Phenotypic, genetic and symbiotic characterization of Erythrina velutina rhizobia from Caatinga dry forest

Abstract Erythrina velutina ("mulungu") is a legume tree from Caatinga that associates with rhizobia but the diversity and symbiotic ability of "mulungu" rhizobia are poorly understood. The aim of this study was to characterize "mulungu" rhizobia from Caatinga. Bacteria were obteined from Serra Talhada and Caruaru in Caatinga under natural regeneration. The bacteria were evaluated to the amplification of nifH and nodC and to metabolic characteristics. Ten selected bacteria identified by 16S rRNA sequences. They were tested in vitro to NaCl and temperature tolerance, auxin production and calcium phosphate solubilization. The symbiotic ability were assessed in an greenhouse experiment. A total of 32 bacteria were obtained and 17 amplified both symbiotic genes. The bacteria showed a high variable metabolic profile. Bradyrhizobium (6), Rhizobium (3) and Paraburkholderia (1) were identified, differing from their geographic origin. The isolates grew up to 45 °C to 0.51 mol L-1 of NaCl. Bacteria which produced more auxin in the medium with l-tryptophan and two Rhizobium and one Bradyrhizobium were phosphate solubilizers. All bacteria nodulated and ESA 90 (Rhizobium sp.) plus ESA 96 (Paraburkholderia sp.) were more efficient symbiotically. Diverse and efficient rhizobia inhabit the soils of Caatinga dry forests, with the bacterial differentiation by the sampling sites.

Characterization of rhizobia isolates obtained from nodules of wild genotypes of common bean

Abstract This study aimed to evaluate the tolerance to salinity and temperature, the genetic diversity and the symbiotic efficiency of rhizobia isolates obtained from wild genotypes of common bean cultivated in soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were subjected to different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28 °C, 33 °C, 38 °C, 43 °C and 48 °C). Genotypic characterization was performed based on BOX-PCR, REP-PCR markers and 16S rRNA sequencing. An evaluation of symbiotic efficiency was carried out under greenhouse conditions in autoclaved Leonard jars. Among 98 isolates about 45% of them and Rhizobium freirei PRF81 showed a high tolerance to temperature, while 24 isolates and Rhizobium tropici CIAT899 were able to use all of the carbon sources studied. Clustering analysis based on the ability to use carbon sources and on the tolerance to salinity and temperature grouped 49 isolates, R. tropici CIAT899 and R. tropici H12 with a similarity level of 76%. Based on genotypic characterization, 65% of the isolates showed an approximately 66% similarity with R. tropici CIAT899 and R. tropici H12. About 20% of the isolates showed symbiotic efficiency similar to or better than the best Rhizobium reference strain (R. tropici CIAT899). Phylogenetic analysis of the 16S rRNA revealed that two efficient isolates (ALSG5A1 and JPrG6A8) belong to the group of strains used as commercial inoculant for common bean in Brazil and must be assayed in field experiments.

Caracterización de rizobacterias promotoras de crecimiento en plántulas de Eucalyptus nitens / Characterization of growth-promoting rhizobacteria in Eucalyptus nitens seedlings

Se aislaron bacterias rizosféricas y endófitas a partir de rizósfera y tejidos de raíz de árboles de Eucalyptus nitens con el objetivo de evaluar su capacidad de promover el crecimiento en plántulas de la misma especie en condiciones de invernadero. Los aislamientos que incrementaron el crecimiento de las plántulas fueron identificados y caracterizados por su capacidad de producir ácido indolacético (AIA), solubilizar fosfato y expresar la 1-aminociclopropano-1-carboxilato (ACC) desaminasa. Los 105 aislamientos obtenidos fueron morfológicamente diferentes y solo 15 promovieron significativamente el crecimiento de plántulas de E. nitens. Los máximos incrementos observados fueron en el peso seco aéreo (142 %) y de la raíz (135 %); también aumentaron la altura de las plantas (50 %) y el largo de raíces (45 %) de las mismas. Las rizobacterias pertenecieron a los géneros Arthrobacter, Lysinibacillus, Rahnella y Bacillus. Los aislados identificados como A. phenanthrenivorans 21 y B. cereus 113 incrementaron la emergencia de E. nitens a los 12 días en un valor promedio de 3,15 veces con relación al control. R. aquatilis aislado 78 presentó la mayor producción de AIA (97,5 ± 2,87 μg/ml) en presencia de triptófano y el mayor índice de solubilización de fósforo (2,4). B. amyloliquefaciens aislado 60 fue positivo para la actividad ACC desaminasa. Los resultados obtenidos indican el potencial de las rizobacterias estudiadas como promotoras de emergencia y crecimiento de plántulas de E. nitens y su posible uso como inoculantes, ya que presentan más de un mecanismo de acción asociado a la promoción del crecimiento.

Rhizospheric and endophytic bacteria were isolated from the rizosphere and root tissue of Eucalyptus nitens. The objective of this work was to evaluate their capacity to promote growth in seedlings of the same species under greenhouse conditions. The isolates that improved seedling growth were identified and characterized by their capacity to produce indoleacetic acid (IAA), solubilize phosphates and increase 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. One hundred and five morphologically different strains were isolated, 15 of which promoted E. nitens seedling growth, significantly increasing the height (50%), root length (45%) as well as the aerial and root dry weight (142% and 135% respectively) of the plants. Bacteria belonged to the genus Arthrobacter, Lysinibacillus, Rahnella and Bacillus. Isolates A. phenanthrenivorans 21 and B. cereus 113 improved 3.15 times the emergence of E. nitens after 12 days, compared to control samples. Among isolated R. aquatilis, 78 showed the highest production of IAA (97.5±2.87 μg/ml) in the presence of tryptophan and the highest solubilizer index (2.4) for phosphorus, while B. amyloliquefaciens 60 isolate was positive for ACC deaminase activity. Our results reveal the potential of the studied rhizobacteria as promoters of emergence and seedling growth of E. nitens, and their possible use as PGPR inoculants, since they have more than one mechanism associated with plant growth promotion.

Phenotypic and Genotypic Characteristics of Rhizobia Isolated from Meknes-tafilalet Soils and Study of Their Ability to Nodulate Bituminaria bituminosa.

Aims: The objectives were to isolate and characterize phenotypically and genotypically the rhizobial strains from the soils belonging to the Meknes-Tafilalet region in order to select strains that are able to nodulate Bituminaria bituminosa. Study Design: An experimental study. Place and Duration of Study: Department of biology (Soil & Environment Microbiology Unit) Faculty of Sciences, Moulay Ismail University and Technical Support Unit for Scientific Research, CNRST in Rabat; between January and August 2010. Methodology: Samples from 23 different sites belonging to the Meknes-Tafilalet region were collected in order to select rhizobial strains that are able to nodulate Bituminaria bituminosa. The morphological, cultural and phenotypic parameters of isolated strains were studied. The phenotypic characteristics include colony morphology, growth speed, tolerances to temperature, salt and pH. To assess the genotypic diversity among the isolates, molecular characteristics based on 16S rDNA gene sequencing were performed. Results: The majority of the isolated strains showed fast-growing capacity (75%). Most strains tolerate neutral to alkaline pH, however some strains (18%) showed weak growth capacity at pH 4. All isolates were tolerant to high salt stress ([NaCl] = 3%). The genotypic characterization based on16S rDNA gene sequencing of the twelve strains showed a high diversity between the isolates. Conclusion: Taken together, our results highlight the important biodiversity of the isolated rhizobial strains and open opportunities for the development of new bio-fertilizer.

Rhizobium radiobacter recovered from blood of a pediatric patient with stem cell transplantation: a case report and characterization of antimicrobial susceptibility profile

Rhizobium radiobacter is an uncommon agent of infection and has been associated with indwelling intravascular devices such as catheter in immunocompromised patients. Here, we report a case of R. radiobacter recovered from blood cultures in stem cell transplantation in a pediatric patient and present an extensive characterization of its antimicrobial susceptibility profile. The isolate presented low MICs to many antimicrobial agents, but high MICs to ceftazidime, piperacillin-tazobactam, aztreonam, and fosfomycin.

Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant

The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.

Caracterización fenotípica y genotípica de doce rizobios aislados de diversas regiones geográficas de Venezuela

Phenotypic and genotypic characterization of twelve rhizobial isolates from different regions of Venezuela. Rhizobial taxonomy and systematics have progressed substantially, nevertheless, few studies have been developed on venezuelan species. This study evaluated the phenotypic and genetic variation between 12 venezuelan indigenous rhizobial isolates and 10 international referential strains, by phenotypical traits and DNA molecular markers. In this regard, a PCR-RFLP of the 16S rDNA gene, the presence of large plasmids, metabolic assays in solid media, salinity resistance, pH and temperature growth conditions, and intrinsic antibiotic resistance were assayed. In reference to the phenotypic attributes, we recognized three main groups: A group I, which comprised all the strains metabolizing between 67.5%-90% of the C and N sources. They were also acid-tolerant, as well as acid producers, capable of growing at 40ºC and in high salinity conditions (2-2.5% NaCl). With regard to the antibiotic sensitivity, this group was susceptible to a 30% of the antibiotic assayed. Strains belonging to Group II exhibited a lower salt tolerance (0.1-1.5%NaCl), as well as a lower acid tolerance, since they grew well at pH values equal or higher than 5.0. This group appeared to be resistant to all of the antibiotics assayed and only metabolized between 52.5%-82.5% of the C and N sources. Group III was represented by a single bacterial strain: it has a extremely low salt tolerance (0.1% NaCl). This strain grew at a pH equal or higher than 5.6, was susceptible to 50% of the antibiotics assayed and metabolized 72% of the C and N sources. On the basis of a PCR- RFLP of the 16S rDNA, three groups were also obtained. Members of the group A showed a close resemblance to Rhizobium tropici CIAT 899 and Sinorhizobium americanum CFN-EI 156, while Group B was closely related to Bradyrhizobium spp. Group C, was also represented by only one isolate. The Trebol isolate, was the only one strain able to form nodules and does not appear to be related to any of the referential rhizobial strains, suggesting a possible symbiotic horizontal gene transfer. Finally, in this work, there are evidences of a genetic diversity in the venezuelan rhizobial strains. A different geographical origin is perhaps an important factor affecting the diversity of the indigenous rhizobia in this study. Rev. Biol. Trop. 59 (3): 1017-1036. Epub 2011 September 01.

Rasgos fenotípicos y marcadores moleculares de ADN se utilizaron para investigar la variación fenotípica y genética entre 12 aislados rizobianos venezolanos y 10 cepas de referencia internacionales. Para ello, se realizó un PCR-RFLP del gen rDNA 16S, la presencia de plásmidos grandes, análisis metabólicos en medios sólidos, resistencia a la salinidad, condiciones del crecimiento a diferentes pH y temperaturas y la resistencia intrínseca a antibióticos. En referencia a las cualidades fenotípicas, se diferenciaron tres grupos principales, un grupo I que abarcó a todas aquellas cepas que metabolizaban entre 67.5% y 90% de las fuentes de C y de N. También eran tolerantes a la acidez y productoras de ácido, capaces de crecer a 40ºC y a altas condiciones de salinidad (NaCl 2-2.5%). Con respecto a la sensibilidad a antibióticos, este grupo era susceptible a un 30% de los antibióticos empleados. Las cepas que pertenecen al grupo II exhibieron una tolerancia salina más baja (0.1- 1.5%NaCl), así como una menor tolerancia a la acidez, puesto que crecieron bien en valores de pH iguales o mayores a 5.0. Este grupo era resistente a todos los antibióticos probados y metabolizaban solamente entre 52.5%-82.5% de las fuentes de C y de N. Una sola cepa bacteriana representó al grupo III, con una baja tolerancia salina (0.1% NaCl). Este aislado creció a un pH mayor o igual a 5.6, era susceptible a 50% de los antibióticos probados y metabolizó el 72% de las fuentes de C y de N. Al tener como base el PCR-RFLP del 16S rDNA, se diferenciaron también tres grupos. Los miembros del grupo A demostraron una estrecha relación con Rhizobium tropici CiAT 899 y Sinorhizobium americanum CFN-Ei156, mientras que el grupo B está estrechamente vinculado a Bradyrhizobium spp. El grupo C, está representado por solo un aislado. El aislado Trebol, fue la única cepa capaz de formar nódulos y no aparece relacionado con ninguna cepa de referencia, y sugiere una transferencia horizontal de genes simbióticos. Finalmente, en este trabajo se evidencia una diversidad genética en las cepas rizobianas venezolanas. El origen geográfico diverso de estas cepas, quizás sea un factor importante que influencie la diversidad de los rizobios indígenas utilizados en este estudio.

Succession and growth strategy of a spring microbial community from kezhou sinter in China

The succession and growth strategy of a spring microbial community under earthquake action were investigated. The majority of pre-earthquake isolates belonged to the Gammaproteobacteria, including two numerically dominant Stenotrophomonas sp. RB25 and Acinetobacter sp. RB11 (r-strategists). The predominant post-earthquake isolates were Alphaproteobacteria, with Rhizobium sp. RA42 (K-strategists) being dominant among these organisms.

Genetic diversity of indigenous common bean (Phaseolus vulgaris L) rhizobia from the state of Minas Gerais, Brazil

We characterized indigenous common bean rhizobia from five districts of the state of Minas Gerais, Brazil. The isolates were trapped by two common bean varieties, the Mineiro Precoce (Andean origin) and Ouro Negro (Mesoamerican origin). Analysis by BOX-PCR of selected isolates detected a high level of genetic diversity.

Bioproduction of indole acetic acid by Rhizobium strains isolated from root nodules of green manure crop, Sesbania sesban [L.] Merr

Twenty six Rhizobium strains were isolated from root nodules of Sesbania sesban [L.] Merr. collected from different regions of Andhra Pradesh. All the 26 Rhizobium strains produced indole acetic acid [IAA], but maximum amount was produced by only five strains in yeast extract mannitol [YEM] medium supplemented with L-tryptophan. The strains were found to elaborate maximum IAA when fed with 2.5 mg/ml Ltryptophan. Cultural requirements were optimized for maximum growth and IAA production. The strains differ in their growth and production of IAA on different carbon and nitrogen sources. Addition of cell wall affecting agents increased the IAA production over controls. The compound was extracted, purified and structurally confirmed as IAA

Genetic transformation and hairy root induction in Holostemma ada-kodien K. Schum--a vulnerable medicinal plant.

Hairy roots were induced from shoot buds and seedling hypocotyls of Holostemma by infection with agropine type Agrobacterium rhizogenes strains. Type of explant, Agrobacterium rhizogenes strain used for infection, co-culture time and photoperiod influenced the transformation frequencies. Hairy roots were induced from seedling hypocotyls and shoot bud explants upon infection with agropine type Agrobacterium rhizogenes strains. The hairy roots were thin, whitish in colour and showed negatively geotropic growth. The transformed nature of hairy roots was confirmed by opine analysis.

Extracellular polysaccharide production by Azorhizobium caulinodans from stem nodules of leguminous emergent hydrophyte Aeschynomene aspera.

The Azorhizobium caulinodans isolated from the stem nodules of a leguminous emergent hydrophyte, Aeschynomene aspera, produced a large amount of extracellular polysaccharides (EPS) in yeast extract basal medium. Maximum EPS production was at the stationary phase of growth. EPS production was increased by 919% over control when the medium was supplemented with sucrose (1.5%), D-biotin (1 microgram/ml) and casamino acid (0.1%). EPS contained rhamnose and arabinose. Possible role of the azorhizobial EPS production in the stem nodule symbiosis is discussed.

Interactions between VA mycorrhizal fungus glomus mosseae, bradyrhizobium japonicum and pseudomonas syringae in soybean plants

The interactions between VA mycorrhizal fungus Glomus mosseae, the pathogenic bacterium Pseudomonas syringae, in the presence or absence of Bradyrhizobium japonicum on some physiological parameters of soybean plants were investigated. The results revealed that co- inoculation of G. mosseae with B. japonicum increased the shoot dry weight, nodulation, accumulated nitrogen and phosphorus as well as nitrate reductase activity in soybean plants. G. mosseae prevented P. syringae from infecting soybean plants and improved the plants growth. Nitrogen and phosphorus accumulation and nitrate reductase activity. The presence of the mycorrhizal fungus suppressed the population density of the pathogenic bacterium in the rhizosphere of soybean plants. The mycorrhiza increased the plant resistance to P. syringae more prominently in the presence of B. japonicum

Estudio microbiológico de un grupo de cepas de Rhizobium phaseoli aisladas en la Meseta Central (México) / Microbiological study of a group of Rhizobium phaseoli strains from the Central Plateau, México

Ciento veinte cepas de Rhizobium phaseoli fueron aisladas de los Estados de México, Tlaxcala, Hidalgo y Puebla. Estas fueron caracterizadas por la morfología macroscópica y microscópica, además de sus propiedades bioquímicas como tolerancia a diferentes concentraciones de NaCl en donde se encontró que el 25.2% crecieron en una concentración de 5% de NaCl; por outro lado el 9.24% fue tolerante a la acidez (pH = 4.5), el mayor porciento de las cepas se comportó como ácido productoras; no hubo cambios notorios al crecer en la leche tornasolada; y sólo el 20.8% de las cepas tuvieron la capacidad de reducir la sal de tetrazolium, coniderándose por esta característica Hup+. Los resultados obtenidos permitieron agrupar varias cepas con propiedades fisiológicas que se pusieron de manifiesto a nivel de invernadero y campo