A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.

First detection of Waddlia chondrophila in Africa using SYBR Green real-time PCR on veterinary samples.

Waddlia chondrophila is a strict intracellular microorganism belonging to the order Chlamydiales that has been isolated twice from aborted bovine fetuses, once in USA and once in Germany. This bacterium is now considered as an abortigenic agent in cattle. However, no information is available regarding the presence of this bacterium in Africa. Given the low sensitivity of cell culture to recover such an obligate intracellular bacterium, molecular-based diagnostic approaches are warranted. This report describes the development of a quantitative SYBR Green real-time PCR assay targeting the recA gene of W. chondrophila. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing pathogens that can cause abortion in cattle. The PCR exhibited a good intra-run and inter-run reproducibility. This real-time PCR was then applied to 150 vaginal swabs taken from Tunisian cows that have aborted. Twelve samples revealed to be Waddlia positive, suggesting a possible role of this bacterium in this setting. This new real-time PCR assay represents a diagnostic tool that may be used to further study the prevalence of Waddlia infection.

Rhizobium strains competitiveness on bean nodulation in Cerrado soils

The objective of this work was to identify the most competitive and effective Rhizobium strains in order to increase common bean yield by nitrogen fixation as alternative or complementation to the nitrogen fertilization. Competitiveness tests were lead in axenic conditions, in Cerrado soil pots and in three field experiments, with native Rhizobium strains that were previously identified, according to their effectiveness and genetic variability. The identification of strains in nodules was performed using serological tests (axenic conditions) - agglutination and enzyme linked immunosorbent (Elisa) assays - and random amplified polymorfic DNA (RAPD) (Cerrado soil). Plant yield was determined using the dry weight (greenhouse conditions), total N and grain yield (field experiments). Among the analyzed Rhizobium strains, native strain SLA 2.2 and commercial strain CIAT 899 were the dominant nodules in plants of the most productive plots, presenting yield productivity similar or higher to those obtained in treatments where 20 kg ha-1 of N were applied.

Ultrastructural changes of the internal limiting membrane removed during indocyanine green assisted peeling versus conventional surgery for an idiopathic epimacular membrane

Purpose:To evaluate the histological features of cellular retinal fragments on the internal limiting membrane (ILM) removed during epiretinal membrane peeling surgery with and without the aid of ICG diluted in 5% glucose Methods:ILM specimens removed from 88 eyes undergoing vitrectomy and membrane peeling surgery for idiopathic epiretinal membrane between 1995 and 2003 were reviewed retrospectively. Surgery was performed in all cases by the same surgeon using the same technique. ICG was diluted with 5% glucose. Histological analysis focused on the presence and characteristics of retinal structures on the retinal surface of the ILM. Statistical analysis compared the results between group I (conventional surgery without ICG) and group II (ICG-assisted peeling) Results:Seventy-one eyes underwent EMM surgery without the aid of ICG (group I) and seventeen underwent EMM ICG-assisted surgery assisted using ICG (group II). The amount of Muller cell debris on the retinal surface of the ILM was more significant in the group I (no ICG) than in the group II (ICG) (40.8 versus 11.8; p = 0.024). Large fragments of Muller cells were more frequently observed in the group I (no ICG) than in the group II (ICG) (63.4% versus 23.5%; p= 0.003).The presence of larger retinal elements such as neural axons and vessels were observed attached to retinal face of the ILM in 5 (7%) cases of the no-ICG group. No such retinal elements were detected in any of the histological ILM specimens of the ICG-assisted group Conclusions:The use of ICG diluted with 5% glucose in the aid of ILM removal during epiretinal membrane surgery was associated with less retinal debris attached to retinal face of the ILM compared to surgery in which ICG was not used. Our findings contradict previous reports in the literature, in which use of ICG diluted with balanced salt solution (BSS) was associated with more retinal fragments attached to the retinal face of the ILM. According to our results, we hypothesize that diluting ICG with 5% glucose may decrease the adhesion of the ILM to the underlying retinal layers such that less retinal debris is removed with peeling of the ILM.

Microsatellites for genetic studies and breeding programs in common bean

Twenty microsatelitte loci were identified and characterized in common bean. Microsatellites were tested in 14 genotypes. The allele number ranged from 1 to 3, and the polymorphism information content (PIC) was between 0.14 and 0.65. These polymorphic markers are available to be used for breeding programs.

Method for evaluation of root hairs of common bean genotypes

The objective of this work was to test a simple method for root hair evaluation of 21 common bean (Phaseolus vulgaris) genotypes, most of them used in breeding programs in Brazil. Hairs of basal and primary roots of 5-day old seedlings, produced on germination paper with no phosphorus addition, were visually evaluated by a rating scale after staining with 0.05% trypan blue. The method reveals variability among the genotypes, and the standard error of the mean is relatively low.

Recovery of a persistent Canine distemper virus expressing the enhanced green fluorescent protein from cloned cDNA.

Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.

Root traits of common bean genotypes used in breeding programs for disease resistance

The objective of this work was to assess root traits of 19 common bean genotypes, used in breeding programs for disease resistance. Genotypes DOR 364 and G 19833 were used as deep and shallow basal root checks, respectively. The number of whorls and basal roots were assessed on five-day old seedlings grown in germination paper. Growth pouch studies were conducted to evaluate basal root gravitropism and lateral root length from primary roots, in seven-day old seedlings. The following root gravitropic traits were estimated: basal growth angle, shallow basal root length (localized in the top 2 cm), and relative shallow basal root growth. Number of whorls varied from 1.47 to 3.07, and number of basal roots ranged from 5.67 (genotype TO) to 12.07 (cultivar Jalo MG-65). Cultivars BRS MG Talismã, Carioca, BRS Pioneiro, and Diamante Negro exhibited shallow basal roots, while genotypes Vi-10-2-1, TU, AB 136, and México 54 showed deep basal roots. Cultivar Jalo MG-65 showed more lateral roots from the primary root than the other genotypes. Genotypes used on common bean breeding programs for disease resistance have great variability on basal and primary root traits.

New microsatellite markers developed from an enriched microsatellite common bean library

The objective of this work was to develop new microsatellite markers in common bean. Ninety nine new microsatelitte loci were developed from a microsatellite enriched library for (CT)8 and (GT)8 motifs, from CAL-143 line. The majority of microsatellite sequences (51%) was related to cellular metabolism. The remaining sequences were associated to transcription functions. Only 17.2% of the sequences presented some level of similarity with other plant species genes.

Combining ability and heterosis in common bean cultivars

The objective of this work was to determine the combining ability and heterosis, for productivity and yield components, in diallel hybrids derived from crossings between BRSMG-Talismã, IPR Uirapuru, FT Soberano, BRS Campeiro, IAC Tybatã, and IPR Juriti parent cultivars. Fifteen hybrids were generated from diallel crosses, excluding reciprocals. The general and specific combining abilities were significant for plant height, number of pods per plant, number of seeds per plant, number of seeds per pod, 50-seed weight, and grain yield, indicating the occurrence of both additive and nonadditive genetic effects. The best strategy to be adopted is the use of BRS Campeiro, FT Soberano and BRSMG-Talismã cultivars in common bean breeding programs involving selection. The most promising combinations were 'IPR Uirapuru' x 'IAC Tybatã', 'IPR Uirapuru' x 'FT Soberano', 'BRS Campeiro' x 'IPR Juriti', and 'BRS Campeiro' x 'IAC Tybatã'. The parents of these hybrids presented high estimates of specific combining abilities. Hybridization of cultivars belonging to distinguished commercial groups propitiates higher heterosis values in the segregant population.

Genetic characterization and nitrogen fixation capacity of Rhizobium strains on common bean

This study aimed to genetically characterize four new Rhizobium strains, and to evaluate their nodulation and fixation capacity compared to commercial strains and to native rhizobia population of a Brazilian Rhodic Hapludox. Two experiments were carried out in randomized blocks design, under greenhouse conditions, in 2007. In the first experiment, the nodulation and nitrogen fixation capacity of new strains were evaluated, in comparison to the commercial strains CIAT-899 and PRF-81 and to native soil population. It was carried out in plastic tubes filled with vermiculite. DNA extractions and PCR sequencing of the intergenic space were made from the isolated pure colonies, in order to genetically characterize the strains and the native rhizobia population. In the second experiment, the nodulation and productivity of common beans Perola cultivar were determined, with the use of evaluated strains, alone or in mixture with PRF-81 strain. It was carried out in pots filled with soil. The native soil population was identified as Rhizobium sp. and was inefficient in nitrogen fixation. Three different Rhizobium species were found among the four new strains. The LBMP-4BR and LBMP-12BR new strains are among the ones with greatest nodulation and fixation capacity and exhibit differential responses when mixed to PRF-81.

Toxicity of neem oil to Bemisia tabaci biotype B nymphs reared on dry bean

The objective of this work was to determine the most susceptible nymphal stage of Bemisia tabaci biotype B to neem (Azadirachta indica A. Juss.) oil applied to dry bean (Phaseolus vulgaris L.) in a screenhouse. A solution of commercial oil (Dalneem) extracted from neem seeds was sprayed directly on each nymphal instar at 0, 0.1, 0.25, 0.5, 1 and 2% concentrations for lethal concentration (LC) determination, and at 0, 0.5 and 1% concentrations for lethal time (LT) determination. The number of living and dead nymphs was recorded five days after spraying for LC determination, and daily during six days for LT determination. The LC50 estimated for fourth instar nymphs occurred at 0.56% concentration. For all instars, LC50 and LC95 were estimated at 0.32 and 2.78% concentrations, respectively. The estimated values of LT50 at 1% concentration were 2.46, 4.45, 3.02 and 6.98 days for the first to fourth instars, respectively. The LT50 occurred at five days for 0.5% and at four days for 1% concentration in all instars. A mortality rate of over 80% was observed on the 6th day for the first to third instars at 1% concentration. The first three nymphal stages were more susceptible to neem oil when compared to the fourth nymphal stage.