Influences of plant organ, genotype, and cultivation site on the endophytic bacteriome of maize (Zea mays L.) in the semi-arid region of Pernambuco, Brazil.

Endophytic bacteria play a crucial role in plant development and adaptation, and the knowledge of how endophytic bacteria assemblage is influenced by cultivation site and plant genotype is an important step to achieve microbiome manipulation. This work aimed to study the roots and stems of endophytic bacteriome of four maize genotypes cultivated in two regions of the semi-arid region of Pernambuco - Brazil. Our hypothesis is that the endophytic community assemblage will be influenced by plant genotypes and cultivation region. Metabarcoding sequencing data revealed significant differences in alfa diversity in function of both factors, genotypes, and maize organs. Beta diversity analysis showed that the bacterial communities differ mainly in function of the plant organ. The most abundant genera found in the samples were Leifsonia, Bacillus, Klebsiella, Streptomyces, and Bradyrhizobium. To understand ecological interactions within each compartment, we constructed co-occurrence network for each organ. This analysis revealed important differences in network structure and complexity and suggested that Leifsonia (the main genera found) had distinct ecological roles depending on the plant organ. Our data showed that root endophytic maize bacteria would be influenced by cultivation site, but not by genotype. We believe that, collectively, our data not only characterize the bacteriome associated with this plant and how different factors shape it, but also increase the knowledge to select potential bacteria for bioinoculant production.

Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes.

The soil environment adjacent to plant roots, termed the rhizosphere, is home to a wide variety of microorganisms that can significantly affect the physiology of nearby plants. Microbes in the rhizosphere can provide nutrients, secrete signaling compounds, and inhibit pathogens. These processes could be manipulated with synthetic biology to enhance the agricultural performance of crops grown for food, energy, or environmental remediation, if methods can be implemented in these nonmodel microbes. A common first step for domesticating nonmodel organisms is the development of a set of genetic engineering tools, termed a synthetic biology toolbox. A toolbox comprises transformation protocols, replicating vectors, genome engineering (e.g., CRISPR/Cas9), constitutive and inducible promoter systems, and other gene expression control elements. This work validated synthetic biology toolboxes in three nitrogen-fixing soil bacteria: Azotobacter vinelandii, Stutzerimonas stutzeri (Pseudomonas stutzeri), and a new isolate of Klebsiella variicola. All three organisms were amenable to transformation and reporter protein expression, with several functional inducible systems available for each organism. S. stutzeri and K. variicola showed more reliable plasmid-based expression, resulting in successful Cas9 recombineering to create scarless deletions and insertions. Using these tools, we generated mutants with inducible nitrogenase activity and introduced heterologous genes to produce resorcinol products with relevant biological activity in the rhizosphere.

Arbuscular mycorrhizal fungi associated with the rhizosphere of an endemic terrestrial bromeliad and a grass in the Brazilian neotropical dry forest.

Arbuscular mycorrhizal fungi form symbiotic associations with 80-90% of all known plants, allowing the fungi to acquire plant-synthesized carbon, and confer an increased capacity for nutrient uptake by plants, improving tolerance to abiotic and biotic stresses. We aimed at characterizing the mycorrhizal community in the rhizosphere of Neoglaziovia variegata (so-called `caroa`) and Tripogonella spicata (so-called resurrection plant), using high-throughput sequencing of the partial 18S rRNA gene. Both plants are currently undergoing a bioprospecting program to find microbes with the potential of helping plants tolerate water stress. Sampling was carried out in the Caatinga biome, a neotropical dry forest, located in northeastern Brazil. Illumina MiSeq sequencing of 37 rhizosphere samples (19 for N. variegata and 18 for T. spicata) revealed a distinct mycorrhizal community between the studied plants. According to alpha diversity analyses, T. spicata showed the highest richness and diversity based on the Observed ASVs and the Shannon index, respectively. On the other hand, N. variegata showed higher modularity of the mycorrhizal network compared to T. spicata. The four most abundant genera found (higher than 10%) were Glomus, Gigaspora, Acaulospora, and Scutellospora, with Glomus being the most abundant in both plants. Nonetheless, Gigaspora, Diversispora, and Ambispora were found only in the rhizosphere of N. variegata, whilst Scutellospora, Paraglomus, and Archaeospora were exclusive to the rhizosphere of T. spicata. Therefore, the community of arbuscular mycorrhizal fungi of the rhizosphere of each plant encompasses a unique composition, structure and modularity, which can differentially assist them in the hostile environment.

Impact of Water Shortage on Soil and Plant Attributes in the Presence of Arbuscular Mycorrhizal Fungi from a Harsh Environment.

Arbuscular mycorrhizal fungi (AMF) play a crucial role in plant health due to their ability to improve tolerance to biotic and abiotic stresses. Our aim was to evaluate the effectiveness of a pool of native AMF from a harsh environment on plant performance and changes in soil attributes under different levels of drought. An experiment using maize was established, varying the soil water content to simulate severe drought (30% of the water-holding capacity [WHC]), moderate (50% of the WHC) and no drought (80% of the WHC, control treatment). Soil and plant attributes were measured (enzyme activity, microbial biomass, AMF root colonisation and plant biomass and nutrient uptake). There was a two-fold increase in plant biomass under moderate drought when compared to no drought treatment, but there was no difference in nutrient uptake. Under severe drought, there were the highest enzyme activities related to phosphorus (P) cycling and P microbial biomass, indicating higher P microbial immobilization. The increase in AMF root colonisation was observed in plants under moderate and no drought. Our findings demonstrated that the better use of the AMF inoculum varied according to drought levels, with better performance under moderate drought due to the increase in plant biomass.

Bacilli Rhizobacteria as Biostimulants of Growth and Production of Sesame Cultivars under Water Deficit.

A strategy using bacilli was adopted aiming to investigate the mitigation of the effects of water deficit in sesame. An experiment was carried out in a greenhouse with 2 sesame cultivars (BRS Seda and BRS Anahí) and 4 inoculants (pant001, ESA 13, ESA 402, and ESA 441). On the 30th day of the cycle, irrigation was suspended for eight days, and the plants were subjected to physiological analysis using an infrared gas analyzer (IRGA). On the 8th day of water suspension, leaves were collected for analysis: superoxide dismutase, catalase, ascorbate peroxidase, proline, nitrogen, chlorophyll, and carotenoids. At the end of the crop cycle, data on biomass and vegetative growth characteristics were collected. Data were submitted for variance analysis and comparison of means by the Tukey and Shapiro-Wilk tests. A positive effect of inoculants was observed for all characteristics evaluated, contributing to improvements in plant physiology, induction of biochemical responses, vegetative development, and productivity. ESA 13 established better interaction with the BRS Anahí cultivar and ESA 402 with BRS Seda, with an increase of 49% and 34%, respectively, for the mass of one thousand seeds. Thus, biological indicators are identified regarding the potential of inoculants for application in sesame cultivation.

Not just passengers, but co-pilots! Non-rhizobial nodule-associated bacteria promote cowpea growth and symbiosis with (brady)rhizobia.

AIMS: To isolate and characterize non-rhizobial nodule-associated bacteria (NAB) from cowpea root-nodules regarding their performance of plant-growth-promoting mechanisms and their ability to enhance cowpea growth and symbiosis when co-inoculated with bradyrhizobia. METHODS AND RESULTS: Sixteen NAB were isolated, identified, and in vitro evaluated for plant growth promotion traits. The ability to promote cowpea growth was analyzed when co-inoculated with Bradyrhizobium pachyrhizi BR 3262 in sterile and non-sterile substrates. The 16S rRNA gene sequences analysis revealed that NAB belonged to the genera Chryseobacterium (4), Bacillus (3), Microbacterium (3), Agrobacterium (1), Escherichia (1), Delftia (1), Pelomonas (1), Sphingomonas (1), and Staphylococcus (1). All strains produced different amounts of auxin siderophores and formed biofilms. Twelve out of the 16 strains carried the nifH, a gene associated with nitrogen fixation. Co-inoculation of NAB (ESA 424 and ESA 29) with Bradyrhizobium pachyrhizi BR 3262 significantly promoted cowpea growth, especially after simultaneous inoculation with the three strains. CONCLUSIONS: NAB are efficient cowpea growth promoters and can improve the efficiency of the symbiosis between cowpea and the N2-fixing microsymbiont B. pachyrhizi BR 3262, mainly under a specific triple microbial association.

Molecular and biochemical responses of sesame (Sesame indicum L.) to rhizobacteria inoculation under water deficit.

Introduction: Water scarcity is a challenge for sesame cultivation under rainfed conditions. In this scenario, a potential strategy to alleviate the water deficit is the application of plant growth-promoting bacteria. The objective of this study was to analyze the interaction of rhizobacteria with sesame cultivation under water deficit conditions. Methods: An experiment was conducted in pots in a greenhouse using the BRS Morena sesame cultivar. The experimental design was completely randomized in a factorial scheme: 2 (irrigation regimes - daily irrigation and water deficit by suspending irrigation until 90% stomatal closure) x 6 (treatments with nitrogen or inoculants), with 5 replications. The types of fertilization were characterized by the addition of nitrogen (ammonium sulfate; 21% N), inoculants based on Bacillus spp. (pant001, ESA 13, and ESA 402), Agrobacterium sp. (ESA 441), and without nitrogen (control). On the fifth day after the suspension of irrigation, plant material was collected for gene expression analysis (DREB1 and HDZ7), activities of antioxidant enzymes (superoxide dismutase and catalase), relative proline content, and photosynthetic pigments. At the end of the crop cycle (about 85 days), production characteristics (root dry matter, aboveground dry matter, number of capsules, and thousand seed weight), as well as leaf nitrogen (N) and phosphorus (P) content, were evaluated. Results and Discussion: There was a positive effect on both production and biochemical characteristics (proline, superoxide dismutase, catalase, and photosynthetic pigments). Regarding gene expression, most of the inoculated treatments exhibited increased expression of the DREB1 and HDZ7 genes. These biological indicators demonstrate the potential of rhizobacteria for application in sesame cultivation, providing nutritional supply and reducing the effects of water deficit.

Elite Bradyrhizobium strains boost biological nitrogen fixation and peanut yield in tropical drylands.

Peanut (Arachis hypogaea L.) is an important crop for the family-based systems in the tropics, mainly in Brazil. In the Brazilian drylands, peanuts are cropped in low technological systems, and cheap and efficient technologies are needed to improve crop yield and sustainability. Despite this importance, few data are available on selecting efficient peanut rhizobia in experiments under different edaphoclimatic conditions. This work evaluated the agronomic efficiency and the biological nitrogen fixation (BNF) by two elite Bradyrhizobium strains under four different fields in the Brazilian semiarid region. We compared a new efficient strain Bradyrhizobium sp. ESA 123 with the reference strain B. elkanii SEMIA 6144, currently used in peanut rhizobial inoculants in Brazil. Besides the inoculated treatments, two uninoculated controls were assessed (with and without 80 kg ha-1 of N-urea). The BNF was estimated by the δ15N approach in three out of four field assays. BNF contribution was improved by inoculation of both Bradyrhizobium strains, ranging from 42 to 51% in Petrolina and 43 to 60% in Nossa Senhora da Glória. Peanuts' yields benefited from the inoculation of both strains and N fertilization in all four assays. Nevertheless, the results showed the efficiency of both strains under different edaphoclimatic conditions, indicating the native strain ESA 123 as a potential bacterium for recommendation as inoculants for peanuts in Brazil, mainly in drylands.

Potential of Bacterial Strains Isolated from Ironstone Outcrops Bromeliads to Promote Plant Growth Under Drought Conditions.

Plant growth-promoting bacteria (PGPB) are bacteria that have mechanisms that facilitate plant growth in stress conditions such as drought. The objective of this study was to characterize bacterial strains isolated from bromeliads roots in ironstone outcrops (Urucum Residual Plateau, Mato Grosso do Sul, Brazil) for plant growth-promoting under drought conditions. Firstly, we screened isolates with the presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Then, all isolates were tested for tolerance to drought, exopolysaccharides (EPS) production, indole-3-acetic acid (IAA)-producing abilities, phosphate and zinc solubilization, production of catalase and hydrolytic enzymes (amylase, cellulase, and protease). Germination assay and a pot experiment with maize plants submitted to well-watered and drought conditions were performed with the strains most promising (VBN11 and VBE23). Briefly, Bacillus cereus VBE23 showed in vitro higher ACC deaminase activity (3.83 and 2.52 µmol α-KB mg-1 h-1 in non-drought and drought conditions, respectively), tolerance to drought, EPS production and other mechanisms of plant growth promotion: solubilization of phosphate and zinc, ammonia production, catalase activity and production of hydrolytic enzymes (amylase, cellulase, and protease). Inoculation of strain VBE23 in maize seeds submitted to drought conditions showed higher germination concerning uninoculated seeds and inoculated with VBN11. Also, the results indicated that the isolate VBE23 provided higher values of fresh and dry biomass compared to the control of uninoculated treatment and inoculated with VBN11 under drought conditions. This is the first report on the PGPB from ironstone outcrops of Urucum Residual Plateau, Mato Grosso do Sul, Brazil. Thus, this bacterial isolate could be used as a strategy for the facilitation of plant growth in drought environments.

Paraburkholderia spp. are the main rhizobial microsymbionts of Mimosa tenuiflora (Willd.) Poir. in soils of the Brazilian tropical dry forests (Caatinga biome).

Mimosa tenuiflora (Willd.) Poir. is widespread in southern and central American drylands, but little information is available concerning its associated rhizobia. Therefore, this study aimed to characterize M. tenuiflora rhizobia from soils of the tropical dry forests (Caatinga) in Pernambuco State, Brazil, at the molecular and symbiotic levels. Soil samples of pristine Caatinga areas in four municipalities were used to grow M. tenuiflora. First, the bacteria from root nodules were subjected to nodC/nifH gene amplification, and the bacteria positive for both genes had the 16S rRNA gene sequenced. Then, ten strains were evaluated using recA, gyrB, and nodC gene sequences, and seven of them had their symbiotic efficiency assessed. Thirty-two strains were obtained and 22 of them were nodC/nifH positive. Twenty strains clustered within Paraburkholderia and two within Rhizobium by 16S rRNA gene sequencing. The beta-rhizobia were similar to P. phenoliruptrix (12) and P. diazotrophica (8). Both alpha-rhizobia were closely related to R. miluonense. The recA + gyrB phylogenetic analysis clustered four and five strains within the P. phenoliruptrix and P. diazotrophica branches, respectively, but they were somewhat divergent to the 16S rRNA phylogeny. For Rhizobium sp. ESA 637, the recA + gyrB phylogeny clustered the strain with R. jaguaris. The nodC phylogeny indicated that ESA 626, ESA 629, and ESA 630 probably represented a new symbiovar branch. The inoculation assay showed high symbiotic efficiency for all tested strains. The results indicated high genetic diversity and efficiency of M. tenuiflora rhizobia in Brazilian drylands and included P. phenoliruptrix-like bacteria in the list of efficient beta-rhizobia in the Caatinga biome.

Vigna spp. Root-Nodules Harbor Potentially Pathogenic Fungi Controlled By Co-habiting Bacteria.

This study aimed to isolate, identify, and evaluate the pathogenicity of nodule-borne fungi of asymptomatic Vigna spp. plants, grown in soils from preserved tropical dry forests (Caatinga) areas and identify the occurrence of co-habiting bacteria from these plants, and which have potential to control the co-occurring pathogenic fungi. Fungi and bacteria were isolated from three Vigna species (V. unguiculata, V. radiata, and V. mungo), grown in soil samples collected in five preserved Caatinga areas (Northeastern, Brazil). All fungi and selected bacteria were phylogenetically characterized by the sequencing of ITS1-5.8S-ITS2, and the 16S rRNA gene, respectively. The pathogenicity of fungi in cowpea seeds germination was evaluated throughout the inoculation experiment in Petri dishes and pots containing sterile substrate. The potential of nodule-borne bacteria to control pathogenic fungi in cowpea was assessed in a pot experiment with a sterilized substrate by the co-inoculation of fungi and bacteria isolated from the respective individual plants and soils. The 23 fungal isolates recovered were classified within the genera Fusarium, Macrophomina, Aspergillus, Cladosporium, and Nigrospora. The inoculation of fungi in cowpea seeds reduced the emergence of seeds in Petri dishes and pots. Twenty-four bacteria (Agrobacterium sp., Bradyrhizobium sp., Bacillus sp., Enterobacter sp., Pseudomonas sp., Paraburkholderia sp., and Rhizobium sp.) inhibited the harmful effects of Macrophomina sp. and Fusarium sp., increasing the germination and emergency of potted cowpea plants, highlighting the strains Agrobacterium sp. ESA 686 and Pseudomonas sp. ESA 732 that controlled, respectively, the Fusarium sp. ESA 771 and Macrophomina sp. ESA 786 by 100 and 84.6% of efficiency.

Co-inoculation of two symbiotically efficient Bradyrhizobium strains improves cowpea development better than a single bacterium application.

The co-inoculation of Bradyrhizobium with other non-bradyrhizobial strains was already assessed on cowpea, but the co-inoculation of two Bradyrhizobium strains was not tested up to now. This study aimed to evaluate the cowpea growth, N accumulation, and Bradyrhizobium competitiveness of the elite strain B. pachyrhizi BR 3262 when co-inoculated with other efficient Bradyrhizobium from the Brazilian semiarid region. Three potted-plant experiments were carried out. In the first assay, 35 efficient Bradyrhizobium isolates obtained from the semiarid region of Brazil were co-inoculated with the elite strains B. pachyrhizi BR 3262. The experiment was conducted in gnotobiotic conditions. The plant growth, nodulation, N nutritional variables, and nodular occupation were assessed. Under gnotobiotic and non-sterile soil conditions, ten selected bacteria plus the elite strain B. yuanmingense BR 3267 were used at the second and third experiments, respectively. The cowpea was inoculated with the 11 bacteria individually or co-inoculated with BR 3262. The plant growth and N nutritional variables were assessed. A double-layer medium spot method experiment was conducted to evaluate the interaction among the co-inoculated strains in standard and diluted YMA media. The co-inoculation treatments showed the best efficiency when compared to the treatments inoculated solely with BR 3262. This strain occupied a low amount of cowpea nodules ranging from 5 to 67.5%. The treatments with lower BR 3262 nodule occupancy showed the best results for the shoot nitrogen accumulation. The culture experiment showed that four bacteria inhibited the growth of BR 3262. In contrast, seven strains from the soils of Brazilian semiarid region were benefited by the previous inoculation of this strain. In the second and third experiments, the results indicated that all 11 co-inoculated treatments were more efficient than the single inoculation, proofing the best performance of the dual inoculation of Bradyrhizobium on cowpea.

Inoculation of plant growth-promoting bacteria attenuates the negative effects of drought on sorghum.

Microbial inoculants are suitable cost-effective technology to help plants endure drought. For the development of commercial inoculants, screening of efficient plant growth-promoting bacteria (PGPB) is a crucial step. The aim of this study was to evaluate the performance of PGPB to modulate drought resistance in Sorghum bicolor. A pot experiment with sorghum was conducted to access the role of previously selected PGPB strains. In addition, two non-inoculated control treatments (with and without urea fertilization) were also evaluated. For comparison, a fully irrigated treatment (FIT) was also assessed. All plants were fully irrigated for 47 days when the water supply was completely suspended for the drought-stressed treatments. When the soil moisture was close to zero, the irrigation was resumed. During dehydration and rehydration process, the leaf gas exchange (LGE) was evaluated. The parameters of plant growth and nitrogen nutrition were assessed 8 days after reirrigation. Comparing to the FIT, all treatments reduced the LGE rates, but in the presence of Bacillus sp. ESA 402 photosynthesis rate was less reduced. Some inoculation treatments promoted better recovery of photosynthesis, comparable to the FIT, 6 days after rehydration. The plant growth and nitrogen nutrition were negatively affected by the drought, but the inoculation of different bacteria reduced some negative effects. The nitrogen accumulation in the shoots was increased by all strains, suggesting their diazotrophic ability even under drought. Overall, the inoculation of Bacillus sp. ESA 402 was the best bacterium with potential for future field trials.

Beneficial Plant-Associated Microorganisms From Semiarid Regions and Seasonally Dry Environments: A Review.

Semiarid regions are apparently low biodiversity environments; however, these environments may host a phylogenetically diverse microbial community associated with plants. Their microbial inhabitants are often recruited to withstand stressful settings and improve plant growth under harsh conditions. Thus, plant-associated microorganisms isolated from semiarid and seasonally dry environments will be detailed in the present review, focusing on plant growth promotion potential and the microbial ability to alleviate plant abiotic stress. Initially, we explored the role of microbes from dry environments around the world, and then, we focused on seasonally dry Brazilian biomes, the Caatinga and the Cerrado. Cultivable bacteria from semiarid and seasonally dry environments have demonstrated great plant growth promotion traits such as plant hormone production, mobilization of insoluble nutrients, and mechanisms related to plant abiotic stress alleviation. Several of these isolates were able to improve plant growth under stressful conditions commonly present in typical semiarid regions, such as high salinity and drought. Additionally, we highlight the potential of plants highly adapted to seasonal climates from the Caatinga and Cerrado biomes as a suitable pool of microbial inoculants to maintain plant growth under abiotic stress conditions. In general, we point out the potential for the exploitation of new microbial inoculants from plants growing in dry environments to ensure a sustainable increase in agricultural productivity in a future climate change scenario.

Screening of plant growth promotion ability among bacteria isolated from field-grown sorghum under different managements in Brazilian drylands.

Sorghum [Sorghum bicolor (L.) Moench] is a multipurpose grass cultivated in drylands due to its adaptation to drought. However the characteristics of sorghum-associated bacteria are not known in the Brazilian drylands. The aim of this study was to isolate and evaluate the plant growth promotion potential bacteria from field-grown sorghum under two irrigation and manure application levels in a Brazilian semi-arid reagion. Sorghum was irrigated with 3 or 1 mm day-1 and fertilized or not with liquid goat manure. Bacteria were obtained from surface-disinfected roots applying two nitrogen-free semi-solid media. The bacteria were evaluated for the presence of nifH gene, 16S rRNA sequences, calcium-phosphate solubilization, production of auxins and siderophores and for sorghum growth promotion. We obtained 20 out of 24 positive bacteria for nifH. The isolates were classified as in six different genera. All isolates produced auxins "in vitro", six bacteria produced siderophores and three Enterobacteriaceae solubilized calcium-phosphate. At least ten bacteria resulted in the increased total N content in the sorghum shoots, comparable to fertilization with 50 mg N plant-1 week-1 and to inoculation with Azospirillum brasilense Ab-V5. Enterobacter sp. ESA 57 was the best sorghum plant-growth promoting bacteria isolated in this study.

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.

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

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 0.51molL-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.

Azospirillum spp. from native forage grasses in Brazilian Pantanal floodplain: biodiversity and plant growth promotion potential.

A sustainable alternative to improve yield and the nutritive value of forage is the use of plant growth-promoting bacteria (PGPB) that release nutrients, synthesize plant hormones and protect against phytopathogens (among other mechanisms). Azospirillum genus is considered an important PGPB, due to the beneficial effects observed when inoculated in several plants. The aim of this study was to evaluate the diversity of new Azospirillum isolates and select bacteria according to the plant growth promotion ability in three forage species from the Brazilian Pantanal floodplain: Axonopus purpusii, Hymenachne amplexicaulis and Mesosetum chaseae. The identification of bacterial isolates was performed using specific primers for Azospirillum in PCR reactions and partial sequencing of the 16S rRNA and nifH genes. The isolates were evaluated in vitro considering biological nitrogen fixation (BNF) and indole-3-acetic acid (IAA) production. Based on the results of BNF and IAA, selected isolates and two reference strains were tested by inoculation. At 31 days after planting the plant height, shoot dry matter, shoot protein content and root volume were evaluated. All isolates were able to fix nitrogen and produce IAA, with values ranging from 25.86 to 51.26 mg N mL-1 and 107-1038 µmol L-1, respectively. The inoculation of H. amplexicaulis and A. purpusii increased root volume and shoot dry matter. There were positive effects of Azospirillum inoculation on Mesosetum chaseae regarding plant height, shoot dry matter and root volume. Isolates MAY1, MAY3 and MAY12 were considered promising for subsequent inoculation studies in field conditions.

The damage caused by Callosobruchus maculatus on cowpea grains is dependent on the plant genotype.

BACKGROUND: Beans from cowpea cultivars fertilized with mineral N or inoculated with various rhizobium strains may contain different nitrogen concentrations and nitrogen metabolite composition, which affects the beans' defense mechanisms against pests. In this study, the population growth of Callosobruchus maculatus reared on beans from four cowpea cultivars fertilized with different nitrogen sources was evaluated. The factors tested were beans from four cowpea cultivars and seven different nitrogen sources: mineral N fertilization, inoculation with five strains of symbiotic diazotrophic bacteria, and soil nitrogen (absolute control). RESULTS: BRS Tapaihum and BRS Acauã cultivars had lower cumulative emergence and instantaneous rate of population growth of the insects compared with other cultivars, indicating antixenosis resistance against C. maculatus. Inoculation of BRS Acauã cultivar with the diazotrophic bacteria strain BR 3299 resulted in higher mortality of C. maculatus. For BRS Tapaihum cultivar, inoculation with diazotrophic bacteria strains BR3267, BR 3262 and BR 3299, and nitrogen fertilization resulted in higher mortality among C. maculatus. CONCLUSION: BRS Tapaihum and BRS Acauã cultivars showed the lowest cumulative insect emergence and instantaneous rates of population growth, and the highest insect mortality, mainly when the grains were obtained from plants inoculated with rhizobial strains. © 2016 Society of Chemical Industry.

Diazotrophic bacteria isolated from wild rice Oryza glumaepatula (Poaceae) in the Brazilian Amazon / Bacterias diazotróficas aisladas de arroz silvestre Oryza glumaepatula (Poaceae) en la Amazonia brasileña

The association of wild grasses with diazotrophic bacteria in Brazilian biomes is poorly understood. The isolation and characterization of bacteria associated with wild grasses can contribute to understand the diazotrophic ecology as well as to identify bacteria with biotechnological applications. In this study, we isolated and characterized diazotrophic bacterial isolates from Oryza glumaepatula collected in Cerrado and Forest areas of the Amazon in Roraima State, Brazil. Healthy O. glumepatula plants were collected at five sampling sites at Forest and seven at Cerrado, respectively. The plants were collected at the Cerrado areas in September 2008 while the Forest plants were collected in June/2008 and April/2009. The plants and the soil adhering to the roots were transferred to pots and grown for 35 days in greenhouse conditions. During the harvest, the shoots and the roots were crushed separately in a saline solution; the suspension was diluted serially and inoculated in Petri dishes containing Dyg’s medium. All distinct bacterial colonies were purified in the same medium. The diazotrophic capacity of each bacterium in microaerophilic conditions was assessed in semisolid BMGM medium. In addition, the pellicles forming bacterial isolates were also evaluated by PCR amplification for nifH gene. The diversity of nifH+ bacteria was analyzed by Box-PCR fingerprinting. For selected strains, the growth promoting capacity of O. sativa as a model plant was also evaluated. A total of 992 bacterial isolates were obtained. Fifty- one bacteria were able to form pellicles in the semisolid medium and 38 also positively amplified the 360bp nifH gene fragment. Among the 38 nifH+ isolates, 24 were obtained from the shoots, while 14 originated from the roots. The Box-PCR profiles showed that the bacterial isolates obtained in this study presented a low similarity with the reference strains belonging to the Herbaspirillum, Azospirillum and Burkholderia genus. The growth- promoting ability was confirmed for at least five isolates. For these bacteria, the root and shoot growing results showed higher increases when compared to those observed in plants inoculated with the evaluated reference strains. These results indicate that O. glumaepatula is colonized by a high diverse diazotrophic community in the Brazilian Amazon. Further investigations are now being carried out to determine the taxonomic positions of these isolates and their growth promoting mechanisms.

La asociación de gramíneas silvestres con bacterias diazotróficas en los biomas brasileños es poco conocida. El aislamiento y caracterización de las bacterias asociadas con gramíneas silvestres puede contribuir a entender la ecología de las diazotróficas y bacterias con aplicaciones biotecnológicas. En este estudio, caracterizamos aislamientos bacterianos de diazotróficas de Oryza glumaepatula recolectadas en Cerrado y zonas boscosas de la Amazonía en el estado de Roraima, Brasil. Plantas sanas de O. glumepatula fueron recolectadas en cinco zonas boscosas y siete en Cerrado. Las plantas de Cerrado fueron recolectadas en septiembre 2008, mientras que las del bosque en Junio 2008 y Abril 2009. Las plantas y el suelo adherido a las raíces se transfirieron a macetas y se cultivaron durante 35 días en condiciones de invernadero. Durante la cosecha, los brotes y las raíces se trituraron por separado en una solución salina, la suspensión se diluyó en serie y se inocularon en placas Petri que contenían medio Dyg. Todas las colonias de bacterias se purificaron en el mismo medio. Se evaluó la capacidad diazotrófica de cada bacteria en condiciones microaerofílicas en medio semisólido BMGM. Además, los aislamientos bacterianos que formaron películas se evaluaron también mediante amplificación por PCR para el gen nifH. La diversidad de bacterias nifH+ se analizó por Huella Genética utilizando la Reacción en Cadena de la Polimerasa. Para las cepas seleccionadas, la capacidad de promover el crecimiento de O. sativa como modelo de planta también se evaluó. Se obtuvo un total de 992 cepas bacterianas. Cincuenta y un bacterias fueron capaces de formar películas en el medio semisólido y 38 amplificaron positivamente el fragmento 360bp del gen nifH. De los 38 aislamientos de nifH+, 24 fueron obtenidos de los brotes, mientras que 14 se originaron a partir de las raíces. Los perfiles de PCR-Box mostraron que los aislamientos bacterianos obtenidos en este estudio presentaron una baja similitud con las cepas de referencia pertenecientes a Herbaspirillum, Azospirillum y el género Burkholderia. La capacidad promotora del crecimiento fue confirmada por al menos cinco aislamientos. Para esta bacteria, la raíz y brote mostraron resultados de crecimiento mayores en comparación con los observados en las plantas inoculadas con las cepas de referencia. Estos resultados indican que O. glumaepatula es colonizada por una muy diversa comunidad diazotrófica en la Amazonia brasileña. Se están llevando a cabo otras investigaciones para esclarecer la taxonomía de estas cepas y sus mecanismos para promover el crecimiento.