(2) concentrations and community structures and abundances of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in intertidal sediments were analyzed by the combined use of a 16S rRNA gene-based molecular approach and microelectrodes. The microelectrode measurements performed in an experimental system developed in an aquarium showed direct evidence of O(2) transport down to a depth of 350 mm of the sediment through a burrow. The 16S rRNA gene-cloning analysis revealed that the betaproteobacterial AOB communities in the sediment surface and the burrow walls were dominated by Nitrosomonas sp. strain Nm143-like sequences, and most of the clones in Nitrospira-like NOB clone libraries of the sediment surface and the burrow walls were related to the Nitrospira marina lineage. Furthermore, we investigated vertical distributions of AOB and NOB in the infaunal burrow walls and the bulk sediments by real-time quantitative PCR (Q-PCR) assay. The AOB and Nitrospira-like NOB-specific 16S rRNA gene copy numbers in the burrow walls were comparable with those in the sediment surfaces. These numbers in the burrow wall at a depth of 50 to 55 mm from the surface were, however, higher than those in the bulk sediment at the same depth. The microelectrode measurements showed higher NH(4)(+) consumption activity at the burrow wall than those at the surrounding sediment. This result was consistent with the results of microcosm experiments showing that the consumption rates of NH(4)(+) and total inorganic nitrogen increased with increasing infaunal density in the sediment. These results clearly demonstrated that the infaunal burrows stimulated O(2) transport into the sediment in which otherwise reducing conditions prevailed, resulting in development of high NH(4)(+) consumption capacity. Consequently, the infaunal burrow became an important site for NH(4)(+) consumption in the intertidal sediment.

Ammonia↗
PubMed1990

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment.

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.

DNA, Bacterial↗
PubMed1993

Conditional suicide system of Escherichia coli released into soil that uses the Bacillus subtilis sacB gene.

The sacB gene from Bacillus subtilis confers sucrose sensitivity upon gram-negative bacteria. The gene was investigated for use as a potential conditional suicide system for Escherichia coli released into soil. To ensure against the loss of the cell death function encoded under nonselective conditions, the nptI-sacR-B suicide cassette was inserted into the E. coli chromosome by using a circular nonreplicative integration vector. Stability studies yielded no loss of the suicide cassette in the integrated E. coli EL1026 strain. sacB induction in the absence of a selective pressure resulted in a lysis efficiency of up to 99.9%. The microcosm experiments confirmed the ability of the suicide cassette to limit the growth and reduce the survival of E. coli strains released into soil. Sucrose addition to sterile soil resulted in a 10(-3)-fold reduction of the final E. coli population density. sacB induction prevented the proliferation and triggered the rapid disappearance of E. coli from natural soil. Mutation to sucrose tolerance occurred at a frequency of 10(-5), making E. coli EL1026 a potential counterselectable donor strain for gene transfer studies. Specificity and potential adaptability to a wide range of gram-negative bacteria are additional conveniences of this conditional suicide system for the containment and counterselection of engineered microorganisms.

Bacillus subtilis↗
PubMed1995

Effect of parental growth on dynamics of conjugative plasmid transfer in the pea spermosphere.

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.

Journal Article↗
PubMed1999

Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis.

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1. 4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.

DNA Primers↗