Investigating the mechanisms underlying phytoprotection by plant growth-promoting rhizobacteria in Spartina densiflora under metal stress.

Pollution of coasts by toxic metals and metalloids is a worldwide problem for which phytoremediation using halophytes and associated microbiomes is becoming relevant. Metal(loid) excess is a constraint for plant establishment and development, and plant growth promoting rhizobacteria (PGPR) mitigate plant stress under these conditions. However, mechanisms underlying this effect remain elusive. The effect of toxic metal(loid)s on activity and gene expression of ROS-scavenging enzymes in roots of the halophyte Spartina densiflora grown on real polluted sediments in a greenhouse experiment was investigated. Sediments of the metal-polluted joint estuary of Tinto and Odiel rivers and control, unpollutred samples from the Piedras estuary were collected and submitted to ICP-OES. Seeds of S. densiflora were collected from the polluted Odiel marshes and grown in polluted and unpolluted sediments. Rhizophere biofilm-forming bacteria were selected based on metal tolerance and inoculated to S. densiflora and grown for 4 months. Fresh or frozen harvested plants were used for enzyme assays and gene expression studies, respectively. Metal excess induced SOD (five-fold increase), whereas CAT and ascorbate peroxidase displayed minor induction (twofold). A twofold increase of TBARs indicated membrane damage. Our results showed that metal-resistant PGPR (P. agglomerans RSO6 and RSO7 and B. aryabhattai RSO25) contributed to alleviate metal stress, as deduced from lower levels of all antioxidant enzymes to levels below those of non-exposed plants. The oxidative stress index (OSI) decreased between 50 and 75% upon inoculation. The results also evidenced the important role of PAL, involved in secondary metabolism and/or lignin synthesis, as a pathway for metal stress management in this halophyte upon inoculation with appropriate PGPR, since the different inoculation treatments enhanced PAL expression between 3.75- and five-fold. Our data confirm, at the molecular level, the role of PGPR in alleviating metal stress in S. densiflora and evidence the difficulty of working with halophytes for which little genetic information is available.

Repeatability of gait analysis measurements in Thoroughbreds in training.

BACKGROUND: With the view of implementing gait symmetry measurements in Thoroughbreds in training for early detection of injuries, repeatability of inertial measurement unit (IMU) gait parameters needs to be established. OBJECTIVES: To assess the variation of head and pelvis movement symmetry in Thoroughbreds in training. STUDY DESIGN: Repeated observations in horses in race training. METHODS: Daily and weekly repeat gait assessments were conducted in 14 Thoroughbreds equipped with IMUs on poll, sacrum and right (RTC) and left (LTC) tuber coxae. Gait was assessed in trot, in-hand, on a level concrete surface. Difference between vertical displacement minima and maxima and range of motion (ROM) were obtained. Ranges containing 50% (median), 75, 90 and 95% of absolute daily and weekly differences were calculated and intraclass correlation coefficients (ICC) calculated for daily and weekly repeats. RESULTS: Median absolute daily differences ranged from 4 to 7 mm and median weekly differences from 4 to 8 mm. 90% of daily differences were between 9 and 16 mm and 90% of weekly differences between 11 and 19 mm. ICC values were found on average across sensors and gait parameters as 0.73 (ranging from 0.40 to 0.92 across parameters) for daily repeats and as 0.65 (0.27 to 0.91) for weekly repeats. MAIN LIMITATIONS: Horses were of varying training and movement asymmetry levels, and no veterinary lameness examination was conducted. CONCLUSIONS: Daily and weekly repeat gait assessments in this group of Thoroughbreds in training show lower ICC values than previously reported from within-day repeats in horses during lameness examinations. We recommend conducting repeatability studies for specific groups of horses when planning long-term studies aiming at identifying horses at risk of injury.

Head, withers and pelvic movement asymmetry and their relative timing in trot in racing Thoroughbreds in training.

BACKGROUND: Horses show compensatory head movement in hindlimb lameness and compensatory pelvis movement in forelimb lameness but little is known about the relationship of withers movement symmetry with head and pelvic asymmetry in horses with naturally occurring gait asymmetries. OBJECTIVES: To document head, withers and pelvic movement asymmetry and timing differences in horses with naturally occurring gait asymmetries. STUDY DESIGN: Retrospective analysis of gait data. METHODS: Head, withers and pelvic movement asymmetry and timing of displacement minima and maxima were quantified from inertial sensors in 163 Thoroughbreds during trot-ups on hard ground. Horses were divided into 4 subgroups using the direction of head and withers movement asymmetry. Scatter plots of head vs. pelvic movement asymmetry illustrated how the head-withers relationship distinguishes between contralateral and ipsilateral head-pelvic movement asymmetry. Independent t test or Mann-Whitney U test (P<0.05) compared pelvic movement asymmetry and timing differences between groups. RESULTS: The relationship between head and withers asymmetry (i.e. same sided or opposite sided asymmetry) predicts the relationship between head and pelvic asymmetry in 69-77% of horses. Pelvic movement symmetry was significantly different between horses with same sign vs. opposite sign of head-withers asymmetry (P<0.0001). Timing of the maximum head height reached after contralateral ('sound') stance was delayed compared to withers (P = 0.02) and pelvis (P = 0.04) in horses with contralateral head-withers asymmetry. MAIN LIMITATIONS: The clinical lameness status of the horses was not investigated. CONCLUSION: In the Thoroughbreds with natural gait asymmetries investigated here, the direction of head vs. withers movement asymmetry identifies the majority of horses with ipsilateral and contralateral head and pelvic movement asymmetries. Withers movement should be further investigated for differentiating between forelimb and hindlimb lame horses. Horses with opposite sided head and withers asymmetry significantly delay the upward movement of the head after 'sound' forelimb stance.

Tumores neuroendocrinos de origen pulmonar: reporte de un caso / Neuroendocrine tumors of lung’s origin, a case report

Introduction or case story: Young female patient (24 years-old), without known morbid precedents. She comes for a ten-days period of symptoms characterized by fever of up to 38.5 °C and a dyspnoea grade III. Physical exam showed decreased vesicular murmur on the right pulmonary base with dullness and positive vocal vibrations. Exams: Thorax X-ray: Atelectasis condensation on the right pulmonary base. CT chest scan without contrast: Nodule located in an intermediate bronchus which generates atelectasis in the basal bronchi. Fibro-bronchoscopy (FOB): A tumour-like injury blocking 100% of the right intermediate bronchus’ duct. Biopsy: Pulmonary tissue with haemorrhagic areas, granular tissue with small cellular clusters of lobular disposal and glandular shape with eccentric central nuclei cells, with homogenous chromatin and without atypical mitosis. Immunohistochemistry: Intensely positive cells to synaptophysin and CD56. Diagnosis: Neuroendocrine Typical Carcinoid Tumor. Comments: The patient evolves without progression of dyspnoea, she is waiting for a surgical resolution of tumour at National Institute of Thorax.

Introducción o historia del caso: Mujer joven de 24 años de edad, sin antecedentes mórbidos, acudió por cuadro de 10 días de evolución, de fiebre de hasta 38,5 °C y disnea grado III. Al examen físico destacó a nivel pulmonar: murmullo pulmonar disminuido en base pulmonar derecha, matidez de la misma zona y vibraciones vocales positivas. Exámenes: Radiografía de Tórax: Condensación atelectásica en base pulmonar derecha. TAC de Tórax sin contraste: Imagen nodular a nivel de bronquio intermedio, que genera atelectasia en bronquios basales. Fibrobroncoscopía (FBC): Lesión tumoral que ocluye el 100% del lumen para bronquio intermedio derecho. Biopsia: Tejido pulmonar con áreas de hemorragia, tejido granulatorio y tumor con acúmulos celulares de disposición lobular y glanduliforme, con núcleos centrales excéntricos, cromatina homogénea, sin atipias. Inmunohistoquímica: Células intensamente positivas para sinaptofisina, y CD-56. Diagnóstico: Tumor Neuroendocrino Carcinoide típico Comentarios: Paciente evoluciona sin progresión de su disnea, esperando resolución quirúrgica del tumor en Instituto Nacional del Tórax.

Assessing the role of endophytic bacteria in the halophyte Arthrocnemum macrostachyum salt tolerance.

There is an increasing interest to use halophytes for revegetation of salt affected ecosystems, as well as in understanding their mechanisms of salt tolerance. We hypothesized that bacteria from the phyllosphere of these plants might play a key role in its high tolerance to excessive salinity. Eight endophytic bacteria belonging to Bacillus and closely related genera were isolated from phyllosphere of the halophyte Arthrocnemum macrostachyum growing in salty agricultural soils. The presence of plant-growth promoting (PGP) properties, enzymatic activities and tolerance towards NaCl was determined. Effects of inoculation on seeds germination and adult plant growth under experimental NaCl treatments (0, 510 and 1030 mM NaCl) were studied. Inoculation with a consortium including the best performing bacteria improved considerably the kinetics of germination and the final germination percentage of A. macrostachyum seeds. At high NaCl concentrations (1030 mM), inoculation of plants mitigated the effects of high salinity on plant growth and physiological performance and, in addition, this consortium appears to have increased the potential of A. macrostachyum to accumulate Na+ in its shoots, thus improving sodium phytoextraction capacity. Bacteria isolated from A. macrostachyum phyllosphere seem to play an important role in plant salt tolerance under stressing salt concentrations. The combined use of A. macrostachyum and its microbiome can be an adequate tool to enhance plant adaptation and sodium phytoextraction during restoration of salt degraded soils.

Isolation of plant-growth-promoting and metal-resistant cultivable bacteria from Arthrocnemum macrostachyum in the Odiel marshes with potential use in phytoremediation.

Arthrocnemum macrostachyum is a halophyte naturally growing in southwest coasts of Spain that can tolerate and accumulate heavy metals. A total of 48 bacteria (30 endophytes and 18 from the rhizosphere) were isolated from A. macrostachyum growing in the Odiel River marshes, an ecosystem with high levels of contamination. All the isolates exhibited plant-growth-promoting (PGP) properties and most of them were multiresistant to heavy metals. Although the presence of heavy metals reduced the capability of the isolates to exhibit PGP properties, several strains were able to maintain their properties or even enhance them in the presence of concrete metals. Two bacterial consortia with the best-performing endophytic or rhizospheric strains were selected for further experiments. Bacterial inoculation accelerated germination of A. macrostachyum seeds in both the absence and presence of heavy metals. These results suggest that inoculation of A. macrostachyum with the selected bacteria could ameliorate plant establishment and growth in contaminated marshes.

Scouting contaminated estuaries: heavy metal resistant and plant growth promoting rhizobacteria in the native metal rhizoaccumulator Spartina maritima.

Spartina maritima is a native endangered heavy metal rhizoaccumulator cordgrass naturally growing in southwest coasts of Spain, where is used as a biotool to rehabilitate degraded salt marshes. Fifteen bacterial strains were isolated from the rhizosphere of S. maritima growing in the estuary of the Tinto River, one of the most polluted areas in the world. A high proportion of bacteria were resistant towards several heavy metals. They also exhibited multiple plant growth promoting (PGP) properties, in the absence and the presence of Cu. Bacillus methylotrophicus SMT38, Bacillusaryabhattai SMT48, B. aryabhattai SMT50 and Bacilluslicheniformis SMT51 were selected as the best performing strains. In a gnobiotic assay, inoculation of Medicago sativa seeds with the selected isolates induced higher root elongation. The inoculation of S. maritima with these indigenous metal-resistant PGP rhizobacteria could be an efficient method to increase plant adaptation and growth in contaminated estuaries during restoration programs.

Prospecting metal-resistant plant-growth promoting rhizobacteria for rhizoremediation of metal contaminated estuaries using Spartina densiflora.

In the salt marshes of the joint estuary of Tinto and Odiel rivers (SW Spain), one of the most polluted areas by heavy metals in the world, Spartina densiflora grows on sediments with high concentrations of heavy metals. Furthermore, this species has shown to be useful for phytoremediation. The total bacterial population of the rhizosphere of S. densiflora grown in two estuaries with different levels of metal contamination was analyzed by PCR denaturing gradient gel electrophoresis. Results suggested that soil contamination influences bacterial population in a greater extent than the presence of the plant. Twenty-two different cultivable bacterial strains were isolated from the rhizosphere of S. densiflora grown in the Tinto river estuary. Seventy percent of the strains showed one or more plant growth-promoting (PGP) properties, including phosphate solubilization and siderophores or indolacetic acid production, besides a high resistance towards Cu. A bacterial consortium with PGP properties and very high multiresistance to heavy metals, composed by Aeromonas aquariorum SDT13, Pseudomonas composti SDT3, and Bacillus sp. SDT14, was selected for further experiments. This consortium was able to two-fold increase seed germination and to protect seeds against fungal contamination, suggesting that it could facilitate the establishment of the plant in polluted estuaries.

Taxonomic and symbiotic diversity of bacteria isolated from nodules of Acacia tortilis subsp. raddiana in arid soils of Tunisia.

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana nodules from various arid soils in Tunisia was analyzed for their diversity at both taxonomic and symbiotic levels. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at 40 °C. Genetic characterization emphasized that most of the strains (42/50) belong to the genus Ensifer, particularly the species Ensifer meliloti, Ensifer garamanticus, and Ensifer numidicus. Symbiotic properties of isolates showed diversity in their capacity to nodulate their host plant and to fix atmospheric nitrogen. The most effective isolates were closely related to E. garamanticus. Nodulation tests showed that 3 strains belonging to Mesorhizobium genus failed to renodulate their host plant, which is surprising for symbiotic rhizobia. Furthermore, our results support the presence of non-nodulating endophytic bacteria belonging to the Acinetobacter genus in legume nodules.

Self-bioremediation of cork-processing wastewaters by (chloro)phenol-degrading bacteria immobilised onto residual cork particles.

Cork manufacturing is a traditional industry in Southern Europe, being the main application of this natural product in wine stoppers and insulation. Cork processing begins at boiling the raw material. As a consequence, great volumes of dark wastewaters, with elevated concentrations of chlorophenols, are generated, which must be depurated through costly physicochemical procedures before discarding them into public water courses. This work explores the potential of bacteria, isolated from cork-boiling waters storage ponds, in bioremediation of the same effluent. The bacterial population present in cork-processing wastewaters was analysed by DGGE; low bacterial biodiversity was found. Aerobic bacteria were isolated and investigated for their tolerance against phenol and two chlorophenols. The most tolerant strains were identified by sequencing 16S rDNA. The phenol-degrading capacity was investigated by determining enzyme activities of the phenol-degrading pathway. Moreover, the capacity to form biofilms was analysed in a microtitre plate assay. Finally, the capacity to form biofilms onto the surface of residual small cork particles was evaluated by acridine staining followed by epifluorescence microscopy and by SEM. A low-cost bioremediation system, using phenol-degrading bacteria immobilised onto residual cork particles (a by-product of the industry) is proposed for the remediation of this industrial effluent (self-bioremediation).

Characterization of root-nodulating bacteria associated to Prosopis farcta growing in the arid regions of Tunisia.

Diversity of 50 bacterial isolates recovered from root nodules of Prosopis farcta grown in different arid soils in Tunisia, was investigated. Characterization of isolates was assessed using a polyphasic approach including phenotypic characteristics, 16S rRNA gene PCR--RFLP and sequencing, nodA gene sequencing and MLSA. It was found that most of isolates are tolerant to high temperature (40°C) and salinity (3%). Genetic characterization emphasizes that isolates were assigned to the genus Ensifer (80%), Mesorhizobium (4%) and non-nodulating endophytic bacteria (16%). Forty isolates belonging to the genus Ensifer were affiliated to Ensifer meliloti, Ensifer xinjiangense/Ensifer fredii and Ensifer numidicus species. Two isolates belonged to the genus Mesorhizobium. Eight isolates failing to renodulate their host plant were endophytic bacteria and belonged to Bacillus, Paenibacillus and Acinetobacter genera. Symbiotic properties of nodulating isolates showed a diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Isolate PG29 identified as Ensifer meliloti was the most effective one. Ability of Prosopis farcta to establish symbiosis with rhizobial species confers an important advantage for this species to be used in reforestation programs. This study offered the first systematic information about the diversity of microsymbionts nodulating Prosopis farcta in the arid regions of Tunisia.

Plasmid Transfer Detection in Soil using the Inducible lPR System Fused to Eukaryotic Luciferase Genes.

We report a model system for plasmid transfer analysis using the regulated lambda phage right promoter, lPR, fused to luc and lucOR as reporter genes. We have demonstrated that the systems cI857-lPR::luc and cI857-lPR::lucOR are temperature-inducible in Escherichia coli but not in other Gram-negative bacteria analyzed, enabling detection of luminescence when plasmids were mobilized from E. coli to those Gram-negative backgrounds. Using light for the detection, we have observed plasmid transfer from E. coli harboring RK2 and R388 derived plasmids to Pseudomonas putida KT2440 (co-introduced with donors) and to indigenous microorganisms, in vitro and in nonsterile soil microcosms. The importance of nutrients for an efficient plasmid transfer in nonsterile soil microcosms has been confirmed. When plasmid transfer experiments were carried out into nonsterile soil microcosms, significant populations of indigenous transconjugants arose. This system provides efficient marker genes and avoids the use of antibiotics for the selection of transconjugants.

Structure of the extracellular polysaccharide secreted by Rhizobium leguminosarum var. phaseoli CIAT 899.

The structure of the extracellular polysaccharide secreted by Rhizobium leguminosarum var. phaseoli CIAT 899 has been studied by methylation analysis. 1H-n.m.r. spectroscopy, and partial acid hydrolysis. The repeating unit is an octasaccharide made up of D-glucose, D-galactose, pyruvic acid, and acetic acid in the molar ratios 6:2:1.5:1.5. Half of the terminal Gal groups are 4,6-substituted by pyruvic acid acetal groups and the other half by O-acetyl groups at position 3. Also, one of the 3-linked glucosyl residues carries a pyruvic acid 4,6-acetal group and one of the 4-linked glucosyl residues is acetylated at position 6.

Use of plasmid R68.45 for constructing a circular linkage map of the Rhizobium trifolii chromosome.

Plasmid R68.45 was used to promote conjugal transfer of chromosomal markers in Rhizobium trifolii RS55. Analysis of two-factor and three-factor crosses among R. trifolii strains enabled construction of a circular linkage map of the R. trifolii chromosome, containing 17 nutritional and resistance markers.