Sulfadiazine uptake and effects in common hazel (Corylus avellana L.).

Soil contamination by antibiotics is a possible consequence of animal husbandry waste, sewage sludge, and reclaimed water spreading in agriculture. In this study, 1-year-old hazel plants (Corylus avellana L.) were grown in pots for 64 days in soil spiked with sulfadiazine (SDZ) in the range 0.01-100 mg kg(-1) soil. Leaf gas exchanges, fluorescence parameters and plant growth were measured regularly during the experiment, whereas plant biomass, sulfonamide concentrations in soil and plant tissues, and the quantitative variation of culturable bacterial endophytes in leaf petiole were analyzed at the end of the trial. During the experiment, photosynthesis and leaf transpiration as well as fluorescence parameters were progressively reduced by the antibiotic. Effects were more evident for leaf transpiration and for the highest SDZ spiking concentrations, whereas growth analyses did not reveal negative effects of the antibiotic. At the end of the trial, a high number of culturable endophytic bacteria in the leaf petiole of plants treated with 0.1 and 0.01 mg kg(-1) were observed, and SDZ was extractable from soil and plant roots for spiking concentrations ≥1 mg kg(-1). Inside plants, the antibiotic was mainly stored at the root level with bioconcentration factors increasing with the spiking dose, and the hydroxylated derivate 4-OH-SDZ was the only metabolite detected. Overall results show that 1-year-old hazel plants can contribute to the reduction of sulfonamide concentrations in the environment, however, sensitive reactions to SDZ can be expected at the highest contamination levels.

Quorum vs. diffusion sensing: a quantitative analysis of the relevance of absorbing or reflecting boundaries.

The consequences of the boundary conditions (signal reflecting vs. signal adsorbing) on bacterial intercellular communication were addressed by a combined physics and microbiology approach. A predictive biophysical model was devised that considered system size, diffusion from given points, signal molecule decay and boundary properties. The theoretical predictions were tested with two experimental agarose-gel-based set-ups for reflecting or absorbing boundaries. N-acyl homoserine lactone (AHL) concentration profiles were measured using the Agrobacterium tumefaciens NTL4 bioassay and found to agree with model predictions. The half-life of AHL was estimated to be 7 days. The absorbing vs. reflecting nature of the boundaries drastically changed AHL concentration profiles. The effect of a single nonreflecting boundary side was equivalent to a 100-fold lower cell concentration. Results suggest that the kinetics of signal accumulation vs. signal removal and their threshold-mediated phenotypic consequences are directly linked to the properties of biofilm boundaries, stressing the relevance of the diffusion sensing component in bacterial communication.

Microbiological features and bioactivity of a fermented manure product (preparation 500) used in biodynamic agriculture.

The fermented manure derivative known as Preparation 500 is traditionally used as a field spray in biodynamic agriculture for maintaining and increasing soil fertility. This work aimed at characterizing the product from a microbiological standpoint and at assaying its bioactive properties. The approach involved molecular taxonomical characterization of the culturable microbial community; ARISA fingerprints of the total bacteria and fungal communities; chemical elemental macronutrient analysis via a combustion analyzer; activity assays for six key enzymes; bioassays for bacterial quorum sensing and chitolipooligosaccharide production; and plant hormonelike activity. The material was found to harbor a bacterial community of 2.38 × 10(8) CFU/g dw dominated by Grampositives with minor instances of Actinobacteria and Gammaproteobacteria. ARISA showed a coherence of bacterial assemblages in different preparation lots of the same year in spite of geographic origin. Enzymatic activities showed elevated values of beta-glucosidase, alkaline phosphatase, chitinase, and esterase. The preparation had no quorum sensing-detectable signal, and no rhizobial nod gene-inducing properties, but displayed a strong auxin-like effect on plants. Enzymatic analyses indicated a bioactive potential in the fertility and nutrient cycling contexts. The IAA activity and microbial degradation products qualify for a possible activity as soil biostimulants. Quantitative details and possible modes of action are discussed.

Evidence for calcium-mediated perception of plant symbiotic signals in aequorin-expressing Mesorhizobium loti.

BACKGROUND: During the interaction between rhizobia and leguminous plants the two partners engage in a molecular conversation that leads to reciprocal recognition and ensures the beginning of a successful symbiotic integration. In host plants, intracellular Ca(2+) changes are an integral part of the signalling mechanism. In rhizobia it is not yet known whether Ca(2+) can act as a transducer of symbiotic signals. RESULTS: A plasmid encoding the bioluminescent Ca(2+) probe aequorin was introduced into Mesorhizobium loti USDA 3147(T) strain to investigate whether a Ca(2+) response is activated in rhizobia upon perception of plant root exudates. We find that M. loti cells respond to environmental and symbiotic cues through transient elevations in intracellular free Ca(2+) concentration. Only root exudates from the homologous host Lotus japonicus induce Ca(2+) signalling and downstream activation of nodulation genes. The extracellular Ca(2+) chelator EGTA inhibits both transient intracellular Ca(2+) increase and inducible nod gene expression, while not affecting the expression of other genes, either constitutively expressed or inducible. CONCLUSION: These findings indicate a newly described early event in the molecular dialogue between plants and rhizobia and highlight the use of aequorin-expressing bacterial strains as a promising novel approach for research in legume symbiosis.

Consequences of relative cellular positioning on quorum sensing and bacterial cell-to-cell communication.

Cell-to-cell bacterial communication via diffusible signals is addressed and the conceptual framework in which quorum sensing is usually described is evaluated. By applying equations ruling the physical diffusion of the autoinducer molecules, one can calculate the gradient profiles that would occur either around a single cell or at the center of volumes of increasing size and increasing cell densities. Water-based matrices at 25 degrees C and viscous biofilms at colder temperatures are compared. Some basic consequences relevant for the field of microbial signalling arise. As regards induction, gradient-mixing dynamics between as little as two cells lying at a short distance appears to be sufficient for the buildup of a concentration reaching the known thresholds for quorum sensing. A straight line in which the highest concentrations occur is also created as a consequence of the gradient overlap geometry, providing an additional signal information potentially useful for chemotactic responses. In terms of whole population signalling, it is shown how the concentration perceived by a cell in the center is critically dependent not only on the cell density but also on the size of the biofilm itself. Tables and formulas for the practical prediction of N-acyl homoserine lactones concentrations at desired distances in different cell density biofilms are provided.

Monitoring a genetically modified Pseudomonas sp. released on pine leaves reveals concerted successional patterns of the bacterial phyllospheric community.

The fate of a biocontrol agent released on pine phyllosphere in a greenhouse-confined trial was followed over 102 days. The microorganism used, a Pseudomonas sp., isolated from Pinus nigra, carries the cry9a toxin gene from Bacillus thuringiensis. In order to detect the GMM, specific primers were used, and a previously defined protocol for DNA isolation from bacteria colonizing pine needles was applied. The method, based on vortexing in a suspension of glass beads followed by microcolumn extractions, allowed sensitive PCR monitoring of the target transgenes. The presence of the released organism was recorded throughout the trial and compared with its entomocidal performance towards larvae of the pine processionary caterpillar Thaumetopoea pityocampa. At the same time the dynamics of the released Pseudomonas within the whole epiphytic bacterial community, was followed by amplifying 16S rDNA pools and comparing ARDRA profiles at seven sampling points. The resulting dendrogram allowed to follow the time-dependent progressive blending of the Pseudomonas profile into those of the resident biota. PCR-dominance of the released bacterium in the community was extended until 21 days from release while its activity against insect larvae lasted for over 3 months. The prokaryotic epiphytic population, irrespective of any particular impact from the released strain, showed no resilience but a general successional trend, which, remarkably, appeared synchronous on all trees tested, including non-inoculated controls. This observation suggests interesting patterns of concerted environmental shifts by phyllospheric microorganisms.

Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes.

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.

Construction of a Pseudomonas sp. derivative carrying the cry9Aa gene from Bacillus thuringiensis and a proposal for new standard criteria to assess entomocidal properties of bacteria.

An isolate of Pseudomonas sp. (16S rDNA sequence 98% homologous to P. graminis and P. lutea) was isolated from the phyllosphere of black pine in northern Italy and used as a host for the gene encoding the Cry9Aa entomocidal toxin from Bacillus thuringiensis subsp. galleriae. An expression system featuring a synthetic highest-consensus promoter specifically tailored for the regulated induction of cloned genes over a broad range of Gram-negative bacteria was used to drive the production of the introduced toxin. The construct showed effective toxicity toward larvae of the greater wax moth (Galleria mellonella), which was also used as a model insect for establishing a number of newly proposed toxicity indices (LC50 cellular efficiency, toxin cellular efficiency, GMO efficiency, lethal cellular intake). These were devised in order to express toxicities of entomocidal bacteria in a standard fashion enabling the fine tuning of biocontrol treatments as well as the comparative evaluation of different reports.