The outstanding diversity of rhizobia microsymbionts of common bean (Phaseolus vulgaris L.) in Mato Grosso do Sul, central-western Brazil, revealing new Rhizobium species.

Common bean is considered a legume of great socioeconomic importance, capable of establishing symbioses with a wide variety of rhizobial species. However, the legume has also been recognized for its low efficiency in fixing atmospheric nitrogen. Brazil is a hotspot of biodiversity, and in a previous study, we identified 13 strains isolated from common bean (Phaseolus vulgaris) nodules in three biomes of Mato Grosso do Sul state, central-western Brazil, that might represent new phylogenetic groups, deserving further polyphasic characterization. The phylogenetic tree of the 16S rRNA gene split the 13 strains into two large clades, seven in the R. etli and six in the R. tropici clade. The MLSA with four housekeeping genes (glnII, gyrB, recA, and rpoA) confirmed the phylogenetic allocation. Genomic comparisons indicated eight strains in five putative new species and the remaining five as R. phaseoli. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparing the putative new species and the closest neighbors ranged from 81.84 to 92.50% and 24.0 to 50.7%, respectively. Other phenotypic, genotypic, and symbiotic features were evaluated. Interestingly, some strains of both R. etli and R. tropici clades lost their nodulation capacity. The data support the description of the new species Rhizobium cerradonense sp. nov. (CNPSo 3464T), Rhizobium atlanticum sp. nov. (CNPSo 3490T), Rhizobium aureum sp. nov. (CNPSo 3968T), Rhizobium pantanalense sp. nov. (CNPSo 4039T), and Rhizobium centroccidentale sp. nov. (CNPSo 4062T).

Genomes of two type strains of the Rhizobium tropici group: R. calliandrae CCGE524T and R. mayense CCGE526T.

The genome sequences of two nitrogen-fixing type strains of the Rhizobium tropici group were obtained: Rhizobium calliandrae CCGE524T and R. mayense CCGE526T. Genomic analyses confirmed their taxonomic position and identified three complete sequences of the repABC genes, indicative of three plasmids, one of them carrying symbiotic genes.

Genome Sequencing of Efficient Plant Growth-Promoting Bacteria for Lowland Rice.

The genomes of five elite strains identified as growth promoters of lowland rice (Oryza sativa L.) in Brazil were sequenced. They ranged in size from 3,695,387 bp to 5,682,101 bp, encompassing genes of saprophytic ability and stress tolerance. Genome taxonomy enabled their classification as Priestia megaterium, Bacillus altitudinis, and three putative new species of Pseudomonas, Lysinibacillus, and Agrobacterium.

Microbiome of Nodules and Roots of Soybean and Common Bean: Searching for Differences Associated with Contrasting Performances in Symbiotic Nitrogen Fixation.

Biological nitrogen fixation (BNF) is a key process for the N input in agriculture, with outstanding economic and environmental benefits from the replacement of chemical fertilizers. However, not all symbioses are equally effective in fixing N2, and a major example relies on the high contribution associated with the soybean (Glycine max), contrasting with the low rates reported with the common bean (Phaseolus vulgaris) crop worldwide. Understanding these differences represents a major challenge that can help to design strategies to increase the contribution of BNF, and next-generation sequencing (NGS) analyses of the nodule and root microbiomes may bring new insights to explain differential symbiotic performances. In this study, three treatments evaluated in non-sterile soil conditions were investigated in both legumes: (i) non-inoculated control; (ii) inoculated with host-compatible rhizobia; and (iii) co-inoculated with host-compatible rhizobia and Azospirillum brasilense. In the more efficient and specific symbiosis with soybean, Bradyrhizobium presented a high abundance in nodules, with further increases with inoculation. Contrarily, the abundance of the main Rhizobium symbiont was lower in common bean nodules and did not increase with inoculation, which may explain the often-reported lack of response of this legume to inoculation with elite strains. Co-inoculation with Azospirillum decreased the abundance of the host-compatible rhizobia in nodules, probably because of competitiveness among the species at the rhizosphere, but increased in root microbiomes. The results showed that several other bacteria compose the nodule microbiomes of both legumes, including nitrogen-fixing, growth-promoters, and biocontrol agents, whose contribution to plant growth deserves further investigation. Several genera of bacteria were detected in root microbiomes, and this microbial community might contribute to plant growth through a variety of microbial processes. However, massive inoculation with elite strains should be better investigated, as it may affect the root microbiome, verified by both relative abundance and diversity indices, that might impact the contribution of microbial processes to plant growth.

Genetic variation in symbiotic islands of natural variant strains of soybean Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens differing in competitiveness and in the efficiency of nitrogen fixation.

Soybean is the most important legume cropped worldwide and can highly benefit from the biological nitrogen fixation (BNF) process. Brazil is recognized for its leadership in the use of inoculants and two strains, Bradyrhizobium japonicum CPAC 15 (=SEMIA 5079) and Bradyrhizobium diazoefficiens CPAC 7 (=SEMIA 5080) compose the majority of the 70 million doses of soybean inoculants commercialized yearly in the country. We studied a collection of natural variants of these two strains, differing in properties of competitiveness and efficiency of BNF. We sequenced the genomes of the parental strain SEMIA 566 of B. japonicum, of three natural variants of this strain (S 204, S 340 and S 370), and compared with another variant of this group, strain CPAC 15. We also sequenced the genome of the parental strain SEMIA 586 of B. diazoefficiens, of three natural variants of this strain (CPAC 390, CPAC 392 and CPAC 394) and compared with the genome of another natural variant, strain CPAC 7. As the main genes responsible for nodulation (nod, noe, nol) and BNF (nif, fix) in soybean Bradyrhizobium are located in symbiotic islands, our objective was to identify genetic variations located in this region, including single nucleotide polymorphisms (SNPs) and insertions and deletions (indels), that could be potentially related to their different symbiotic phenotypes. We detected 44 genetic variations in the B. japonicum strains and three in B. diazoefficiens. As the B. japonicum strains have gone through a longer period of adaptation to the soil, the higher number of genetic variations could be explained by survival strategies under the harsh environmental conditions of the Brazilian Cerrado biome. Genetic variations were detected in genes enconding proteins such as a dephospho-CoA kinase, related to the CoA biosynthesis; a glucosamine-fructose-6-phosphate aminotransferase, key regulator of the hexosamine biosynthetic pathway; a LysR family transcriptional regulator related to nodulation genes; and NifE and NifS proteins, directly related to the BNF process. We suggest potential genetic variations related to differences in the symbiotic phenotypes.

Microbiological quality analysis of inoculants based on Bradyrhizobium spp. and Azospirillum brasilense produced "on farm" reveals high contamination with non-target microorganisms.

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.

Combining microorganisms in inoculants is agronomically important but industrially challenging: case study of a composite inoculant containing Bradyrhizobium and Azospirillum for the soybean crop.

The increasing global perception of the importance of microbial inoculants to promote productivity and sustainability in agriculture prompts the adoption of bio-inputs by the farmers. The utilization of selected elite strains of nitrogen-fixing and other plant-growth promoting microorganisms in single inoculants creates a promising market for composite inoculants. However, combining microorganisms with different physiological and nutritional needs requires biotechnological development. We report the development of a composite inoculant containing Bradyrhizobium diazoefficiens and Azospirillum brasilense for the soybean crop. Evaluation of use of carbon sources indicates differences between the microbial species, with Bradyrhizobium growing better with mannitol and glycerol, and Azospirillum with malic acid and maleic acid, allowing the design of a formulation for co-culture. Species also differ in their growth rates, and the best performance of both microorganisms occurred when Azospirillum was inoculated on the third day of growth of Bradyrhizobium. The composite inoculant developed was evaluated in five field trials performed in Brazil, including areas without and with naturalized populations of Bradyrhizobium. The composite inoculant resulted in symbiotic performance comparable to the application of the two microorganisms separately. In comparison to the single inoculation with Bradyrhizobium, co-inoculation resulted in average increases of 14.7% in grain yield and 16.4% in total N accumulated in the grains. The performance of the composite inoculant was similar or greater than that of the non-inoculated control receiving a high dose of N-fertilizer, indicating the importance of the development and validation of inoculants carrying multiple beneficial microorganisms.

Impact of pesticides in properties of Bradyrhizobium spp. and in the symbiotic performance with soybean.

Soybean [Glycine max (L.) Merr.] has great economic and nutritional importance mainly due to its high protein content. All plant's N needs can be met by the symbiosis with elite Bradyrhizobium strains applied as inoculants to the seeds at sowing time; however, the increasing use of pesticides in seed treatments can impair the contribution of the biological nitrogen fixation. In this study, we report decreases in cell survival of two strains, B. japonicum SEMIA 5079 and B. elkanii SEMIA 587 in seeds inoculated and treated with StandakTop™, composed of the fungicides pyraclostrobin and thiophanate-methyl and the insecticide fipronil, the pesticides most used in soybean seed treatment in several countries. Cell death was enhanced with the time of exposure to the pesticides, and B. elkanii was less tolerant, with almost no detectable viable cells after 15 days. Change in colony morphology with smaller colonies was observed in the presence of the pesticides, being more drastic with the time of exposure, and attributed to an adaptive response towards survival in the presence of the abiotic stress. However, morphological changes were reversible after elimination of the stressing agent and symbiotic performance under controlled greenhouse conditions was similar between strains that had been or not exposed to the pesticides. In addition, no changes in DNA profiles (BOX-PCR) of both strains were observed after the contact with the pesticides. In two field experiments, impacting effects of the pesticides were observed mainly on the total N accumulated in grains of plants relying on both N2-fixation and N-fertilizer. Our data indicate that StandakTop® affects parameters never reported before, including colony morphology of Bradyrhizobium spp. and N metabolism and/or N remobilization to soybean grains.

Soybean tolerance to drought depends on the associated Bradyrhizobium strain.

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.

Towards sustainable yield improvement: field inoculation of soybean with Bradyrhizobium and co-inoculation with Azospirillum in Mozambique.

The effects of sole inoculation of soybean (Glycine max L. Merrill) with Bradyrhizobium and co-inoculation with Bradyrhizobium and Azospirillum on nodulation, plant growth and yields were investigated in the 2013/2014 and 2014/2015 cropping seasons under field conditions in Mozambique. The treatments included (1) Control (non-inoculated control, with symbiosis depending on indigenous rhizobia), (2) Urea (non-inoculated, receiving 200 kg ha-1 of N), (3) Sole inoculation with B. diazoefficiens strain USDA 110, and (4) Co-inoculation with B. diazoefficiens strain USDA 110 and A. brasilense strains Ab-V5 and Ab-V6, evaluated in a randomized complete block design with five replications. Nodule number and dry weight, shoot dry weight, biological and grain yields, grain dry weight, and harvest index were evaluated. In general, both sole inoculation and co-inoculation enhanced nodulation in relation to control. Sole inoculation increased grain yield by 22% (356 kg ha-1), the same enhancement magnitude attained under mineral N treatment, suggesting that Bradyrhizobium inoculation provides ecological and economic sustainability to the soybean crop in Mozambique or other countries with similar agro-climatic conditions. Co-inoculation did not increase grain yields in relation to neither the control nor sole inoculation, indicating that further research with adapted and high yielding soybean varieties along with effective rhizobial strains is required in Mozambique to attune the beneficial Azospirillum-plant cultivar-rhizobia interactions that have been reported in other countries for several legumes, including soybean.

Microbial inoculants: reviewing the past, discussing the present and previewing an outstanding future for the use of beneficial bacteria in agriculture.

More than one hundred years have passed since the development of the first microbial inoculant for plants. Nowadays, the use of microbial inoculants in agriculture is spread worldwide for different crops and carrying different microorganisms. In the last decades, impressive progress has been achieved in the production, commercialization and use of inoculants. Nowadays, farmers are more receptive to the use of inoculants mainly because high-quality products and multi-purpose elite strains are available at the market, improving yields at low cost in comparison to chemical fertilizers. In the context of a more sustainable agriculture, microbial inoculants also help to mitigate environmental impacts caused by agrochemicals. Challenges rely on the production of microbial inoculants for a broader range of crops, and the expansion of the inoculated area worldwide, in addition to the search for innovative microbial solutions in areas subjected to increasing episodes of environmental stresses. In this review, we explore the world market for inoculants, showing which bacteria are prominent as inoculants in different countries, and we discuss the main research strategies that might contribute to improve the use of microbial inoculants in agriculture.

Draft Genome Sequence of Bradyrhizobium elkanii Strain SEMIA 938, Used in Commercial Inoculants for Lupinus spp. in Brazil.

Due to its high capacity for nitrogen fixation, strain SEMIA 938 is used in commercial inoculants for lupins in Brazil. Its genome was estimated at 8,780,064 bp and indicates that it belongs to the Bradyrhizobium elkanii species, while the analysis of nodulation genes classifies the strain in the symbiovar sojae.

Draft Genome Sequence of Agrobacterium deltaense Strain CNPSo 3391, Isolated from a Soybean Nodule in Mozambique.

Agrobacterium deltaense strain CNPSo 3391 was isolated from a soybean nodule in Mozambique. Its genome size was estimated at 4,926,588 bp. This isolate carries several coding sequences for stress tolerance, but no identifiable nodulation or virulence genes. Possible ecological roles of bacteria isolated from legume nodules and closely related to Agrobacterium are discussed.

Outstanding impact of soil tillage on the abundance of soil hydrolases revealed by a metagenomic approach

ABSTRACT 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.

Feasibility of transference of inoculation-related technologies: A case study of evaluation of soybean rhizobial strains under the agro-climatic conditions of Brazil and Mozambique.

The soybean-Bradyrhizobium symbiosis can be very effective in fixing nitrogen and supply nearly all plant's demand on this nutrient, obviating the need for N-fertilizers. Brazil has been investing in research and use of inoculants for soybean for decades and with the expansion of the crop in African countries, the feasibility of transference of biological nitrogen fixation (BNF) technologies between the continents should be investigated. We evaluated the performance of five strains (four Brazilian and one North American) in the 2013/2014 and 2014/2015 crop seasons in Brazil (four sites) and Mozambique (five sites). The experimental areas were located in relatively similar agro-climatic regions and had soybean nodulating rhizobial population ranging from ≪ 10 to 2 × 105 cells g-1 soil. The treatments were: (1) NI, non-inoculated control with no N-fertilizer; (2) NI + N, non-inoculated control with 200 kg of N ha-1; and inoculated with (3) Bradyrhizobium japonicum SEMIA 5079; (4) B. diazoefficiens SEMIA 5080; (5) B. elkanii SEMIA 587; (6) B. elkanii SEMIA 5019; (7) B. diazoefficiens USDA 110; (8) SEMIA 5079 + 5080 (only tested in Brazil). The best inoculation treatments across locations and crop seasons in Brazil were SEMIA 5079 + 5080, SEMIA 5079 and USDA 110, with average grain yield gains of 4-5% in relation to the non-inoculated treatment. SEMIA 5079, SEMIA 5080, SEMIA 5019 and USDA 110 were the best strains in Mozambique, with average 20-29% grain yield gains over the non-inoculated treatment. Moreover, the four best performing strains in Mozambique resulted in similar or better yields than the non-inoculated + N treatment, confirming the BNF as an alternative to N-fertilizers. The results also confirm the feasibility to transfer soybean inoculation technologies between countries, speeding up the establishment of sustainable cropping systems.

Antioxidant activity and induction of mechanisms of resistance to stresses related to the inoculation with Azospirillum brasilense.

We investigated the effects of Azospirillum brasilense strains Ab-V5 and Ab-V6 in the induction of mechanisms of systemic acquired resistance (SAR) and induced system resistance (ISR) on maize (Zea mays L.) plants. Under normal growth conditions, the treatments consisted of the standard inoculation of cells at sowing, and leaf spray of cells or their metabolites at the V2.5 growth stage; under saline stress (170 mM NaCl), the treatment consisted of standard single and co-inoculation of A. brasilense and Rhizobium tropici. The main compounds in the Azospirillum metabolites were identified as indole-3-acetic acid (IAA) and salicylic acid (SA). Under normal conditions, A. brasilense cells applied at sowing or by leaf spray increased the activities of catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) in leaves, and of ascorbate peroxidase (APX) in roots; however, interestingly, in general the highest activities were observed by leaf spray of metabolites. Under normal conditions, the highest levels of salicylic acid (SA) and jasmonic acid (JA) were achieved in leaves by leaf spray of metabolites, of SA in roots by leaf spray of cells, and of JA in roots by standard inoculation and leaf spray of metabolites. Under saline stress, plant protection occurred via SA and abscisic acid (ABA), but not JA. In general, inoculation resulted in further increases in SA in leaves and roots, and ABA in leaves. We hypothesize that A. brasilense confers protection to maize plants by simultaneous induction of JA and SA pathways, and, under saline stressing conditions, by SA and ABA pathways.

Draft Genome Sequences of Azospirillum brasilense Strains Ab-V5 and Ab-V6, Commercially Used in Inoculants for Grasses and Legumes in Brazil.

Azospirillum brasilense strains Ab-V5 and Ab-V6 are largely used in commercial inoculants for grasses and legumes in Brazil. Their genomes were estimated at 6,934,595 and 7,197,196 bp, respectively, and encompass genes related to nitrogen fixation, synthesis of phytohormones, and environmental adaptation. Although the strains differ in phenotypic properties, their genomes are highly similar.

Outstanding impact of soil tillage on the abundance of soil hydrolases revealed by a metagenomic approach.

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.

Revealing strategies of quorum sensing in Azospirillum brasilense strains Ab-V5 and Ab-V6.

Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

Preinoculation of Soybean Seeds Treated with Agrichemicals up to 30 Days before Sowing: Technological Innovation for Large-Scale Agriculture.

The cultivation of soybean in Brazil experienced an expressive growth in the last decades. Soybean is highly demanding on nitrogen (N) that must come from fertilizers or from biological fixation. The N supply to the soybean crop in Brazil relies on the inoculation with elite strains of Bradyrhizobium japonicum, B. elkanii, and B. diazoefficiens, which are able to fulfill the crop's N requirements and enrich the soil for the following crop. The effectiveness of the association between N2-fixing bacteria and soybean plants depends on the efficacy of the inoculation process. Seed treatment with pesticides, especially fungicides or micronutrients, may rapidly kill the inoculated bacteria, affecting the establishment and outcome of the symbiosis. The development of technologies that allow inoculation to become a successful component of industrial seed treatment represents a valuable tool for the seed industry, as well as for the soybean crop worldwide. In this article, we report the results of new technologies, developed by the company Total Biotecnologia Indústria e Comércio S/A of Brazil, for preinoculation of soybean seeds with bradyrhizobia, in the presence of agrichemicals. Our results demonstrate improved bacterial survival for up to 30 days after inoculation, without compromising nodulation, N2-fixation, and yield in the field.