Search

A planta de soja tem a associação simbiôntica com micro-organismos que têm a capacidade de fixar nitrogênio (N) e liberá-lo para as plantas. Nas cultivares de soja de alto potencial produtivo, a demanda por N estaria sendo atendida através da fixação biológica de nutrientes (FBN) ou haveria a necessidade de complementação deste nutriente para a cultura. Este trabalho teve como objetivo verificar a resposta da cultivar de soja Desafio inoculada com bactérias Bradyrhizobium japonicum e Bradyrhizobium elkanii, e com adubação nitrogenada suplementar na fase reprodutiva, via solo e via foliar. Os tratamentos constituíram da testemunha (sem aplicação de N), 30 e 60 kg de N ha-1 via solo e solução a 2% de N aplicados nas fases R1 e R5.3. O delineamento foi de blocos casualizados com quatro repetições e as sementes foram inoculadas com bactérias das espécies Bradyrhizobium japonicum e Bradyrhizobium elkanii (estirpes Semia 587 e Semia 5019). A aplicação tardia de N em R1 e em R5.3 proporcionou aumento na produtividade de grãos de 478,6 kg ha-1 e 472,8 kg ha-1, respectivamente. A aplicação via solo de N em R1 apresenta melhor índice de colheita, quando comparada com a R5.3. A aplicação tardia de solução de 2% de N, via foliar, não proporciona aumento na produtividade da cultura da soja. Palavras-chave: Glycine max. Produtividade. Fixação Biológica. Adubação Nitrogenada. Abstract The soybean plant has the symbiotic association with microorganisms that has the ability to fix nitrogen (N) and release it to the plants. Soybean cultivars with high productive potential, N demand would be met through biological nutrient fixation (BNF) or there would be a need to complement this nutrient for the crop. The objective of this work was to verify the response of soybean inoculated with bacteria of the genus Rhizobium and Bradyrhizobium, the supplementary nitrogen fertilization in the reproductive phase, soil and leaf pathway. The treatments consisted of control (without application of N), 30 and 60 kg of N ha-1 via soil and 2 % N solution applied in phases R1 and R5.3. The design was randomized blocks with 4 replications and the seeds were inoculated with bacteria of the species Bradyrhizobium japonicum and Bradyrhizobium elkanii (strains Semia 587 and Semia 5019). The late application of N in R1 and R5.3 increased grain yield of 478.6 kg ha-1 and 472.8 kg ha-1, respectively. The application, via soil, of N in R1 presents a better harvest index, when compared to R5.3. The late application of 2% N solution, via leaf, did not increase the soybean crop yield Keywords: Glycine max. Productivity. Biological Fixation. Nitrogen Fertilization

Actually, in Republic of Congo, rhizobia have poorly phenotypically and biochemically characterized. This study aimed to characterize native rhizobia. Rhizobia strains were isolated using nodule roots collected on Milletia laurentii, Acacia spp., Albizia lebbeck, and Vigna unguiculata. The strains isolated were characterized microbiologically, biochemically, physiologically, and molecularly identified using 16S rRNA method. The results reported in this study are only for six strains of all 77 isolated: RhA1, RhAc4, RhAc15, RhAc13, RhW1, and RhV3. All native strains were positive to urease activity, negative to cellulase and pectinase activity except for one isolate that showed a positive cellulase activity. Moreover, isolates have grown at 12% of NaCl. On different effects of temperatures, isolates were able to grow up to 44°C and showed good growth at pH from 7 to 9 and the ability to use ten different carbon hydrates sources. The strains were identified as Rhizobium tropici, Rhizobium sp., Mesorhizobium sp. Bradyrhizobium yuanmingense and Bradyrhizobium elkanii. The phylogenetically analysis of the 16S rRNA genes, using a clustering method, allowed us to have a history that is both ancient and stable of four clades among genes with similar patterns. Expanding our awareness of the new legume-rhizobia will be a valuable resource for incorporating an alternative nitrogen fixation approach to consolidate the growth of legumes. These germs can be used in Congolese agriculture to improve yield of crops.

Lanthanide (Ln)-dependent XoxF-type methanol dehydrogenase (MDH) genes can be found in bacteria that are not believed to be methylotrophs, and studies on their methylotrophic pathways and their use of Ln are now emerging. Ln-dependent methanol utilization in Bradyrhizobium sp. strain Ce-3, which belongs to the Bradyrhizobium elkanii superclade (clade II), was investigated in this study. Strain Ce-3 was able to grow in a media containing methanol as a sole carbon source and light Ln (L-Ln, i.e., La3+, Ce3+, Pr3+, and Nd3+), whereas the strain did not show any growth with Ca2+ or the heavy Ln, Sm3+. We found that the uptake of L-Ln is enhanced mainly by methanol and L-Ln species, and the strain incorporates each L-Ln species evenly into the cell. The genome of strain Ce-3 encodes the xox cluster for Ln-dependent methanol dehydrogenase (xoxF) and the enzymes participating in the methanol oxidation pathway (xoxG, fldA, and gfaA) and regulation (xoxR), but the gene encoding formate dehydrogenase (FDH) was not found in the cluster. MDH, formaldehyde dehydrogenase, and FDH activities were induced by methanol/Ln. Moreover, expression of the genes on the xox cluster was upregulated by methanol/Ln. Based on these results, we concluded that strain Ce-3 possesses a complete L-Ln-dependent methanol oxidation pathway, which is dissimilar to plant phyllospheric bacteria, Methylobacterium species, with a transport system for L-Ln species.

Bradyrhizobium is a genus of plant growth-promoting rhizobacteria (PGPR) that have been studied for several decades mainly for the ability to fix diazotrophic nitrogen after having been established endosymbiotically inside root nodules of the legumes of Fabaceae. The aim of this work was to evaluate the capability of Bradyrhizobium to promote the growth of crops belonging to other families, in this case, rice (Oryza sativa), both in laboratory and in field trials. For laboratory test, surface-sterilized rice seeds were soaked with cultures of each strain and planted in pots. Plant length and dry weight were measured after 35 days. For the field test, rice seeds of varieties Yerua La Plata and Guri INTA were inoculated with the three best strains observed in the laboratory test and planted in plots. After 60 days of growth, plant length and dry weight were measured. At harvest time, we measured the dry weight of the aerial part, yield and thousand-grain weight. Inoculation with any of the three species described provoked significant increments compared to the uninoculated control at least in one of the parameters measured, both in the laboratory and in the field tests. Bradyrhizobium japonicum E109 was the strain that promoted rice growth the most in the lab while Bradyrhizobium elkanii SEMIA 587 was the strain that promoted rice growth the most in the field, with increments in yield of approximately 1000 kg/ha. Data obtained suggest that the Bradyrhizobium species promoted all rice growth and yield., Centro de Investigaciones en Fitopatología

There is little information that supports the new technology of inoculants with biological protectors and indecates the results in soybean crop. In order to evaluate the morphophysiological responses of soybean crop to conventional inoculants, inoculants with protectors, co-inculation with Azospirillum brasilense and at increasing doses of N. The treatments consisted of: Control; Bradyrhizobium elkanii + biological protector; Bradhyrizobium elkanii + Bradhyrizobium japonicum + biological protector; Bradyrhizobium elkanii; Bradyrhizobium elkanii + co-inoculation with Azospirillum brasilense; Bradyrhizobium elkanii + biological protector + co-inoculation with Azospirillum brasilense; Bradyrhizobium elkanii + Bacillus amyloliquefaciens + biological protector; Dose 20 kg ha-1 of N; Dose N 40 kg ha-1 and Dose N 60 kg ha-1 of N. Nodulation, chlorophyll a, b and total content, nitrogen content in leaf tissue and grains were evaluated thousand grain mass and productivity were evaluated. The biological agents tested in the experiment provided an increase in the number of nodules and an increase in the mass of 1,000 grains when compared nitrogen fertilization. The treatments with nitrogen fertilization, compared to the treatments with inoculation, were higher for chlorophyll a, b and total contents. Long-life inoculants, in the absence and presence of coinoculation in seed treatment, demonstrated efficiency in number of nodules and in the supply of N in soybean crop, with superior responses on the non-inoculated control, even in soil with initial concentration 1,510 10-4 bacteria cells per gram of soil. Long-life inoculants offer greater practicality to the rural producer and demonstrated efficiency in the supply of N to soybean crop.

Bradyrhizobium elkanii, a rhizobium with a relatively wide host range, possesses a functional type III secretion system (T3SS) that is involved in symbiotic incompatibility against Rj4-genotype soybean (Glycine max) and some accessions of mung bean (Vigna radiata). To expand our knowledge on the T3SS-mediated partner selection mechanism in the symbiotic legume-rhizobia association, we inoculated three Lotus experimental accessions with wild-type and T3SS-mutant strains of B. elkanii USDA61. Different responses were induced by T3SS in a host genotype-dependent manner. Lotus japonicus Gifu inhibited infection; L. burttii allowed infection, but inhibited nodule maturation at the post-infection stage; and L. burttii and L. japonicus MG-20 both displayed a nodule early senescence-like response. By conducting inoculation tests with mutants of previously reported and newly identified effector protein genes of B. elkanii USDA61, we identified NopF as the effector protein triggering the inhibition of infection, and NopM as the effector protein triggering the nodule early senescence-like response. Consistent with these results, the B. elkanii USDA61 gene for NopF introduced into the Lotus symbiont Mesorhizobium japonicum induced infection inhibition in L. japonicus Gifu, but did not induce any response in L. burttii or L. japonicus MG-20. These results suggest that Lotus accessions possess at least three checkpoints to eliminate unfavorable symbionts, including the post-infection stage, by recognizing different T3SS effector proteins at each checkpoint.

Black gram (Vigna mungo L. Hepper) is one of the main leguminous crops that provide chief source of food. Several Bradyrhizobium species are able to induce effective nodules in black gram cultivars. In the present study, we characterized forty isolates of indigenous black gram bradyrhizobia from Myanmar based on the sequence analysis of the bacterial 16S rRNA gene. The sequence analysis confirmed that all isolates were categorized and identified as the genus Bradyrhizobium and they were conspecific with B. elkanii, B. sp., B. liaoningense, B. japonicum and B. yunamingense. Almost all the collected isolates from major black gram growing regions of Nyaunglebin Bago Regio, Chaungzon Mon State, Sittwe Rakhine State, Danubyu Ayeyarwady Region and Launglon Tanintharyi Region were identified as B. liaoningense. At Danubyu Ayeyarwady Region and Pyinmanar Nay Pyi Taw Region, most of the strains were identified as B. japonicum. On the other hand, more or less all the isolates from Launglon Tanintharyi Region and Hpa-an Kayin State were related to B. elkanii. However, all B. sp. strains were found in Salingyi Sagaing Region black gram growing region. This is the first report describing Bradyrhizobium strains that were isolated from soil samples of major black gram growing areas in Myanmar. Evaluation of the effectiveness of Myanmar Bradyrhizobim strains isolated from soil samples of major black gram growing areas of Myanmar for plant growth and nitrogen fixation was studied in pot experiments with completely randomized design and three replicates. The nodule dry weight, shoot dry weight and acetylene reduction activity of the plant inoculated with Bradyrhizobium elkanii LauBG38 were significantly higher in ARA per plant, nodule and shoot dry weights than the other tested isolates in both Yezin-4 and Yezin-7 black gram varieties. We expect that Myanmar Bradyrhizobium elkanii LauBG38 will be able to use as Biofertilizer for black gram cultivars.

Africa is known for its rich legume diversity with a significant number of endemic species originating in South Africa. Many of these legumes associate with rhizobial symbionts of the genus Bradyrhizobium, of which most represent new species. Yet, none of the Bradyrhizobium species from South Africa have been described. In this study, phylogenetic analysis of 16S rRNA gene sequences of fourteen strains isolated in southern Africa from root nodules of diverse legumes (i.e., from the tribes Crotalarieae, Acacieae, Genisteae, Phaseoleae and Cassieae) revealed that they belong to the Bradyrhizobium elkanii supergroup. The taxonomic position and possible novelty of these strains were further interrogated using genealogical concordance of five housekeeping genes (atpD, dnaK, glnII, gyrB and rpoB). These phylogenies consistently recovered four monophyletic groups and one singleton within Bradyrhizobium. Of these groups, two were conspecific with Bradyrhizobium brasilense UFLA 03-321T and Bradyrhizobium ivorense CI-1BT, while the remaining three represented novel taxa. Their existence was further supported with genome data, as well as metabolic and physiological traits. Analysis of nodA gene sequences further showed that the evolution of these bacteria likely involved adapting to local legume hosts and environmental conditions through the acquisition, via horizontal gene transfer, of optimal symbiotic loci. We accordingly propose the following names Bradyrhizobium acaciae sp. nov. 10BBT (SARCC 730T = LMG 31409T), Bradyrhizobium oropedii sp. nov. Pear76T (SARCC 731T = LMG 31408T), and Bradyrhizobium altum sp. nov. Pear77T (SARCC 754T = LMG 31407T) to accommodate three novel species, all of which are symbionts of legumes in South Africa.

Bradyrhizobium species are symbiotic partners of soybean plants. However, some Bradyrhizobium bacteria do not form functional nodules on the roots of Rj4 genotype soybean cultivars. Our obj...

Industrial seed treatment of soybean [Glycine max (L.) Merr.] has increased over the last years. New technologies have been developed to allow the inoculation procedure, which is traditionally done at the day of sowing, to be performed with the industrial treatment days or even weeks before sowing. Since little is known about the compatibility of agrochemicals and pre-inoculation, the objective of this study was to determine whether the storage period and the combination of fungicides and insecticides could negatively affect the physiological quality and yield of soybean seeds that were pre-inoculated. Soybean seeds received fourteen seed treatments that consisted of different fungicides and insecticides and were pre-inoculated with Bradyrhizobium elkanii. The seeds were treated and stored for 51 days until it was sown in the field. Every 17 days the physiological quality of the seed was assessed. The results shown that pre-inoculation did not affect the physiological quality of seeds. However, some combinations of agrochemicals, as well as storage period reduced seed vigor and seed germination, while increased abnormal seedlings. The findings of this study indicated that some combinations of fungicides and insecticides can have adverse effect on the physiological quality of seed that is stored for up to 51 days before sowing, but none of them jeopardized the nodulation and soybean yield under field conditions. Key words: Biological nitrogen fixation, Bradyrhizobium, compatibility, fungicide, Glycine max, HiCoat, industrial seed treatment, insecticide, polymer.

Bradyrhizobium elkanii utilizes the type III effector Bel2-5 for nodulation in host plants in the absence of Nod factors (NFs). In soybean plants carrying the Rj4 allele, however, Bel2-5 causes restriction of nodulation by triggering immune responses. Bel2-5 shows similarity with XopD of the phytopathogen Xanthomonas campestris pv. vesicatoria and possesses two internal repeat sequences, two ethylene (ET)-responsive element-binding factor-associated amphiphilic repression (EAR) motifs, a nuclear localization signal (NLS), and a ubiquitin-like protease (ULP) domain, which are all conserved in XopD except for the repeat domains. By mutational analysis, we revealed that most of the putative domains/motifs in Bel2-5 were essential for both NF-independent nodulation and nodulation restriction in Rj4 soybean. The expression of soybean symbiosis- and defense-related genes was also significantly altered by inoculation with the bel2-5 domain/motif mutants compared with the expression upon inoculation with wild-type B. elkanii, which was mostly consistent with the phenotypic changes of nodulation in host plants. Notably, the functionality of Bel2-5 was mostly correlated with the growth inhibition effect of Bel2-5 expressed in yeast cells. The nodulation phenotypes of the domain-swapped mutants of Bel2-5 and XopD indicated that both the C-terminal ULP domain and upstream region are required for the Bel2-5-dependent nodulation phenotypes. These results suggest that Bel2-5 interacts with and modifies host targets via these multiple domains to execute both NF-independent symbiosis and nodulation restriction in Rj4 soybean.

The study of the nitrogen fixation and phylogenetic diversity of nodule microsymbionts of grain legumes in many parts of the globe is often carried out in order to identify legume-rhizobia combinations for agricultural sustainability. Several reports have therefore found that rhizobial species diversity is shaped by edapho-climatic conditions that characterize different geographic locations, suggesting that rhizobial communities often possess traits that aid their adaptation to their habitat. In this study, the soybean-nodulating rhizobia from semi-arid savannahs of Ghana and South Africa were evaluated. The authenticated rhizobial isolates were highly diverse based on their colony characteristics, as well as their BOX-PCR profiles and gene sequences. In the 16S rRNA phylogeny, the isolates were placed in the different clades Bradyrhizobium iriomotense and Bradyrhizobium jicamae together with two superclades Bradyrhizobium japonicum and Bradyrhizobium elkanii. The multilocus (atpD, glnII, gyrB, recA) phylogenetic analyses indicated the dominance of Bradyrhizobium diazoefficiens and putative new Bradyrhizobium species in the semi-arid Ghanaian region. The phylogenetic analyses based on the symbiotic genes (nifH and nodC) clustered the test isolates into different symbiovars (sv. glycinearum, sv. retame and sv. sojae). Principal component analysis (PCA) showed that soil factors played a significant role in favoring the occurrence of soybean-nodulating microsymbionts in the tested local conditions. The results suggested that isolates had marked local adaptation to the prevailing conditions in semi-arid regions but further studies are needed to confirm new Bradyrhizobium species.

Vigna minima is a climbing annual plant widely distributed in barren wilderness, grass land, and shrub bush of China and other countries such as Japan. However, the rhizobia nodulating with this plant has never been systematically studied. In order to reveal the biodiversity of nodulating rhizobia symbiosis with V. minima, a total of 874 rhizobium isolates were obtained from root nodules of the plant spread in 11 sampling sites of Shandong Peninsula, China, and they were designated as 41 haplotypes in the genus Bradyrhizobium based upon recA sequence analyses. By multilocus sequence analysis (MLSA) of five housekeeping genes (dnaK, glnII, gyrB, recA, and rpoB), the 41 strains representing different recA haplotypes were classified into nine defined species and nine novel genospecies. Bradyrhizobium elkanii, Bradyrhizobium ferriligni, and Bradyrhizobium pachyrhizi were the predominant and universally distributed groups. The phylogeny of symbiotic genes of nodC and nifH showed similar topology and phylogenetic relationships, in which all the representative strains were classified into two clades grouped with strains nodulating with Vigna spp., demonstrating that Vigna spp. shared common nodulating groups in the natural environment. All the representative strains formed nodules with V. minima in a nodulation test performed in green house conditions. The correlation between V. minima nodulating rhizobia and soil characteristics analyzed by CANOCO indicates that available nitrogen, total nitrogen, and organic carbon in the soil samples were the main factors affecting the distribution of rhizobia isolated in this study. This study systematically uncovered the biodiversity and distribution characteristics of V. minima nodulating rhizobia for the first time, which provided novel information for the formation of the corresponding rhizobium community.

Growth and competitive infection behaviors of two sets of Bradyrhizobium spp. strains were examined at different temperatures to explain strain-specific soybean nodulation under local climate conditions. Each set consisted of three strains—B. japonicum Hh 16-9 (Bj11-1), B. japonicum Hh 16-25 (Bj11-2), and B. elkanii Hk 16-7 (BeL7), and B. japonicum Kh 16-43 (Bj10J-2), B. japonicum Kh 16-64 (Bj10J-4), and B. elkanii Kh 16-7 (BeL7)—which were isolated from the soybean nodules cultivated in Fukagawa and Miyazaki soils, respectively. The growth of each strain was evaluated in YM liquid medium at 15, 20, 25, 30, and 35 °C with shaking at 125 rpm for one week while measuring their OD660 daily. In the competitive infection experiment, each set of the strains was inoculated in sterilized vermiculite followed by sowing surface-sterilized soybean seeds, and they were cultivated at 20/18 °C and 30/28 °C in a 16/8 h (day/night) cycle in a phytotron for three weeks, then nodule compositions were determined based on the partial 16S-23R rRNA internal transcribes spacer (ITS) gene sequence of the isolates from the nodules. The optimum growth temperatures were at 15–20 °C for all B. japonicum strains, while they were at 25–35 °C for all B. elkanii strains. In the competitive experiment with the Fukagawa strains, Bj11-1 and BeL7 dominated in the nodules at the low and high temperatures, respectively. In the Miyazaki strains, BjS10J-2 and BeL7 dominated at the low and high temperatures, respectively. It can be assumed that temperature of soil affects rhizobia growth in rhizospheres and could be a reason for the different competitive properties of B. japonicum and B. elkanii strains at different temperatures. In addition, competitive infection was suggested between the B. japonicum strains.

The mung bean has a great potential under tropical conditions given its high content of grain protein. Additionally, its ability to benefit from biological nitrogen fixation (BNF) through association with native rhizobia inhabiting nodule microbiome provides most of the nitrogen independence on fertilizers. Soil microbial communities which are influenced by biogeographical factors and soil properties, represent a source of rhizobacteria capable of stimulating plant growth. The objective of this study is to support selection of beneficial bacteria that form positive interactions with mung bean plants cultivated in tropical soils, as part of a seed inoculation program for increasing grain yield based on the BNF and other mechanisms. Two mung bean genotypes (Camaleão and Esmeralda) were cultivated in 10 soil samples. Nodule microbiome was characterized by next-generation sequencing using Illumina MiSeq 16S rRNA. More than 99% of nodule sequences showed similarity with Bradyrhizobium genus, the only rhizobial present in nodules in our study. Higher bacterial diversity of soil samples collected in agribusiness areas (MW_MT-I, II or III) was associated with Esmeralda genotype, while an organic agroecosystem soil sample (SE_RJ-V) showed the highest bacterial diversity independent of genotype. Furthermore, OTUs close to Bradyrhizobium elkanii have dominated in all soil samples, except in the sample from the organic agroecosystem, where just B. japonicum was present. Bacterial community of mung bean nodules is mainly influenced by soil pH, K, Ca, and P. Besides a difference on nodule colonization by OTU sequences close to the Pseudomonas genus regarding the two genotypes was detected too. Although representing a small rate, around 0.1% of the total, Pseudomonas OTUs were only retrieved from nodules of Esmeralda genotype, suggesting a different trait regarding specificity between macro- and micro-symbionts. The microbiome analysis will guide the next steps in the development of an inoculant for mung bean aiming to promote plant growth and grain yield, composed either by an efficient Bradyrhizobium strain on its own or co-inoculated with a Pseudomonas strain. Considering the results achieved, the assessment of microbial ecology parameters is a potent coadjuvant capable to accelerate the inoculant development process and to improve the benefits to the crop by soil microorganisms.

Bradyrhizobium sp. RD5-C2, isolated from soil that is not contaminated with 2,4-dichlorophenoxyacetic acid (2,4-D), degrades the herbicides 2,4-D and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). It possesses tfdAα and cadA (designated as cadA1), which encode 2,4-D dioxygenase and the oxygenase large subunit, respectively. In the present study, the genome of Bradyrhizobium sp. RD5-C2 was sequenced and a second cadA gene (designated as cadA2) was identified. The two cadA genes belonged to distinct clusters comprising the cadR1A1B1K1C1 and cadR2A2B2C2K2S genes. The proteins encoded by the cad1 cluster exhibited high amino acid sequence similarities to those of other 2,4-D degraders, while Cad2 proteins were more similar to those of non-2,4-D degraders. Both cad clusters were capable of degrading 2,4-D and 2,4,5-T when expressed in non-2,4-D-degrading Bradyrhizobium elkanii USDA94. To examine the contribution of each degradation gene cluster to the degradation activity of Bradyrhizobium sp. RD5-C2, cadA1, cadA2, and tfdAα deletion mutants were constructed. The cadA1 deletion resulted in a more significant decrease in the ability to degrade chlorophenoxy compounds than the cadA2 and tfdAα deletions, indicating that degradation activity was primarily governed by the cad1 cluster. The results of a quantitative reverse transcription-PCR analysis suggested that exposure to 2,4-D and 2,4,5-T markedly up-regulated cadA1 expression. Collectively, these results indicate that the cad1 cluster plays an important role in the degradation of Bradyrhizobium sp. RD5-C2 due to its high expression.

Although inoculating soybean with rhizobia for biological nitrogen fixation is a common practice in agriculture, rhizobia are also known to associate with grasses. In this study, we evaluate the potential utility of the rhizobial strains SEMIA 587 and 5019 (Bradyrhizobium elkanii), 5079 (Bradyrhizobium japonicum), and 5080 (Bradyrhizobium diazoefficiens), recommended for Brazilian soybean inoculation, in colonizing black oat plants and promoting growth in black and white oats, and ryegrass. Inoculation of white oats with SEMIA 587 increase the seed germination (SG) by 32.09%, whereas the SG of black oats inoculated with SEMIA 587 and 5019 increased by 40.38% and 37.85%, respectively. Similarly, inoculation of ryegrass with all strains increased SG values between 24.63 and 27.59%. In addition, white oats with SEMIA 587 and 5080 had root areas significantly superior to those in other treatments, whereas inoculation with SEMIA 5079 and 5080 resulted in the highest volume of roots. Likewise, SEMIA 5079 and 5080 significantly increased the length, volume, and area of black oats roots, whereas SEMIA 587 increased the volume, area, and dry mass of roots and shoot. Inoculation in ryegrass with SEMIA 587 significantly increased the root volume. Moreover, most strains transformed with gfp and gus were observed to colonize the roots of black oats. Collectively, the findings of this study indicate that rhizobial strains recommended for inoculation of soybean can also be used to promote the growth of the three assessed grass species, and are able to colonize the roots of black oats.

In natural plant populations, a fine-scale spatial genetic structure (SGS) can result from limited gene flow, selection pressures or spatial autocorrelation. However, limited gene flow is considered the predominant determinant in the establishment of SGS. With limited dispersal ability of bacterial cells in soil and host influence on their variety and abundance, spatial autocorrelation of bacterial communities associated with plants is expected. For this study, we collected genetic data from legume host plants, Chamaecrista fasciculata, their Bradyrhizobium symbionts and rhizosphere free-living bacteria at a small spatial scale to evaluate the extent to which symbiotic partners will have similar SGS and to understand how plant hosts choose among nodulating symbionts. We found SGS across all sampled plants for both the host plants and nodulating rhizobia, suggesting that both organisms are influenced by similar mechanisms structuring genetic diversity or shared habitat preferences by both plants and microbes. We also found that plant genetic identity and geographic distance might serve as predictors of nodulating rhizobia genetic identity. Bradyrhizobium elkanii was the only type of rhizobia found in nodules, which suggests some level of selection by the host plant.

Nitrogen-fixing root nodule symbiosis occurs in ten taxonomic lineages from four related orders -Fagales, Fabales, Rosales and Cucurbitales- that together are called the nitrogen-fixing clade (NFC). Nodulating plants within the NFC are scattered by non-nodulating species, as well as can interact either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. Besides, it is generally assumed that nodulation evolved independently multiple times, though molecular genetic support for this hypothesis is lacking.Parasponia is the only non-legume plant which can establish nitrogen-fixing endosymbiosis with rhizobium, and it is the only nodulating plant within the Cannabaceae. The Parasponia lineage represents five species and phylogenetic analysis shows that this lineage is embedded within the non-nodulating Trema clade. As Parasponia and Trema are closely related, F1 hybrids could be created by crossing of the diploid Parasponia andersonii (2n=20) and the allotetraploid Trema tomentosa (2n=4X=40). Conceptually, P. andersonii x T. tomentosa F1 hybrid plants reflects a diploid T. tomentosa with a haploid genome of P. andersonii introduced. The F1 hybrid between diploid Parasponia andersonii and tetraploid Trema tomentosa can form nodules, whereas it is devoid of intracellular infection when inoculated with either Mesorhizobium plurifarium BOR2 or Bradyrhizobium elkanii WUR3. Based on its genetic composition and symbiotic phenotype, we argue that the F1 hybrid may mimic future engineer results. Therefore, we aimed to obtain a better understanding of the deviation in nodulation phenotype of wild type P. andersonii and F1 hybrid plants. To do so, we compared nodulation efficiencies and intracellular infection within nodule cells upon inoculation with a range of rhizobium strains, as Parasponia can interact with a wide range of rhizobia. This revealed that the host range of hybrid plants is narrower when compared to P. andersonii. We also show that the block in intracellular infection within hybrid nodules is consistent for all nodulating strains identified, cannot be overcome by increased LCO biosynthesis nor by mutating the type III or IV secretion systems of nodulating strains. The hybrid plants can establish arbuscular mycorrhization effectively, suggesting that the block of intracellular infection is rhizobium specific. Taken together, this indicates the occurrence of a yet unknown mechanism leading to an impaired host range and block of intracellular infection of hybrid plants.To answer evolution and genetic basis of nodulation, comparative genomic and transcriptomic analysis has been conducted using Parasponia species (Cannabaceae), the only non-legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that in legumes are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related non-nodulating plant species show evidence of parallel loss in non-nodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION. Parallel loss of these symbiosis genes indicates that these non-nodulating lineages lost the potential to nodulate. By making use of the highly efficient Parasponia transformation platform, we conducted promoter:GUS expression analysis as well as CRISPR-Cas9 mutagenesis. Consistent with legumes, P. andersonii PanNIN and PanNF-YA1 are co-expressed in nodules. By analyzing single, double and higher-order CRISPR-Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF-YA1 are PanNIN-dependent and that PanNF-YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF-YA1 commit conserved symbiotic functions in non-elgume plant species. As Rosales, Fabales and Fagales diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis. Taken together, these results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ~100 million years ago in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

We report here the annotated draft genome sequence of the rhizobium strain BR 2003. This strain is able to establish symbiosis and to fix nitrogen with a broad range of leguminous species. The estimation of the average nucleotide identity confirmed the strain as a member of Bradyrhizobium elkanii.

The symbiosis of soybean with Bradyrhizobium diazoefficiens USDA110, which always competes with other rhizobia in the field, is of great agronomic and environmental importance. Herein, a dual-luciferase reporter assay was utilized to monitor the dynamics of two dominant bradyrhizobia infecting roots of soybean. More explicitly, luciferase-tagged B. diazoefficiens USDA110 (USDA110-FLuc) and Bradyrhizobium elkanii USDA 94 (USDA94-RLuc) were designed, co-inoculated into soybean seeds, and observed for their colonization in root nodules by bioluminescence imaging. The results showed that USDA110-FLuc initiated infection earlier than USDA94-RLuc, but its occupancy in the nodules decreased as the plant grew. A nodulation test showed that nodD1 mutant USDA110 strains, including CRISPR engineered mutants, were less competitive than wild type. I constructed siRNAs to knockdown nodD1 at different target sites and transformed them into the bacteria. Surprisingly, although siRNAs - with 3' end target sites - were able to repress up to 65% of nodD1 expression, the profiling of total RNAs with a bioanalyzer revealed that 23S/16S-rRNA ratios of siRNA-transformed and wild type USDA110 strains were similar, but lower than that of nodD1 mutant. In short, the current work - while reporting the competitiveness of B. diazoefficiens USDA110 in early occupancy of soybean nodules and the gene nodD1 as a key determinant of this infection - gives an insight on siRNA silencing in microbes, and demonstrates a highly efficient imaging approach that could entail many new avenues for many biological research fields.

O tratamento de sementes de soja tem grande adoção e relevância no Brasil. Nesse contexto, o tratamento industrial de sementes vem com a advento de trazer facilidade aos produtores de soja em questões relacionadas à logística, à qualidade e à possibilidade de maximizar a produtividade de grãos. O tratamento industrial de sementes permite a associação de diferentes xenobióticos com inoculantes, porém há muitas dúvidas em relação aos efeitos dessa associação, principalmente entre fungicidas e inoculantes. Com o exposto, o presente trabalho teve por objetivo estudar como o armazenamento de sementes inoculadas e o tratamento de sementes, influencia a fixação biológica de nitrogênio (FBN), o desenvolvimento de plantas e a produtividade na cultura da soja. Um experimento em vaso e dois experimentos em campo foram conduzidos para avaliar a nodulação, concentração de ureídeos, eficiência da fixação biológica de nitrogênio, atividade da nitrogenase, biomassa de plantas, produtividade e massa de mil grãos. Tanto o armazenamento de sementes pré-inoculadas, como o uso de produtos comerciais no tratamento de sementes influenciaram variáveis relacionadas à FBN nos três experimentos. Considerando-se os componentes de produção da cultura, o armazenamento influenciou a massa de mil grãos, enquanto os tratamentos de sementes influenciaram a produtividade apenas aos 30 dias de armazenamento. The treatment of soybean seeds has been widely adopted and is relevant in Brazil. In this context, the industrial treatment of seeds comes with the advantage of making it easier for soybean growers in matters related to logistics, quality and the possibility of maximizing grain yield. Industrial seed treatment allows the association of different xenobiotics with inoculants, but there are many doubts regarding the effects of this association, especially between fungicides and inoculants. With the above, the present work aimed to study how the storage of inoculated seeds and seed treatment influenced the biological nitrogen fixation (BNF), plant development and yield in soybean crop. One pot experiment and two field experiments were conducted to evaluate nodulation, ureides concentration, biological nitrogen fixation efficiency, nitrogenase activity, plant biomass, yield and one thousand grain mass. Both the storage of pre-inoculated seeds and the use of commercial products in seed treatment influenced variables related to BNF in the three experiments. Considering the crop production components, storage influenced the mass of one thousand grains, while seed treatments influenced the yield only at 30 days of storage.

Bradyrhizobium elkanii BLY3-8 does not form nodules on the roots of Rj3-genotype soybean (cultivar D-51). This is a cultivar-specific nodulation restriction. The genes A6X20_40975 and A6X20_41030 in strain BLY3-8 were predicted to encode the transcriptional activator and apparatus of the type III secretion system (T3SS) (the proteins TtsI and RhcJ), respectively. Mutants disrupted in these genes overcame the nodulation restriction. These results suggest that an effector injected via T3SS into Rj3-genotype soybean is involved in nodulation restriction by Rj3-genotype soybean.

In the past thirty years, the biosafety of the aboveground part of crops, including horizontal gene transferal through pollen dispersal and hybridization, has been the focus of research; however, microbial communities in the underground part are attracting increasing attention. In the present study, the soybean root-associated bacterial communities of the G2-EPSPS plus GAT transgenic soybean line Z106, its recipient variety ZH10, and Z106 treated with glyphosate (Z106J) were compared at the seedling, flowering, and seed filling stages by high-throughput sequencing of the V4 hypervariable regions of 16S rRNA gene amplicons using Illumina MiSeq. The results obtained showed no significant differences in the alpha/beta diversities of root-associated bacterial communities at the three stages among ZH10, Z106, and Z106J under field growth conditions; however, the relative abundance of four main nitrogen-fixing bacterial genera significantly differed among ZH10, Z106, and Z106J. Ternary plot results indicated that in the root compartment, the proportional contributions of rhizobial nitrogen-fixing Ensifer fredii and Bradyrhizobium elkanii, which exhibit an extremely broad nodulation host range, markedly differed among the three treatments at the three stages. Thus, the present results indicate that transgenic G2-EPSPS and GAT soybean may induce different changes in functional bacterial species in soil, such as E. fredii and B. elkanii, from ZH10, which were compensated for/enriched at the flowering and seed filling stages, respectively, to some extent through as of yet unknown mechanisms by transgenic soybean treated with glyphosate.

Organisms rely on symbiotic associations for metabolism, protection, and energy. However, these intimate partnerships can be vulnerable to exploitation. What prevents microbial mutualists from parasitizing their hosts? In legumes, there is evidence that hosts have evolved sophisticated mechanisms to manage their symbiotic rhizobia, but the generality and evolutionary origins of these control mechanisms are under debate. Here, we focused on the symbiosis between Parasponia hosts and N2-fixing rhizobium bacteria. Parasponia is the only non-legume lineage to have evolved a rhizobial symbiosis and thus provides an evolutionary replicate to test how rhizobial exploitation is controlled. A key question is whether Parasponia hosts can prevent colonization of rhizobia under high nitrogen conditions, when the contribution of the symbiont becomes nonessential. We grew Parasponia andersonii inoculated with Bradyrhizobium elkanii under four ammonium nitrate concentrations in a controlled growth chamber. We measured shoot and root dry weight, nodule number, nodule fresh weight, nodule volume. To quantify viable rhizobial populations in planta, we crushed nodules and determined colony forming units (CFU), as a rhizobia fitness proxy. We show that, like legumes and actinorhizal plants, P. andersonii is able to control nodule symbiosis in response to exogenous nitrogen. While the relative host growth benefits of inoculation decreased with nitrogen fertilization, our highest ammonium nitrate concentration (3.75 mM) was sufficient to prevent nodule formation on inoculated roots. Rhizobial populations were highest in nitrogen free medium. While we do not yet know the mechanism, our results suggest that control mechanisms over rhizobia are not exclusive to the legume clade.

Bradyrhizobium elkanii, a rhizobium with a relatively wide host range, possesses a functional type III secretion system (T3SS) that is involved in symbiotic incompatibility against Rj4-genotype soybean (Glycine max) and some accessions of mung bean (Vigna radiata). To expand our knowledge on the T3SS-mediated partner selection mechanism in the symbiotic legume-rhizobia association, we inoculated three Lotus experimental accessions with wild-type and T3SS-mutant strains of B. elkanii USDA61. Different responses were induced by T3SS in a host genotype-dependent manner. Lotus japonicus Gifu inhibited infection; L. burttii allowed infection, but inhibited nodule maturation at the post-infection stage; and L. burttii and L. japonicus MG-20 both displayed a nodule early senescence-like response. By conducting inoculation tests with mutants of previously reported and newly identified effector protein genes of B. elkanii USDA61, we identified NopF as the effector protein triggering the inhibition of infection, and NopM as the effector protein triggering the nodule early senescence-like response. Consistent with these results, the B. elkanii USDA61 gene for NopF introduced into the Lotus symbiont Mesorhizobium japonicum induced infection inhibition in L. japonicus Gifu, but did not induce any response in L. burttii or L. japonicus MG-20. These results suggest that Lotus accessions possess at least three checkpoints to eliminate unfavorable symbionts, including the post-infection stage, by recognizing different T3SS effector proteins at each checkpoint.

Cowpea (Vigna unguiculata L. Walp.) is indigenous to Africa, and highly valued for its N2-fixing trait and the nutritional attributes of its grain and leaves. The species’ ability to establish effective symbiosis with diverse rhizobial populations gives it survival and growth advantage in N-limited environments. To explore the functional diversity and phylogenetic positions of rhizobia nodulating cowpea in Africa, nodules were collected from various cowpea varieties grown in soils from the Guinea savanna and Sudano-sahelian agroecologies of Northern Ghana, and from the lowveld and middleveld areas of Mpumalanga Province in South Africa. Box-PCR profiling and multilocus sequence analysis revealed the presence of diverse microsymbionts responsible for cowpea nodulation across the study sites. BOX-PCR amplifications yielded variable band sizes, ranging from 618 bp to 5354 bp, which placed the isolates in six major clusters (Cluster A–F). Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, rpoB, nifH and nodC genes revealed the presence of diverse Bradyrhizobium sp. closely related to Bradyrhizobium daqingense, Bradyrhizobium subterraneum, Bradyrhizobium yuanmingense, Bradyrhizobium embrapense, Bradyrhizobium pachyrhizi, Bradyrhizobium elkanii and novel Bradyrhizobium species in the soils studied, a finding that could be attributed to the unique edapho-climatic conditions of the contrasting environments. The test isolates exhibited distinct symbiotic efficiencies, and also induced variable (p ≤ 0.001) photosynthetic rates, leaf transpiration, total chlorophyll and shoot biomass accumulation on cowpea (their homologous host). Canonical correspondence analysis showed that the distribution of these microsymbionts was influenced by the concentrations of macro- and micronutrients in soils. The pairwise genetic distances derived from phylogenies and nodule functioning showed significant (p Bradyrhizobium symbiosis.

Pre-inoculation of soybean seeds with Bradyrhizobium spp. associated with polymers could be an alternative to the conventional inoculation performed at the day of sowing. The objectives of this study were i) to determine whether the HiCoat® technology (peat inoculant + liquid inoculant + polymer) allows the survival of B. elkanii after storage of the inoculated seeds, and ii) to evaluate whether the pre-inoculation and seed treatment reduces the physiological quality of seed and soybean yield. Soybean seeds were treated with different fungicides and insecticides associated with pre-inoculation using HiCoat® and the seeds were stored at room temperature. The survival of B. elkanii was reduced over time with standard inoculation, however there was above 2 x 106 colony-forming units of B. elkanii per seed at 60 days after inoculation with HiCoat®. Seed germination, number of nodules per plant, mass of nodules, yield, number of grains per plant and number of pods per plants were not significantly affected by the pre-inoculation and seed treatments. Our findings indicated that the fungicide and insecticide associated with HiCoat® can be used to treat and to pre-inoculate seeds that can be stored for up to 71 days without compromising the nodulation of plants and soybean yield. Key words: Industrial seed treatment, biological nitrogen fixation, storage period, fungicide, insecticide, polymer, HiCoat, Glycine max.

Phenotypic and genotypic characteristics of cowpea rhizobia indigenous to soils of Ethiopia are unknown. Forty indigenous cowpea rhizobial isolates were collected from cowpea-growing areas of the country and were characterized for their growth and genetic properties. Based on their cultural characteristics, the isolates were categorized into fast (FG), slow (SG), and extraslow-growings (ESG). The FG, SG, and ESG isolates had mean generation time (h)/colony diameter (mm)/date of turbidity formation (d) in the range of 2.5-7.5/2-4/2-3, 7.5-30/0.5-3.5/3-5, and 30-50/0.5-1/5-7, respectively. Thirty two and sixteen percentages of the isolates were FG and ESG, respectively. Most of the isolates (87%) could grow on culture medium of pH 4.5, but were intolerant of pH 8. The intrinsic antibiotics resistance (IAR) pattern was FG>SG>ESG for ampicillin, ciprofloxacin, chloramphenicol, erythromycin, neomycin sulfate and penicillin, whereas the pattern was FG SG>ESG for dextrin, dextrose, glucose, starch and sucrose whereas it was FG 7.5 h were grouped together at 70% of similarity. Partial sequence analysis of 16S rRNA gene showed the existence of isolates most similar to rhizobial species of Bradyrhizobium species, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium rubi, and Mesorhizobium species. In general, cowpea rhizobial isolates from soils of Ethiopia in this study were mainly SG and sensitive to stress in vitro conditions, but versatile in utilization of varieties of C and N substrates. Such studies are important in Ethiopia to identify rhizobial isolates that could be amendable for use as inoculants to improve cowpea production. Key words: Phylogeny, diversity, isolates, growth categories, cluster analysis.

Four bacterial EPSs extracted from Rhizobium leguminosarum bv. trifolii Rt24.2, Sinorhizobium meliloti Rm1021, Bradyrhizobium japonicum USDA110, and Bradyrhizobium elkanii USDA76 were determined towards their metal ion adsorption properties and possible modification of Cerrena unicolor laccase properties. The highest magnesium and iron ion-sorption capacity (~ 42 and ~ 14.5%, respectively) was observed for EPS isolated from B. japonicum USDA110. An evident influence of EPSs on the stability of laccase compared to the control values (without EPSs) was shown after 30-day incubation at 25 °C. The residual activity of laccases was obtained in the presence of Rh76EPS and Rh1021EPS, i.e., 49.5 and 41.5% of the initial catalytic activity, respectively. This result was confirmed by native PAGE electrophoresis. The EPS effect on laccase stability at different pH (from 3.8 to 7.0) was also estimated. The most significant changes at the optimum pH value (pH 5.8) was observed in samples of laccase stabilized by Rh76EPS and Rh1021EPS. Cyclic voltamperometry was used for analysis of electrochemical parameters of laccase stabilized by bacterial EPS and immobilized on single-walled carbon nanotubes (SWCNTs) with aryl residues. Laccases with Rh76EPS and Rh1021EPS had an evident shift of the value of the redox potential compared to the control without EPS addition. In conclusion, the results obtained in this work present a new potential use of bacterial EPSs as a metal-binding component and a modulator of laccase properties especially stability of enzyme activity, which can be a very effective tool in biotechnology and industrial applications.

Co-inoculation of selected nitrogen-fixing bacteria with plant growth-promoting bacteria is the promising way for the improvement of soybean production through enhancing plant growth, nodulation, and N2 fixation. Therefore, this experiment was conducted to study the effects of co-inoculation of Bradyrhizobium elkanii BLY3-8 with Streptomyces griseoflavus P4 on plant growth, nodulation, N2 fixation, N uptake, and seed yield of Rj4 soybean varieties. Two experiments with completely randomized design and three replicates were done in this study. N2-fixation ability of soybean was evaluated by acetylene reduction activity (ARA) and relative ureide method. In the first experiment, synergetic effect in N2 fixation and nodulation was occurred in co-inoculation treatment (BLY3-8 + P4) in Yezin-3 and Fukuyutaka. Based on these results, co-inoculation effect of BLY3-8 and P4 was assessed on Yezin-3 and Fukuyutaka varieties at three different growth stages, using Futsukaichi soil under natural environmental ...

A total of eighty one (81) rhizobial isolates were recovered from root nodules of cowpea (Vigna unguiculata L. Walp.) and groundnut (Arachis hypogaea L.) grown in soils collected from eight different sites (Hawassa, Wondogenet, Chofa, Badawacho, Bodity, Gofa, Ziway, and Alemtena) in Ethiopia with no known history of inoculation. The test isolates together with seven reference strains belonging to five genera including Rhizobium, Ensifer, Mesorhizobium, Bradyrhizobium and Azorhizobium were characterized using ninety phenotypic traits. Thirty one isolates (38%) were found to be fast growers while fifty isolates (62%) were slow growers. The majority of the isolates showed an intrinsic resistance to antibiotics (µg/ml), Chloramphenicol (5 and 15), Lincomycin (100), Novobiocin (0.5 and 1.5), and Erythromycin (10 and 20) and to heavy metals manganese sulphate (500) and copper chloride (100). Most isolates did not tolerate NaCl concentration >3% (w/v) and high temperature (45°C). Dendrogram was constructed by applying the unweighted pair group method with arithmetic means (UPGMA) using NTSYSpc Version 2.1. They were grouped into seven clusters and eight unclustered positions, when 82% relative similarity was used as a cut point. Fifty eight percent of the test isolates were grouped with Bradyrhizobium japonicum and Bradyrhizobium elkanii superclades, thus indicating that rhizobia nodulating cowpea and groundnut are delineated within a branch that defines Bradyrhizobium genus. To elucidate the precise taxonomic positions of the isolates, further genetic studies are required using modern molecular biological methods. Key words: Groundnut, cowpea, isolates, phenotypic traits, Bradyrhizobium, Rhizobium.