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The phosphate (P)-solubilizing potential of rhizobia isolated from active root nodules of Brazilian native Mimosa and Desmodium was assessed. Out of the 15 strains selected, five Paraburkholderia isolated from Mimosa spp. grown in rocky outcrops stood out. The Ca 3 (PO 4 ) 2 -solubilizing efficiency of these strains ranged from 110.67 to 356.3 mgL -1 , with less expressive results for FePO 4 and Al(H 2 PO 4 ) 3, that might be attributed to the low solubility of these two P compounds. Paraburkholderia strains CNPSo 3281 and CNPSo 3076 were the most efficient siderophore producers (44.17 and 41.87 µMol EDTA) and two of the top FePO 4 solubilizers. Acidification of the culture media was observed for all the strains and P sources. Regarding Ca 3 (PO 4 ) 2 solubilization , the main organic acids detected were glucuronic (an important component of rhizobia exopolysaccharides) and gluconic acids. Genomic analysis of P. nodosa CNPSo 3281 and CNPSo 3076 along with other phosphate-solubilizing Paraburkholderia species of the genus pointed out a conserved gene organization of phoUBR, pstSCAB, ppk and ppx. Greenhouse experiment revealed that P. nodosa CNPSo 3281 and CNPSo 3076 promoted maize growth under low P. Our results indicate the relevance of native rhizobia as multifunctional plant-associated bacteria and the rocky outcrops ecosystems as hotspots for bioprospection., Competing Interests: Declarations. Conflicts of interest: The authors declare no ethical conflicts; the authors declare that they have consented to participate in the manuscript and publish it. All authors have read and approved the final manuscript., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Brazil stands out in research, industrial development, and farmers' use of microbial inoculants, with an emphasis on getting benefits from the biological nitrogen fixation process with the soybean crop. Nowadays, about 140 million doses of inoculants are commercialized annually for the soybean in the country, and strain identification is achieved by rep-PCR, an effective but time-consuming method. Aiming to develop an easy, low-cost, and low-time-consuming method, we used a complete genome-based approach based on the unequivocal identification of unique genes present in the genomes of each of the four Bradyrhizobium strains used in commercial inoculants: Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019, Bradyrhizobium japonicum SEMIA 5079, and Bradyrhizobium diazoefficiens SEMIA 5080. The unique pairs of primers able to amplify genomic regions of different sizes allowed the identification of the four strains in a simple multiplex polymerase chain reaction (PCR). Validation was confirmed by using single colonies, multiple cultures, and commercial inoculants. The number of labor hours of a technician was 3.08 times higher, and the final cost was 3.25 times higher in the rep-PCR than in the multiplex PCR. Most importantly, the results for multiplex PCR were obtained on the same day, in contrast with 15 days in the traditional methodology. The genomic approach developed can be easily applied to a variety of microbial inoculants worldwide, in addition to studies of ecology and evaluation of the competitiveness of the strains., (© 2024. The Author(s).)

The rhizobia-Desmodium (Leguminosae, Papilionoideae) symbiosis is generally described by its specificity with alpha-rhizobia, especially with Bradyrhizobium. Our study aimed to isolate rhizobia from root nodules of native D. barbatum, D. incanum, and D. discolor, collected in remnants of the biomes of Atlantic Forest and Cerrado in protected areas of the Paraná State, southern Brazil. Based on the 16S rRNA phylogeny, 18 out of 29 isolates were classified as Alphaproteobacteria (Bradyrhizobium and Allorhizobium/Rhizobium) and 11 as Betaproteobacteria (Paraburkholderia). Phylogeny of the recA gene of the alpha-rhizobia resulted in ten main clades, of which two did not group with any described rhizobial species. In the 16S rRNA phylogeny of the beta-rhizobia, Paraburkholderia strains from the same host and conservation unity occupied the same clade. Phenotypic characterization of representative strains revealed the ability of Desmodium rhizobia to grow under stressful conditions such as high temperature, salinity, low pH conditions, and tolerance of heavy metals and xenobiotic compounds. Contrasting with previous reports, our results revealed that Brazilian native Desmodium can exploit symbiotic interactions with stress-tolerant strains of alpha- and beta-rhizobia. Stress tolerance can highly contribute to the ecological success of Desmodium in this phytogeographic region, possibly relating to its pioneering ability in Brazil. We propose Desmodium as a promising model for studies of plant-rhizobia interactions., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The use of inoculants carrying diazotrophic and other plant growth-promoting bacteria plays an essential role in the Brazilian agriculture, with a growing use of microorganism-based bioproducts. However, in the last few years, some farmers have multiplied microorganisms in the farm, known as "on farm" production, including inoculants of Bradyrhizobium spp. for soybean (Glycine max L. Merrill.) and Azospirillum brasilense for corn (Zea mays L.) or co-inoculation in soybean. The objective was to assess the microbiological quality of such inoculants concerning the target microorganisms and contaminants. In the laboratory, 18 samples taken in five states were serial diluted and spread on culture media for obtaining pure and morphologically distinct colonies of bacteria, totaling 85 isolates. Molecular analysis based on partial sequencing of the 16S rRNA gene revealed 25 genera of which 44% harbor species potentially pathogenic to humans; only one of the isolates was identified as Azospirillum brasilense, whereas no isolate was identified as Bradyrhizobium. Among 34 isolates belonging to genera harboring species potentially pathogenic to humans, 12 had no resistance to antibiotics, six presented intrinsic resistance, and 18 presented non-intrinsic resistance to at least one antibiotic. One of the samples analyzed with a shotgun-based metagenomics approach to check for the microbial diversity showed several genera of microorganisms, mainly Acetobacter (~ 32% of sequences) but not the target microorganism. The samples of inoculants produced on farm were highly contaminated with non-target microorganisms, some of them carrying multiple resistances to antibiotics., (© 2021. Sociedade Brasileira de Microbiologia.)

Plant growth-limiting factors, such as low nutrient availability and weak pathogen resistance, may hinder the production of several crops. Plant growth-promoting bacteria (PGPB) used in agriculture, which stimulate plant growth and development, can serve as a potential tool to mitigate or even circumvent these limitations. The present study evaluated the feasibility of using bacteria isolated from the maize rhizosphere as PGPB for the cultivation of this crop. A total of 282 isolates were collected and clustered into 57 groups based on their genetic similarity using BOX-PCR. A representative isolate from each group was selected and identified at the genus level with 16S rRNA sequencing. The identified genera included Bacillus (61.5% of the isolates), Lysinibacillus (30.52%), Pseudomonas (3.15%), Stenotrophomonas (2.91%), Paenibacillus (1.22%), Enterobacter (0.25%), Rhizobium (0.25%), and Atlantibacter (0.25%). Eleven isolates with the highest performance were selected for analyzing the possible pathways underlying plant growth promotion using biochemical and molecular techniques. Of the selected isolates, 90.9% were positive for indolepyruvate/phenylpyruvate decarboxylase, 54.4% for pyrroloquinoline quinine synthase, 36.4% for nitrogenase reductase, and 27.3% for nitrite reductase. Based on biochemical characterization, 9.1% isolates could fix nitrogen, 36.6% could solubilize phosphate, 54.5% could produce siderophores, and 90.9% could produce indole acetic acid. Enzymatic profiling revealed that the isolates could degrade starch (90.1%), cellulose (72.7%), pectin (81.8%), protein (90.9%), chitin (18.2%), urea (54.5%), and esters (45.4%). Based on the data obtained, we identified three Bacillus spp. (LGMB12, LGMB273, and LGMB426), one Stenotrophomonas sp. (LGMB417), and one Pseudomonas sp. (LGMB456) with the potential to serve as PGPB for maize. Further research is warranted to evaluate the biotechnological potential of these isolates as biofertilizers under field conditions., (© 2021. Sociedade Brasileira de Microbiologia.)

We evaluated the effect of three different Bradyrhizobium strains inoculated in two soybean genotypes (R01-581F, drought-tolerant, and NA5858RR, drought-sensitive) submitted to drought in two trials conducted simultaneously under greenhouse. The strains (SEMIA 587, SEMIA 5019 (both B. elkanii), and SEMIA 5080 (B. diazoefficiens)) were inoculated individually in each genotype and then submitted to water restriction (or kept well-watered, control) between 45 and 62 days after emergence. No deep changes in plant physiological variables were observed under the moderate water restriction imposed during the first 10 days. Nevertheless, photosynthesis and transpiration decreased after the severe water restriction imposed for further 7 days. Water restriction reduced growth (- 30%) and the number of nodules (- 47% and - 58% for R01-581F and NA5858RR, respectively) of both genotypes, with a negative effect on N-metabolism. The genotype R01-581F inoculated with SEMIA 5019 strain had higher photosynthetic rates compared with NA5858RR, regardless of the Bradyrhizobium strain. On average, R01-581F showed better performance under drought than NA5858RR, with higher number of nodules (51 vs. 38 nodules per plant, respectively) and less accumulation of ureides in petioles (15 μmol g -1 vs. 34 μmol g -1 , respectively). Moreover, plants inoculated with SEMIA 5080 had higher glutamine synthetase activity under severe water restriction, especially in the drought-tolerant R01-518F, suggesting maintenance of N metabolism under drought. The Bradyrhizobium strain affects the host plant responses to drought in which the strain SEMIA 5080 improves the drought tolerance of R01-518F genotype.

Biological nitrogen fixation (BNF) with the soybean crop probably represents the major sustainable technology worldwide, saving billions of dollars in N fertilizers and decreasing water pollution and the emission of greenhouse gases. Accordingly, the identification of strains occupying nodules under field conditions represents a critical step in studies that are aimed at guaranteeing increased BNF contribution. Current methods of identification are mostly based on serology, or on DNA profiles. However, the production of antibodies is restricted to few laboratories, and to obtain DNA profiles of hundreds of isolates is costly and time-consuming. Conversely, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique might represent a golden opportunity for replacing serological and DNA-based methods. However, MALDI-TOF databases of environmental microorganisms are still limited, and, most importantly, there are concerns about the discrimination of protein profiles at the strain level. In this study, we investigated four soybean rhizobial strains carried in commercial inoculants used in over 35 million hectares in Brazil and also in other countries of South America and Africa. A supplementary MALDI-TOF database with the protein profiles of these rhizobial strains was built and allowed the identification of unique profiles statistically supported by multivariate analysis and neural networks. To test this new database, the nodule occupancy by Bradyrhizobium strains in symbiosis with soybean was characterized in a field experiment and the results were compared with serotyping of bacteria by immuno-agglutination. The results obtained by both techniques were highly correlated and confirmed the viability of using the MALDI-TOF MS technique to effectively distinguish bacteria at the strain level.

The soil represents the main source of novel biocatalysts and biomolecules of industrial relevance. We searched for hydrolases in silico in four shotgun metagenomes (4,079,223 sequences) obtained in a 13-year field trial carried out in southern Brazil, under the no-tillage (NT), or conventional tillage (CT) managements, with crop succession (CS, soybean/wheat), or crop rotation (CR, soybean/maize/wheat/lupine/oat). We identified 42,631 hydrolases belonging to five classes by comparing with the KEGG database, and 44,928 sequences by comparing with the NCBI-NR database. The abundance followed the order: lipases>laccases>cellulases>proteases>amylases>pectinases. Statistically significant differences were attributed to the tillage system, with the NT showing about five times more hydrolases than the CT system. The outstanding differences can be attributed to the management of crop residues, left on the soil surface in the NT, and mechanically broken and incorporated into the soil in the CT. Differences between the CS and the CR were slighter, 10% higher for the CS, but not statistically different. Most of the sequences belonged to fungi (Verticillium, and Colletotrichum for lipases and laccases, and Aspergillus for proteases), and to the archaea Sulfolobus acidocaldarius for amylases. Our results indicate that agricultural soils under conservative managements may represent a hotspot for bioprospection of hydrolases., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Bradyrhizobium embrapense CNPSo 2833 T is a nitrogen-fixing symbiont of the legume pasture Desmodium. Its draft genome contains 8,267,832bp and 7876 CDSs. The symbiotic island includes nodulation and nitrogen fixation genes resembling the operon organization of B. japonicum. Several CDSs related to secretion proteins and stress tolerance were also identified., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.

With the aim of a better characterization of the somatic recombination process in Trichoderma pseudokoningii, a progeny from crossings between T. pseudokoningii strains contrasting for auxotroph markers was characterized by RAPD markers and PFGE (electrophoretic karyotype). Cytological studies of the conidia, conidiogenesis and heterokaryotic colonies were also performed. The genotypes of the majority of the recombinant strains analyzed were similar to only one of the parental strains and the low frequency of polymorphic RAPD bands suggested that the nuclear fusions may not occur into the heterokaryon. In some heterokaryotic regions the existence of intensely staining hyphae might be related to cell death. We proposed that a mechanism of somatic recombination other than parasexuality might occur, being related to limited vegetative compatibility after postfusion events, as described for other Trichoderma species.

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.