Strain USDA Research Articles

Genetic variation and genetic complexity of nodule occupancy in soybean inoculated with USDA110 and USDA123 rhizobium strains

BackgroundSymbiotic nitrogen fixation differs among Bradyrhizobium japonicum strains. Soybean inoculated with USDA123 has a lower yield than strains known to have high nitrogen fixation efficiency, such as USDA110. In the main soybean-producing area in the Midwest of the United States, USDA123 has a high nodule incidence in field-grown soybean and is competitive but inefficient in nitrogen fixation. In this study, a high-throughput system was developed to characterize nodule number among 1,321 Glycine max and 69 Glycine soja accessions single inoculated with USDA110 and USDA123.ResultsSeventy-three G. max accessions with significantly different nodule number of USDA110 and USDA123 were identified. After double inoculating 35 of the 73 accessions, it was observed that PI189939, PI317335, PI324187B, PI548461, PI562373, and PI628961 were occupied by USDA110 and double-strain nodules but not by USDA123 nodules alone. PI567624 was only occupied by USDA110 nodules, and PI507429 restricted all strains. Analysis showed that 35 loci were associated with nodule number in G. max when inoculated with strain USDA110 and 35 loci with USDA123. Twenty-three loci were identified in G. soja when inoculated with strain USDA110 and 34 with USDA123. Only four loci were common across two treatments, and each locus could only explain 0.8 to 1.5% of phenotypic variation.ConclusionsHigh-throughput phenotyping systems to characterize nodule number and occupancy were developed, and soybean germplasm restricting rhizobium strain USDA123 but preferring USDA110 was identified. The larger number of minor effects and a small few common loci controlling the nodule number indicated trait genetic complexity and strain-dependent nodulation restriction. The information from the present study will add to the development of cultivars that limit USDA123, thereby increasing nitrogen fixation efficiency and productivity.

Bradyrhizobium inoculants impact on promiscuous nodulating soybeans cultivars in Ghana's farming systems

AbstractSoybean is rarely inoculated with commercial Bradyrhizobium inoculants in Ghana's cropping systems. A 2‐year field study was conducted at Council for Scientific and Industrial Research–Savanna Agricultural Research Institute's experimental field at Nyankpala, Ghana to assess how commercial inoculants affect growth, nodulation, nitrogen fixation, and grain yield of promiscuous nodulating soybean cultivars. The experiment was a split‐plot design with the main plot factor as soybean [tropical Glycine max (L.) Merr. crosses (TGX) cultivars]; Jenguma (TGX1448‐2E), Afayak (TGX1834‐5E), and Songda (TGX 1445‐3E). The subplot factor consisted of three commercial Bradyrhizobium japonicum inoculants, namely, Biofix (strain USDA 110), NoduMax (strain USDA 110), and Legumefix (strain 532c) plus uninoculated control. Shoot biomass and nodulation were assessed at vegetative (V8, 8‐leaf stage), full bloom (R2), beginning to pod (R3), full pod (R4), and full seed (R6) stages, respectively, and N fixation and grain yield at maturity. Shoot dry matter increased with growth stage and peaked at the R6 stage with significant effect associated with Biofix and NoduMax. Nodulation on the upper root and the whole root also peaked at the R4 stage, with pronounced effects associated with Biofix and NoduMax. Enhanced grain yields of up to 30% were achieved with Biofix and NoduMax compared to the uninoculated control in both 2016 and 2017. Regarding the cultivars, shoot biomass increased with growth stage and peaked at R6 stage and Afayak yielded greater shoot biomass. On average (2 years), Afayak produced greater grain yields than Jenguma and Songda. In conclusion, Biofix and NoduMax were the most promising commercial inoculants for soybean production in Northern Ghana.

Symbiotic effectiveness of Bradyrhizobium strains on soybean growth and productivity in Northern Mozambique

Inoculation of soybean [Glycine max(L.) Merr.] with rhizobia strains is a low-cost investment which can increase yields of smallholder farmers in Mozambique. The performance of fourBradyrhizobiumstrains was evaluated to identify the best strain to inoculate soybean grown in different agro-ecologies. Field experiments were conducted in three ecological zones in 2018 and 2019 using soybean variety Zamboane inoculated withBradyrhizobium diazoefficiensstrain USDA 110,B. japonicumstrains USDA 136, USDA 442 and WB74, and a non-inoculated control in a randomized complete block design with four replications. Indigenous rhizobia populations at the sites ranged from 9.0 x 101to 2.2 x 103cells g−1soil. All four strains increased nodulation, but USDA 110 was superior at two sites with low native rhizobia population, whereas USDA 442 and WB74 were the best at the site with relatively high native rhizobia population. On an average, the strains doubled the number of nodules and increased the dry weight up to 5.8-fold. Inoculation increased shoot dry weight and N content at podding, plant biomass, and number of pods plant−1across sites but the effects of the strains on seeds per pod, and 100-seed weight were inconsistent. Shoot N content did not differ among inoculant strains and ranged from 15.70 g kg−1in the control to 38.53 g kg−1across inoculation. All four strains increased soybean grain yield across sites in 2018 but USDA 110 outperformed the other strains and was also the best at one of the two sites in 2019. Grain yield responses associated with USDA 110 ranged from 552 kg ha−1(56%) to 1,255 kg ha−1(76%). Positive correlations between nodule dry weight plant−1and seed yield, and number of pods plant−1and grain yield were observed. The gross margin ranged from $343.50–$606.80 ha−1for the control, but it increased to $688.34–$789.36 when inoculants were applied. On an average, inoculation increased gross margin by $182.57-$395.35 ha−1over that of non-inoculated control in 2018 but drought stress in 2019 reduced the benefit. The results demonstrate that USDA 110 was the best inoculant strain and has the potential of increasing smallholder productivity and net returns.

Isolation and characterization of Bradyrhizobium elkanii as a root nodule symbiont of red sword bean Canavalia gladiata var. gladiata

ABSTRACT Bacterial strains were isolated from root nodules of red sword bean (Canavalia gladiata var. gladiata) cultivated in Shizuoka, Japan, in order to elucidate the taxonomy of the symbionts. Of the 52 bacterial isolates, 28 strains were identified as the genus Bradyrhizobium and 10 as Rhizobium, based on the nucleotide sequences of 16S rRNA genes. Nine Bradyrhizobium isolates, which were phylogenetically selected from the 28 strains, exhibited nucleotide sequences of ITS regions that were 99.9% or 100% identical with known B. elkanii strains. These nine strains shared more than 70% similarity with B. elkanii USDA 76 T or USDA 94 in DNA-DNA hybridization analysis, indicating that the strains are B. elkanii. In a nodule formation experiment using red sword bean seeds treated with mercury chloride solution, all of the plants which were inoculated with each of the B. elkanii strains (the isolate TI06 or MI08, or the type strain USDA 76 T) formed round-type root nodules (234–664 nodules per plant), while no nodules were observed in control plants that were cultivated without inoculating bacterial strains. The bacterial strains, which were isolated from the obtained nodules, exhibited nucleotide sequences of the ITS regions that were identical to those of the corresponding inoculated strains. The root nodules formed in the experiment exhibited acetylene-reducing activity, suggesting the nitrogen-fixation activity of the nodules. We thus conclude that B. elkanii is a root nodule symbiont of red sword bean.

Inoculant, nitrogen and phosphorus improves photosynthesis and water-use efficiency in soybean production

AbstractSoybean yield within the Southern Africa falls below its potential despite similar climatic conditions across some agroecologies, replicable agronomic management practices and introduced improved varieties. Understanding physiological processes and water-use efficiency (WUE) of soybean offer information on bridging this yield gap. A field study was conducted in 2017 and 2018 seasons in two agroecologies (Angonia and Ruace) in Mozambique to evaluate the effects of Bradyrhizobium diazoefficiens strain USDA110 formerly known as Bradyrhizobium japonicum inoculant, nitrogen and phosphorus on nodulation, physiology and yield of non-promiscuous (Safari) and promiscuous (TGx 1740-2F) soybean varieties. Data on transpiration, photosynthesis, leaf area index, radiation interception and WUE from the beginning of flowering to maturity were collected. Transpiration rate varied considerably with interaction between locations, growth stages, varieties and treatments. At podding, phosphorus-treated soybean at Angonia transpired less (6.3 mmol/m2/s) than check plants (6.6 mmol/m2/s). Photosynthesis rate and WUE were distinct with variety, growth stages and inputs within agroecologies. For instance, in Angonia 2018 season, phosphorus fertilized TGx 1740-2F photosynthesized more at flowering (25.3 μmol/m2/s) while the lowest was phosphorus-treated Safari at podding with 17.2 μmol/m2/s. At the same site in 2017, inoculated soybean photosynthesized more at 22.8 μmol/m2/s leading to better WUE of 3.6 that corresponded to 2894 kg/ha yield. Overall, soybean WUE was higher when inoculated than N-treated, while P application yielded better. Results from this study will complement breeders’ effort in developing phosphorus efficient varieties suited for a wide range of changing climatical conditions.

Importance of Bio-Organic Fertilizers on Peanut (Arachis Hypogaea L.) Nutrition Following Organic Farming Approach with Application of 15N Isotope Dilution Concept

Abstract Field trial was conducted under low fertile sand soil conditions to follow up the role of organic additives with presence and absence of compatible bacterial inoculum in enhancement of peanut growth and yield. Bradyrhizobium spp. (foreign strain USDA 3456), inoculation resulted in relative increase of seed yield, as average, by about 5% over the un-inoculated plants despite of organic additive source. Similarly, organic additives had increased the seed yield, in average, over the non-treated plant which relatively accounted for 5%, 16% and 37% as affected by animal manure, leuceana residues and quail feces, respectively. On overall means basis of N uptake by seeds, comparison between organic additives indicated the superiority of animal manure over both of leuceana residues and quail feces. Nitrogen derived from mineral fertilizer seems to be higher in absence of organic manure and tended to decrease with addition of different organic amendments. Accordingly, the portion of fertilizer-N remained in soil after harvest was, in general, very low especially under organic amended soil and in absence of bacterial inoculation. Nitrogen derived from air by different plant parts on the basis of overall means indicated higher records accounted for 45.22 kg ha-1 with treatment leuceana residues followed by quail feces (45.22 kg ha-1) and animal manure (32.89 kg ha-1), respectively but all of them were superior over the un-treated treatment. Portion of N derived from organic sources was higher in seeds and straw than roots and seed cover, respectively.

Effect of rhizobial inoculation combined with phosphorus fertilizer on nitrogen accumulation, growth and yield of soybean in Benin

The objective of this study was to determine the amount of phosphorus application needed for the Bradyrhizobium inoculation to improve soybean N uptake and grain yield. Two factors were investigated: inoculation with Bradyrhizobium diazoefficiens strain USDA 110 and application of phosphorus fertilizer with different doses (0; 50; 100 and 150 kg ha-1 P2O5). Trials were carried out in two agro-ecological zones (ZAE) in Benin with two experimental sites namely Ketou and Ze. The experimental design was a Complete Randomized Block Design. Results showed that the highest mean height values were recorded T1+150P (42.22 cm at Ze and 43.82 cm at Ketou) while the lowest values were recorded with the treatment T0 + 0P. Treatment T0 +100P gave the highest aboveground biomass at 10 weeks after sowing at Ketou (2521.43 kg DM ha-1) while T1+150P recorded the highest aboveground biomass at Ze (3392.75 kg DM ha-1). At Ketou, the grain yield was increased under T1+100P by 98% compared with T0+0P. At Ze, the treatments T1+100P significantly increased the soybean grain yield by 147% compared with T0+0P. Likewise, T1+100P allowed to have the highest total nitrogen yields in the grain which is respectively 144.13 kg ha-1 N and 295.02 ha-1 kg N at Ketou and Ze. Overall, the inoculation with B. diazoefficiens had significantly increased the number of nodules for all of the phosphorus fertilizer amounts. The mycorhization frequency was significantly lower under the treatment T0 + 0P (41.46%) than the other treatments while that was higher under the treatment T1+50P (76.04%). Keywords: Bradyrhizobium diazoefficiens, soybean inoculation, grain yield, Benin.

Effect of salt stress on soybean growth and nodulation under inoculation with soybean rhizobia

ABSTRACT Salinity is one of the major problems when it comes to soil fertility and agricultural productivity. In this regard, this study aimed to elucidate the influence of four salinity levels (0, 20, 50, 100 mM NaCl) on the soybean growth and the nodulation by rhizobia inoculated on the soybean variety CNS. Additionally, the compatibility of soybean varieties with the seven representative strains of rhizobia was also investigated. Through the compatibility test, it was revealed that the Sinorhizobium (Ensifer) fredii USDA 191 and the three bradyrhizobia (Bradyrhizobium japonicum USDA 6T, B. elkanii USDA 31, and B. diazoefficiens USDA 110T) showed compatible nodulation with the Rfg1 genotype soybean, whereas, the S. fredii USDA 192, 193, and 205T showed incompatible nodulation. The inoculation tests with bradyrhizobia (USDA 31, 110T) and sinorhizobia (USDA 191, 192, 193) indicated a better growth of soybean for bradyrhizobia than the sinorhizobia until 50 mM NaCl. Note that even though the symbiotic efficiency at each strain was not different among 0, 20, and 50 mM NaCl treatments, the symbiotic efficiency of bradyrhizobia was higher than the sinorhizobia at each NaCl treatment. The result of the mix inoculation test indicated that the nodule occupancy rate of strain USDA 191 increased significantly along with the NaCl concentration but strain USDA 110T showed an opposite response. These results indicate a positive relationship between the soybean growth and the nitrogen fixation ability of the inoculant under salt-stress conditions. Furthermore, the results obtained from the mix inoculation test partly describe the dominance of sinorhizobia under saline conditions.

EFFECTS OF COMBINED INOCULATION OF BAMBARA GROUNDNUT (Vigna subterranean L. Verdc.) WITH GLOMUS MOSSEA AND BRADYHRHIZOBIUM JAPONICUM ON NITROGEN AND PHOSPHOROUS UPTAKE IN SHOOT, PLANT BIOMASS, LEAF CHLOROPHYLL AND MYCORRHIZAL INOCULATION EFFICIENCY (MIE).

The response of Bambara groundnut to co-inoculation with Abuscular mychorrhizal fungi (G. mossea) and Bradhyrhizobium japonicum (strain USDA110) with regard to leaf chlorophyll, percentage shoot nitrogen and phosphorus, nodule weight and plant biomass was studied. Bambara plants were grown under screen house conditions in pots. Plants were inoculated with 1ml of B. japonicum USDA 110 strain (109 cfu /ml), Mychorrizal was applied to the plants 10g, and 20g (90 spores/g) and water was applied at 10ml, 20 ml and 50ml every other day. The obtained results showed that dual inoculation activity was able to improve both nitrogen and phosphorus in plant shoot, MIE, but did not improve biomass and leaf chlorophyll when compared with plants subjected to single inoculation with only G. mossea and only B. japonicum. More Nitrogen and Phosphorus was retained in the shoot of plants co-inoculated with B. japonicum and 20g G. mossea when given 50ml of water and also had higher biomass. Leaf chlorophyll reduced in plants as flowering approached. B. japonicum was able to positively influence and establish symbiosis with G.mossea and synergistically effectively act as “mycorrhiza helper bacteria” (MHB) when both were co-inoculated in Bambara plant.

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 200kgha-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% (356kgha-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.

Bacterial Consortium and Microbial Metabolites Increase Grain Quality and Soybean Yield

The effects of Bradyrhizobium inoculation on soybean growth and productivity are well known, but plant responses to consortia of other beneficial microbes and microbial molecules have not yet been well explored. Therefore, the main aim of this study was to evaluate the effect of different combinations of beneficial bacteria with and without microbial secondary metabolites (MSM) on two soybean cultivars in three cropping seasons under tropical field conditions. The bacterial consortia consisted of Bradyrhizobium japonicum (strain SEMIA 5079) plus Bradyrhizobium diazoefficiens (strain SEMIA 5080) inoculated with different combinations of Bacillus subtilis (strain QST 713), Azospirillum brasilense (strains Ab-V5 and Ab-V6), and MSM (metabolites enriched in lipo-chitooligosaccharides (LCOs) extracted from B. diazoefficiens (strain USDA 110) and from Rhizobium tropici (strain CIAT 889)). Standard inoculation of Bradyrhizobium combined with Azospirillum brasilense and microbial secondary metabolites increased leaf total N (7.1%), total P (11.1%), and N-ureide (16.5%); nodule number (NN, 26%) and dry weight (NDW, 22%); root (RDW, 15.4%) and shoot dry weight (SDW, 6%); 100-seed weight (3.7%); grain yield (up to 516 kg ha−1); grain crude protein concentration (2.4%); and the agronomic efficiency index (AEI) (11%). Inoculation with bacterial consortia and metabolites increased grain yield and quality, representing a promising technology for sustainable soybean cropping in tropical regions.

Effects of growth‐promoting bacteria on soybean root activity, plant development, and yield

AbstractRhizobia and other plant growth‐promoting rhizobacteria (PGPR) have been broadly used as inoculants in agriculture, resulting in morphofunctional improvements in roots and grain yield. This study was carried out during two cropping seasons under field and greenhouse conditions in Brazil to verify the effects of inoculation of two soybean cultivars with PGPR and secondary microbial metabolites (SMMs) on root activity and nodulation, plant development, and grain yield. Inoculation and co‐inoculation treatments consisted of Bradyrhizobium japonicum strain SEMIA 5079 and B. diazoefficiens strain SEMIA 5080 inoculated together, in combination with Bacillus subtilis strain QST 713, Azospirillum brasilense strains Ab‐V5 and Ab‐V6, and SMMs extracted from B. diazoefficiens strain USDA 110 and Rhizobium tropici strain CIAT 889. Root systems were evaluated by direct (optical reading) and indirect (rubidium nitrate application, 85RbNO3) methods. Increases of up to 1.6% in root diameter (0.01‐ to 0.5‐mm class), 28.5% in length, 19.7% in root volume, 17.8% in root surface area, 29% in the number of nodules, 27.2% in nodule dry weight, 13.5% in root dry weight, and 3.8% in shoot dry weight. Greater exploration and activity within and between rows following inoculation at up to 40 and 10 cm in depth, respectively, were observed in plants co‐inoculated with the standard inoculation (only Bradyrhizobium spp.) + SMMs + A. brasilense, resulting in a yield increase of 485 kg ha−1. The results emphasize the biotechnological potential of using secondary metabolites of rhizobia with inoculants containing rhizobia and PGPR to improve the growth and soybean yield in tropical conditions.

English

Soybean is an important crop in the Democratic Republic of Congo, a country faced with high levels of war induced malnutrition but its productivity is limited by poor soil fertility coupled with low access to nitrogen mineral fertilizers. Commercial rhizobia strains introduced in 2010 failed to adapt and increase soybeans yields at desired level. We studied the performance of six indigenous rhizobia strains in enhancing soybean productivity compared to two commercial strains USDA110 and SEMIA5019. The study was carried out in the greenhouse and field of Kalambo station of International Institute of Tropical Agriculture (IITA), D. R. Congo during 2016/2017 cropping season. The treatments included: (1) N-, control without inoculation and N-fertilizer; (2) N+, non-inoculated control with 80 kg of N ha−1; and inoculated with (3) commercial strain Bradyrhizobium diazoefficiens USDA110; (4) commercial strain Bradyrhizobium elkanii SEMIA5019; (5) local strains Bradyrhizobium japonicum NAC17; (6) NAC22; (7) NAC37, (8) NAC42 (9) NAC 46 and (10) NAC78. Greenhouse and field experiments were laid out as completely randomized design and randomized complete block design respectively. The best inoculation treatments across all experiments were the indigenous strains NAC46 and NAC17 which nodulated equally or better than the commercial strain USDA 110. In the field NAC46 and NAC17 increased soybean grain yield from 2.4 to 3.3 t ha-1 and 3.4 t ha-1; indicating the increase of 68.7 and 70.8% respectively, over the commercial strain USDA110. The results demonstrated that indigenous rhizobia NAC46 and NAC17 would thus be the silver bullet to enhanced BNF and soybean yields in South Kivu province of Democratic Republic of Congo. Key words: Inoculation, local rhizobia; soil fertility, USDA110.

Revealing the Draft Genome Sequence of Bradyrhizobium sp. Strain USDA 3458, an Effective Symbiotic Diazotroph Isolated from Cowpea (Vigna unguiculata) Genotype IT82E-16.

Pairing plants with plant growth-promoting bacteria is critical to the future of agriculture. Bradyrhizobium sp. strain USDA 3458 isolated from Vigna unguiculata (cowpea) paired with cowpea genotype IT82E-16 represents a novel combination in arid regions. Here, we report the draft genome sequence of strain USDA 3458.

Lanthanide-dependent methanol dehydrogenase from the legume symbiotic nitrogen-fixing bacterium Bradyrhizobium diazoefficiens strain USDA110

The legume symbiotic nitrogen-fixing bacterium, B. diazoefficiens strain USDA110, utilizes methanol for growth in the presence of light lanthanides, such as La3+, Ce3+, Pr3+ or Nd3+, and its cells possess significant methanol dehydrogenase (MDH) activity. We purified MDH to homogeneity from B. diazoefficiens strain USDA110 grown in a methanol/Ce3+ medium; the protein was identified as XoxF5-type MDH (blr6213). The purified XoxF contained 0.58 cerium atoms per enzyme subunit. Moreover, the in-solution structure of XoxF was analyzed by small angle X-ray scattering (SAXS) analysis; the radius of gyration (Rg) and maximum particle dimension (Dmax) of XoxF were calculated to be 32.3 and 96.8 Å, respectively, suggesting that XoxF adopts a dimer structure in solution. These results show that B. diazoefficiens strain USDA110 has XoxF, a lanthanides-dependent MDH, which has methanol oxidation activity and is induced by methanol/lanthanaides, and that lanthanide is one of the important factors in methanol utilization by the strain.

An Integrated Systems Approach Unveils New Aspects of Microoxia-Mediated Regulation in Bradyrhizobium diazoefficiens.

The adaptation of rhizobia from the free-living state in soil to the endosymbiotic state comprises several physiological changes in order to cope with the extremely low oxygen availability (microoxia) within nodules. To uncover cellular functions required for bacterial adaptation to microoxia directly at the protein level, we applied a systems biology approach on the key rhizobial model and soybean endosymbiont Bradyrhizobium diazoefficiens USDA 110 (formerly B. japonicum USDA 110). As a first step, the complete genome of B. diazoefficiens 110spc4, the model strain used in most prior functional genomics studies, was sequenced revealing a deletion of a ~202 kb fragment harboring 223 genes and several additional differences, compared to strain USDA 110. Importantly, the deletion strain showed no significantly different phenotype during symbiosis with several host plants, reinforcing the value of previous OMICS studies. We next performed shotgun proteomics and detected 2,900 and 2,826 proteins in oxically and microoxically grown cells, respectively, largely expanding our knowledge about the inventory of rhizobial proteins expressed in microoxia. A set of 62 proteins was significantly induced under microoxic conditions, including the two nitrogenase subunits NifDK, the nitrogenase reductase NifH, and several subunits of the high-affinity terminal cbb3 oxidase (FixNOQP) required for bacterial respiration inside nodules. Integration with the previously defined microoxia-induced transcriptome uncovered a set of 639 genes or proteins uniquely expressed in microoxia. Finally, besides providing proteogenomic evidence for novelties, we also identified proteins with a regulation similar to that of FixK2: transcript levels of these protein-coding genes were significantly induced, while the corresponding protein abundance remained unchanged or was down-regulated. This suggested that, apart from fixK2, additional B. diazoefficiens genes might be under microoxia-specific post-transcriptional control. This hypothesis was indeed confirmed for several targets (HemA, HemB, and ClpA) by immunoblot analysis.

Symbiotic characteristics of Bradyrhizobium diazoefficiens USDA 110 mutants associated with shrubby sophora (Sophora flavescens) and soybean (Glycine max)

Site-specific insertion plasmid pVO155 was used to knockout the genes involved in the alternation of host range of strain Bradyrhizobium diazoefficiens USDA 110 from its original determinate-nodule-forming host soybean (Glycine max), to promiscuous and indeterminate-nodule-forming shrubby legume sophora (Sophora flavescens). Symbiotic phenotypes of these mutants inoculated to these two legumes, were compared to those infected by wild-type strain USDA 110. Six genes of the total fourteen Tn5 transposon mutated genes were broken using the pVO155 plasmid. Both Tn5 and pVO155-inserted mutants could nodulate S. flavescens with different morphologies of low-efficient indeterminate nodules. One to several rod or irregular bacteroids, containing different contents of poly-β-hydroxybutyrate or polyphosphate were found within the symbiosomes in nodulated cells of S. flavescens infected by the pVO155-inserted mutants. Moreover, none of bacteroids were observed in the pseudonodules of S. flavescens, infected by wild-type strain USDA 110. These mutants had the nodulation ability with soybean but the symbiotic efficiency reduced to diverse extents. These findings enlighten the complicated interactions between rhizobia and legumes, i. e., mutation of genes involved in metabolic pathways, transporters, chemotaxis and mobility could alter the rhizobial entry and development of the bacteroid inside the nodules of a new host legume.

Is there a need for Bradyrhizobium yuanmingense and B. japonicum reinoculation in subsequent cropping seasons under smallholder farmers’ conditions?

Reliable information on the persistence of rhizobium in soil in the absence of host between growing periods is important in deciding whether inoculation on the same plot in subsequent seasons is necessary. This study determined the survival of introduced rhizobium strains and predominant factors that influence the declining rates of their populations. Bradyrhizobium yuanmingense (BR 3267) and B. japonicum (USDA 110) were manually incorporated into soils at four different locations (Kpalga, Tanina, Tunayilli and Busa) in northern Ghana at 2.5 × 108 (log10 8.4) and 2.5 × 107 (log10 7.4) cells g−1 peat, respectively, per 6 m2. The populations of surviving cells were estimated at 0, 21, 42, 81, 142 and 296 days using the Most Probable Number (MPN) count technique. Several decline functions were applied to the data with hyperbolic regression function emerging as the option that provides the best fit for B. yuanmingense strain BR 3267 and B. japonicum strain USDA 110 at all locations. There was no significant difference in the declining rates between the different locations; however, there were differences in the declining rates for the sampling times. At 296 days, the numbers of surviving cells of B. yuanmingense strain BR 3267 and B. japonicum strain USDA 110 were log10 1.9 and log10 1.7, respectively. Native rhizobium population and soil moisture were the predominant factors that affected the survival of the introduced strains. It is evident from the studies that these strains can survive in sufficient numbers at least within a year; therefore, re-inoculation may not be necessary for a following season especially when using B. yuanmingense strain BR 3267.

Competition between introduced Bradyrhizobium japonicum strains and indigenous bradyrhizobia in Minnesota organic farming systems

Organic farmers recognize the importance of rhizobial associations with legume plants to help meet N fertility and plant productivity needs. A field experiment was done at three organic fields in Minnesota to assess the effect of indigenous Bradyrhizobium japonicum ORGS3 and ORGS5 and reference USDA 110 strains on the growth and yield performance of soybean. Soybean genotypes MN1505SP and Lambert inoculated with B. japonicum ORGS3 had significantly greater (P < 0.01) nodule numbers (42.1 ± 2.5), herbage N-contents (4.02 ± 0.01%), dry biomass (12.60 ± 1.45 g), and plant populations (117,890 ± 288.13 plant/acre) compared with the un-inoculated control. Grain yields were not affected by inoculation. Most nodules formed on non-inoculated Lambert (70%) and MN1505SP (53%) were occupied by strain ORGS5. The inoculant strains USDA110 and ORGS5 increased nodule occupancy by 10% on MN1505SP and Lambert. In contrast, strain ORGS3, and the combination of strains ORGS5 plus ORGS3, increased nodules occupancy on Lambert by 23 and 20%, respectively, compared with the control. The majority of nodules on Lambert (59%) and MN1505SP (52%) in the Farmington and Lamberton fields, respectively, were occupied by ORGS5. In contrast, 41 and 45% of nodules formed on Lambert and MN1505SP at Rosemount, respectively, were occupied by strain ORGS3. The lowest percentage of nodules formed on Lambert (4%) and MN1505SP (5%), in the Farmington field, were occupied by USDA110. These results showed that Bradyrhizobium strains ORGS3 and ORGS5 can be used to enhance N fixation and productivity of organically-grown soybeans grown in Minnesota fields.

The Soybean Rfg1 Gene Restricts Nodulation by Sinorhizobium fredii USDA193.

Sinorhizobium fredii is a fast-growing rhizobial species that can establish a nitrogen-fixing symbiosis with a wide range of legume species including soybeans (Glycine max). In soybeans, this interaction shows a high level of specificity such that particular S. fredii strains nodulate only a limited set of plant genotypes. Here we report the identification of a dominant gene in soybeans that restricts nodulation with S. fredii USDA193. Genetic mapping in an F2 population revealed co-segregation of the underlying locus with the previously cloned Rfg1 gene. The Rfg1 allele encodes a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance proteins that restricts nodulation by S. fredii strains USDA257 and USDA205, and an allelic variant of this gene also restricts nodulation by Bradyrhizobium japonicum USDA122. By means of complementation tests and CRISPR/Cas9-mediated gene knockouts, we demonstrate that the Rfg1 allele also is responsible for resistance to nodulation by S. fredii USDA193. Therefore, the Rfg1 allele likely provides broad-spectrum resistance to nodulation by many S. fredii and B. japonicum strains in soybeans.