Combination of Culture-Dependent and -Independent Methods Reveals Diverse Acyl Homoserine Lactone-Producers from Rhizosphere of Wetland Plants

The culture-dependent method and a degenerate primer-based culture-independent method were combined in an effort to identify N-acyl homoserine lactone (AHL) producers in rhizosphere of wetland plants, Salix babylonica (willow) and Phragmites australis (reed). Overall, eight potential AHL-producing genera were found, which were Aeromonas, Pseudomonas, Polymorphum, Agrobacterium, Rhizobium, Sinorhizobium, Ensifer, and Pectobacterium. Thin layer chromatograph assay revealed various AHL profiles from cultivable AHL-producers. The degenerate primer pair RAHL352F and RAHL461R was found to cover AHL synthetase genes from families Rhizobiaceae and Rhodobacteraceae. Little overlap was found in taxa of potential AHL-producers obtained by the two methods, indicating that they were well complement to each other. This is the first survey for AHL-producers that employed combined culture-dependent and -independent methods.     

Prey Range Characterization, Ribotyping, and Diversity of Soil and Rhizosphere Bdellovibrio spp. Isolated on Phytopathogenic Bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ PFU g⁻¹. A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

Quorum-sensing regulation in soil pseudomonads

228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two tester systems, for the capacity to produce N-acyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chloroaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide; it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csaI genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation of various metabolic processes in P. chlororaphis was studied.     

Main rhizosphere characteristics of the Cd hyperaccumulator Rorippa globosa (Turcz.) Thell

Aim

This article was aimed to explore the main rhizospherial properties of the Cd hyperaccumulator R. globosa compared to those of the non hyperaccumulator Rorippa palustris (Leyss.) Bess. representing the same genus (Rorippa) of Cruciferae.

Method

Pot culture experiments using soil spiked with Cd as CdCl₂·2.5H₂O and rhizobags were conducted to determine the differences in Cd accumulation vs. pH, dissolved organic carbon (DOC), Cd chemical fractionation, enzyme activities, and microorganism number in the rhizospheres of R. globosa and R. palustris, and in the bulk soils.

Results

Experiments on Cd uptake by R. globosa and R. palustris from soil spiked with different doses of Cd ranging from 0 to 40 mg?kg^?1, confirmed Cd-hyperaccumulating properties of R. globosa (Cd accumulation in the above-ground organs >100 mg kg^?1, enrichment factor EF> 1, translocation factor TF> 1, no significant biomass reduction at Cd doses >10 mg kg^?1) and the lack of such properties in R. palustris, which made these species suitable for comparative studies. The pH value was found to be a constant, specific property of the rhizosphere of R. globosa and R. palustris, and of the bulk soil, independent on the Cd dose, however the differences were rather small: by 0.2 unit lower in the rhizosphere of R. globosa, and only by 0.1 unit lower in the rhizosphere of R.. palustris compared to the bulk soil. Chemical fractionation of Cd, i.e. its affinity to pools of different binding strength, also appeared to be a specific feature of a rhizosphere and soil independent on the Cd dose. It exhibited a unique capability of the rhizosphere of the Cd-hyperaccumulator R. globosa to mobilize Cd, which enriched the most labile exchangeable fraction in 24.4 % and the immobile residual fraction in 42.3 %, compared to 19.3 % and 50.8 % in the bulk soil and in the rhizosphere of the non-hiperaccumulator R.palustris that did not show significant difference (p?<?0.05) from the bulk soil. In turn, DOC concentrations, enzymatic (urease and catalase) activity and microorganism (bacteria, fungi and actinomycetes) growth in rhizosphere soils were largely influenced by different Cd doses, although they were always considerably higher in the rhizosphere soils of R globosa, than in the rhizosphere of R. palustris and in the bulk soil, in particular at Cd doses ≥10 mg kg^?1.

Conclusion

pH and DOC changes in the rhizosphere of the Cd-hyperaccumulator R. globosa were found to be of a minor importance. The alteration of Cd chemical fractionation consisting in substantial reduction of the immobile residual pool and Cd enrichment primarily in the most labile exchangeable fraction, along with over 2-fold higher number of microorganisms was considered to be the driving force of Cd hyperaccumulation.     

Investigation of N-acyl homoserine lactone (AHL) molecule production in Gram-negative bacteria isolated from cooling tower water and biofilm samples

In this study, 99 Gram-negative rod bacteria were isolated from cooling tower water, and biofilm samples were examined for cell-to-cell signaling systems, N-acyl homoserine lactone (AHL) signal molecule types, and biofilm formation capacity. Four of 39 (10 %) strains isolated from water samples and 14 of 60 (23 %) strains isolated from biofilm samples were found to be producing a variety of AHL signal molecules. It was determined that the AHL signal molecule production ability and the biofilm formation capacity of sessile bacteria is higher than planktonic bacteria, and there was a statistically significant difference between the AHL signal molecule production of these two groups (p?<?0.05). In addition, it was found that bacteria belonging to the same species isolated from cooling tower water and biofilm samples produced different types of AHL signal molecules and that there were different types of AHL signal molecules in an AHL extract of bacteria. In the present study, it was observed that different isolates of the same strains did not produce the same AHLs or did not produce AHL molecules, and bacteria known as AHL producers did not produce AHL. These findings suggest that detection of signal molecules in bacteria isolated from cooling towers may contribute to prevention of biofilm formation, elimination of communication among bacteria in water systems, and blockage of quorum-sensing controlled virulence of these bacteria.     

Sensitive Whole-Cell Biosensor Suitable for Detecting a Variety of N-Acyl Homoserine Lactones in Intact Rhizosphere Microbial Communities

To investigate quorum sensing in rhizosphere soil, a whole-cell biosensor, Agrobacterium tumefaciens(pAHL-Ice), was constructed. The biosensor responded to all N-acyl homoserine lactones (AHLs) tested, except C₄ homoserine lactone, with a minimum detection limit of 10⁻¹² M, as well as to both exogenously added AHLs and AHL-producing bacterial strains in soil. This highly sensitive biosensor reveals for the first time the increased AHL availability in intact rhizosphere microbial communities compared to that in bulk soil.     

Indole-3-acetic acid producing root-associated bacteria on growth of Brazil Pine (Araucaria angustifolia) and Slash Pine (Pinus elliottii)

Araucaria forests in Brazil today correspond to only 0.7 % of the original 200 km² of natural forest that covered a great part of the southern and southeastern area of the Atlantic Forest and, although Araucaria angustifolia is an endangered species, illegal exploitation is still going on. As an alternative to the use of hardwoods, Pinus elliottii presents rapid growth and high tolerance to climatic stress and low soil fertility or degraded areas. Thus, the objective of this study was to evaluate the effect of IAA-producing bacteria on the development of A. angustifolia and P. elliottii. We used five bacterial strains previously isolated from the rhizosphere of A. angustifolia, which produce quantities of IAA ranging from 3 to 126 μg mL^?1. Microbiolized seeds were sown in a new gnotobiotic system developed for this work, that allowed the quantification of the plant hormone IAA produced by bacteria, and the evaluation of its effect on seedling development. Also, it was shown that P. elliottii roots were almost as satisfactory as hosts for these IAA producers as A. angustifolia, while different magnitudes of mass increases were found for each species. Thus, we suggest that these microbial groups can be helpful for the development and reestablishment of already degraded forests and that PGPR isolated from Araucaria rhizosphere have the potential to be beneficial in seedling production of P. elliottii. Another finding is that our newly developed gnotobiotic system is highly satisfactory for the evaluation of this effect.     

Carbon Inputs from <Emphasis Type="Italic">Miscanthus</Emphasis> Displace Older Soil Organic Carbon Without Inducing Priming

The carbon (C) dynamics of a bioenergy system are key to correctly defining its viability as a sustainable alternative to conventional fossil fuel energy sources. Recent studies have quantified the greenhouse gas mitigation potential of these bioenergy crops, often concluding that C sequestration in soils plays a primary role in offsetting emissions through energy generation. Miscanthus is a particularly promising bioenergy crop and research has shown that soil C stocks can increase by more than 2 t C ha^?1 yr^?1. In this study, we use a stable isotope (¹³C) technique to trace the inputs and outputs from soils below a commercial Miscanthus plantation in Lincolnshire, UK, over the first 7 years of growth after conversion from a conventional arable crop. Results suggest that an unchanging total topsoil (0–30 cm) C stock is caused by Miscanthus additions displacing older soil organic matter. Further, using a comparison between bare soil plots (no new Miscanthus inputs) and undisturbed Miscanthus controls, soil respiration was seen to be unaffected through priming by fresh inputs or rhizosphere. The temperature sensitivity of old soil C was also seen to be very similar with and without the presence of live root biomass. Total soil respiration from control plots was dominated by Miscanthus-derived emissions with autotrophic respiration alone accounting for ～50 % of CO₂. Although total soil C stocks did not change significantly over time, the Miscanthus-derived soil C accumulated at a rate of 860 kg C ha^?1 yr^?1 over the top 30 cm. Ultimately, the results from this study indicate that soil C stocks below Miscanthus plantations do not necessarily increase during the first 7 years.     

Plant growth promoting traits of phosphate-solubilizing rhizobacteria isolated from apple trees in trans Himalayan region of Himachal Pradesh

Two hundred and six phosphate-solubilizing rhizobacteria (PSB) were isolated from rhizosphere soil (RS) and root endosphere (ER) of apple trees from different sites of four locations viz., Chamba, Shimla, Kinnaur and Kullu of Himachal Pradesh, Northern India, and were screened for plant growth promoting traits (PGPTs) by using culture dependent procedures. Indole acetic acid (IAA) production was detected in 50 isolates (24.2 %), siderophore synthesis in 53 isolates (25.7 %), hydrocyanic acid (HCN) in 40 isolates (19.4 %) and percentage growth inhibition against Dematophora necatrix in 61 isolates (29.6 %). Overall, 54.3 % of PSB isolates from RS and 64.4 % from ER showed none of the PGPTs tested. Among the PSB showing PGPTs, 10.6 % had single trait and 30.6 % had multiple traits showing two (10.7 %), three (14.1 %) and four (5.8 %) types of PGPTs. The Shannon–Weaver diversity index (H′) revealed that PGPT-possessing PSBs in RS were more abundant than ER. Clustering analysis by principal component analysis showed that ER was most important factor influencing the ecological distribution and physiological characterization of PGPT-possessing PSB. There was a positive correlation (0.94, p < 0.05) between HCN and antifungal activity producers, and IAA and antifungal activity producers (0.99, p < 0.05). Significant positive correlation (0.42, p < 0.05) between HCN producers and altitude was also noted.     

The effects of molybdenum and boron on the rhizosphere microorganisms and soil enzyme activities of soybean

This study examined the effects of molybdenum (Mo) and boron (B) on the rhizosphere microorganisms and the soil enzyme activities of soybean. The soybeans were treated with seven different Mo and B supplements (control: without Mo and B) Mo1 (0.0185 g kg^?1), B1 (0.08 g kg^?1), Mo1 + B1 (0.0185 + 0.08 g kg^?1), Mo2 (0.185 g kg^?1), B2 (0.3 g kg^?1) and Mo2 + B2 (0.185 + 0.3 g kg^?1) throughout the plants’ four growth stages. The results showed that Mo, B, and combined Mo and B treatments increased the soil microbial populations, stimulated the rhizosphere metabolisms, and improved the soil enzyme activities. These stimulatory effects varied in intensity among the treatment groups. The Mo and B combination treatments were more beneficial for the soybean rhizosphere soil than that of Mo-only or the B-only treatments, which suggests that the two elements have complementary functions in the biological processes of the soybean rhizosphere.     

Oxygen input controls the spatial and temporal dynamics of arsenic at the surface of a flooded paddy soil and in the rhizosphere of lowland rice (Oryza sativa L.): a microcosm study

The impact of oxygen (O₂) input at the soil surface and in the rhizosphere of rice (Oryza sativa L.) on the spatial and temporal dynamics of arsenic (As) was investigated in a flooded paddy soil. A soil microcosm and root-mat technique were designed to mimic submerged conditions of paddy fields. Water-filled containers with (planted) or without (unplanted) 27-day-old rice seedlings were fitted for 20 days on top of microcosms containing an As-affected soil (Bangladesh). After the initial establishment of strongly reduced conditions (?230 mV) in both planted and unplanted soils, the redox potential gradually increased until the day 8 to reach?+?50 mV at 2 mm from the surface of unplanted soils only. This oxidation was associated with an accumulation of NH₄-oxalate extractable As (25.7 mg kg^?1) in the 0.5-mm top layer, i.e. at levels above the initial total content of As in the soil (14 mg kg^?1) and a subsequent depletion of As in soil solution at 2 mm from soil surface. Root O₂-leakage induced the formation of an iron (Fe) plaque in root apoplast, with no evidence of outer rhizosphere oxidation. Arsenic content reached 173 mg kg^?1 in the Fe plaque. This accumulation induced a depletion of As in soil solution over several millimetres in the rhizosphere. Arsenic contents in root symplast and shoots (112 and 2.3 mg kg^?1, respectively) were significantly lower than in Fe plaque. Despite a large As concentration in soil solution, Fe plaque appeared highly efficient to sequester As and to restrict As acquisition by rice. The oxidation-mediated accumulation of As in the Fe plaque and in the oxidised layer at the top of the soil mobilised 21 and 3% of the initial amount of As in the planted and unplanted soils, respectively. Soil solution As concentration steadily decreased during the last 16 days of the soil stage, likely indicating a decrease in the ability of the soil to re-supply As from the solid-phase to the solution. The driving force of As dynamic in soil was therefore attributed to the As diffusion from reduced to oxidised soil layers. These results suggest a large mobility of As in the soil during the flooded period, controlled by the setting of oxic/anoxic interfaces at the surface of soil in contact with flooding water and in the rhizosphere of rice.     

Diversity of culturable bacterial communities in the intestinal tracts of goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>) and their ability to produce <Emphasis Type="Italic">N</Emphasis>-acyl homoserine lactone

Intestinal bacteria isolated from goldfish (Carassius auratus) were identified based on 16 ribosomal RNA (rRNA) gene sequences and screened for their ability to produce N-acyl homoserine lactone (AHL), an autoinducer of the quorum sensing (QS) system. The 230 aerobes/facultative anaerobes that were isolated comprised members of the genera Aeromonas (184 isolates), Citrobacter (11), Enterobacter (2), Shewanella (28), Vagococcus (1), and Vibrio (4). Among these genera, the two most abundant species were Aeromonas veronii (163 isolates) and Shewanella xiamenensis (27). In addition, 142 obligate anaerobes consisting of Cetobacterium somerae (139 isolates), Clostridium frigidicarnis (2), and Cetobacterium sp. (1) were also isolated. One hundred seventy isolates (74.2%) belonging to the genera Aeromonas, Citrobacter, Enterobacter, Shewanella, and Vibrio produced AHL, while 155 (67.7%) and 91 (39.7%) isolates possessed the luxR and luxI gene homologs, respectively. None of the obligate anaerobes produced AHL or possessed luxRI homologs. Total viable counts ranged from 1.2 × 10⁷ to 2.2 × 10⁹ CFU/g, which were accounted for 0.8 to 15.2% of direct counts. Aeromonas veronii, S. xiamenensis, and C. somerae were detected from five goldfish at densities ranging from 4.0 × 10⁶ to 1.7 × 10⁹ CFU/g, indicating that these bacteria are dominant components of the culturable gut flora in goldfish. In addition, members of the genera Aeromonas and Shewanella appeared to communicate with each other by using the QS system to some extent when the concentration of AHL reaches a certain threshold. It is therefore suggested that bacteria with the ability to disrupt AHL secretion in intestinal environments are potential candidates for probionts for preventing opportunistic infections in freshwater fish such as goldfish.     

Isolation and characterization of acyl homoserine lactone–producing bacteria during an urban river biofilm formation

The presence and diversity of acyl homoserine lactone (AHL)-producers in an urban river biofilm were investigated during 60-day biofilm formation. AHL biosensors detected the presence of AHL-producers in 1–60-day river biofilms. Screening for AHL-producers resulted in 17 Aeromonas spp., 3 Pseudomonas spp., 3 Ensifer spp., and 1 Acinetobacter sp. Among these isolates, six of them were closely related to Acinetobacter tjernbergiae, Aeromonas allosaccharophila, Aeromonas aquariorum, Aeromonas jandaei, Pseudomonas panipatensis, and Ensifer adhaerens and represented novel AHL-producing species. Thin layer chromatography revealed that C4-homoserine lactone was prevailing in Aeromonas spp., whereas C6- and C8-homoserine lactones and their derivatives were prevailing in other strains. Using degenerate primers, novel AHL synthetase genes from the three Ensifer spp. were successfully amplified. This study reports for the first time the diversity of AHL-producers from a river biofilm and the variety of novel AHL synthetase genes in Ensifer group.     

Root exudate components change litter decomposition in a simulated rhizosphere depending on temperature

The release of root exudates into the rhizosphere is known to enhance soil biological activity and alter microbial community structure. To assess whether root exudates also stimulated litter decomposition, in a rhizosphere model system we continuously injected solutions of glucose, malate or glutamate through porous Rhizon^® soil solution samplers into the soil at rhizosphere concentrations. The effect of these substances on the decomposition of ¹⁴C-labelled Lolium perenne shoot residues present in the soil was evaluated by monitoring ¹⁴CO₂ evolution at either 15°C or 25°C. The incorporation of the ¹⁴C into the microbial biomass and appearance in the dissolved organic matter (DOM) pool was estimated after 32 d incubation. The presence of malate and glutamate increased the mineralization of L. perenne residues by approximately 20% relative to the soil without their addition at 15°C, however, no significant effects on residue decomposition were observed at 25°C. The incorporation of the ¹⁴C-label into the microbial biomass and DOM pool was not affected by the addition of either glucose, malate or glutamate. Although nearly the same amount of L. perenne residues were mineralized at either temperature after 32 d, less ¹⁴C was recovered in the microbial biomass and DOM pools at 25°C compared to 15°C. Alongside other results, this suggests that the rate of microbial turnover is greater at 25°C compared to 15°C. We conclude that the addition of labile root exudate components to the rhizosphere induced a small but significant increase on litter decomposition but that the magnitude of this effect was regulated by temperature.     

13C isotope fractionation during rhizosphere respiration of C3 and C4 plants

Stable carbon isotopes are used extensively to partition total soil CO₂ efflux into root-derived rhizosphere respiration or autotrophic respiration and soil-derived heterotrophic respiration. However, it remains unclear whether CO₂ from rhizosphere respiration has the same δ¹³C value as root biomass. Here we investigated the magnitude of ¹³C isotope fractionation during rhizosphere respiration relative to root biomass in six plant species. Plants were grown in a carbon-free sand-perlite medium inoculated with microorganisms from a farm soil for 62 days inside a greenhouse. We measured the δ¹³C value of rhizosphere respiration using a closed-circulation 48-hour CO₂ trapping method during 40~42 and 60~62 days after sowing. We found a consistent depletion in ¹³C (0.9~1.7‰) of CO₂ from rhizosphere respiration relative to root biomass in three C₃ species (Glycine max L. Merr., Helianthus annuus L. and Triticum aestivum L.), but a relatively large depletion in ¹³C (3.7~7.0‰) in three C₄ species (Amaranthus tricolor L., Sorghum bicolor (L.) Moench and Zea mays L. ssp. mays). Overall, our results indicate that CO₂ from rhizosphere respiration is more ¹³C-depleted than root biomass. Therefore, accounting for this ¹³C fractionation is required for accurately partitioning total soil CO₂ efflux into root-derived and soil-derived components using natural abundance stable carbon isotope methods.     

Incorporation into the transplant soil plug of the plant protective agent Paenibacillus alvei strain K165 confers protection to melon against Fusarium wilt

Fusarium oxysporum f.sp. melonis (FOM) is a plant pathogen affecting melon production worldwide. An environmental friendly disease management strategy is the use of biocontrol agents (BCAs). Towards this direction, two BCA release strategies, seed coating and amendment of the transplant soil plug with the BCA strain Paenibacillus alvei K165 at various ratios, were evaluated against FOM in planta and in vitro. A reduction in Fusarium wilt symptom development was observed in melon plants, after mixing the transplant soil plug with K165 (10⁷ cfu g^?1 powder) at a ratio of 10 % (v/v). The monitoring of K165 rhizosphere population in the different treatments revealed a possible existence of a threshold population level that has to be attained before suppression of disease occurs. The data of the present study suggest that K165 plant protective activity against FOM can be possibly attributed to antibiosis and the triggering of Chit1 and Pal1 gene expression.     

Evaluation of molecular techniques for identification and enumeration of <Emphasis Type="Italic">Raoultella terrigena</Emphasis> ATCC 33257 in water purifier efficacy testing

Raoultella terrigena ATCC 33257, a representative of the coliform group, is commonly used as a challenge organism in water purifier efficacy testing. In addition to being time consuming, traditional culturing techniques and metabolic identification systems (including automated systems) also fail to accurately differentiate this organism from its closely related neighbors belonging to the Enterobacteriaceae group. Molecular-based techniques, such as real-time quantitative polymerase chain reaction (qPCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting, are preferred methods of detection because of their accuracy, reproducibility, specificity, and sensitivity, along with shorter turnaround time. ERIC-PCR performed with the 1R primer set demonstrated stable unique banding patterns (~800, ~300 bp) for R. terrigena ATCC 33257 different from patterns observed for R. planticola and R. ornithinolytica. The primer pair developed from gyraseA (gyrA) sequence of R. terrigena for the SYBR Green qPCR assay using the AlleleID^® 7.0 primer probe design software was highly specific and sensitive for the target organism. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cells and 4.7 fg with genomic DNA. The primer pair was successful in determining the concentration (5.5 ± 0.3 × 10⁶ CFU/ml) of R. terrigena from water samples spiked with equal concentration of Escherichia coli and R. terrigena. Based on these results from the ERIC-PCR and the SYBR Green qPCR assay, these molecular techniques can be efficiently used for rapid identification and quantification of R. terrigena during water purifier testing.     

Development and Padronization of Three Multiplex PCRs for the Diagnosis of Chlamydia trachomatis, Toxoplasma gondii, Herpes Simplex Viruses 1 and 2, and Cytomegalovirus

To develop multiplex PCRs (mPCRs) that allows simultaneous diagnosis of the infectious agents Chlamydia trachomatis, Toxoplasma gondii, HSV 1/2, and Cytomegalovirus (CMV). The study included patients with clinical suspicion of these agents, and clinical samples were blood, cerebrospinal fluid, urine, vaginal swabs, and amniotic fluid. After the extraction of DNA, this was used as a template in amplification by PCR of selected genes. The following conditions were tested: primer concentration, MgCl₂ concentration, and annealing temperature. Three mPCRs were developed: multiplex I (CMV, HSV 1/2), multiplex II (CMV, HSV 1/2, T. gondii), and multiplex III (C. trachomatis, T. gondii, HSV 1/2, and CMV). The primer pairs used were shown to be specific for each infectious agent, and the specificity of mPCR assays was 100 %. Both the reactions of the monoplex PCR and mPCR produced a detection limit of 2 × 10^?5 to 6 × 10^?7 ng/μl of different DNAs. Upon conclusion, amplified products of expected size were obtained in 3 different reactions, and all the infectious agents were detected simultaneously in each mPCR. The concordant results of the study suggest that mPCR can be a powerful tool to improve the diagnostics of infectious diseases.     

Potential of a 16S rRNA-Based Taxonomic Microarray for Analyzing the Rhizosphere Effects of Maize on Agrobacterium spp. and Bacterial Communities

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.