Hopanoids Confer Robustness to Physicochemical Variability in the Niche of the Plant Symbiont Bradyrhizobium diazoefficiens.

Rhizobia are a group of bacteria that increase soil nitrogen content through symbiosis with legume plants. The soil and symbiotic host are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the Bradyrhizobium clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens. Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in symbioses with success similar to that of the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the growth defects of extended-hopanoid-deficient mutants can be at least partially compensated for by the physicochemical environment, specifically, by optimal osmotic and divalent cation concentrations. Through biophysical measurements of lipid packing and membrane permeability, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in-culture and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environments within root nodules. To improve the legume-rhizobium symbiosis through either bioengineering or strain selection, it will be important to consider the full life cycle of rhizobia, from soil to symbiosis. IMPORTANCE Rhizobia, such as B. diazoefficiens, play an important role in the nitrogen cycle by making nitrogen gas bioavailable through symbiosis with legume plants. As climate change threatens soil health, this symbiosis has received increased attention as a more sustainable source of soil nitrogen than the energy-intensive Haber-Bosch process. Efforts to use rhizobia as biofertilizers have been effective; however, long-term integration of rhizobia into the soil community has been less successful. This work represents a small step toward improving the legume-rhizobium symbiosis by identifying a cellular component-hopanoid lipids-that confers robustness to environmental stresses rhizobia are likely to encounter in soil microenvironments as sporadic desiccation and flooding events become more common.

Potential of Bradyrhizobia inoculation to promote peanut growth and beneficial Rhizobacteria abundance.

AIMS: To investigate the effects of three symbiotic Bradyrhizobium strains on peanut growth and on rhizobacterial communities in flowering and harvest stages in an organic farm, also to evaluate the role of plant development in influencing peanut rhizobacterial microbiota and correlations among the inoculants, rhizobacterial communities and plant growth. METHODS AND RESULTS: Peanut seeds were inoculated with three individual Bradyrhizobium strains, plant growth performance was measured in two developmental stages and rhizobacterial communities were analysed by Illumina sequencing of rpoB gene amplicons from peanut rhizosphere. The three bradyrhizobial inoculants significantly increased the nodule numbers and aboveground fresh weight of peanut plants regardless of the different growth stages, and the pod yields were increased to some extent and significantly positively correlated with Bradyrhizobium abundances in rhizosphere. Principal coordinate analysis indicated that the rhizobacterial communities were strongly influenced by the inoculation and peanut developmental stages. The bradyrhizobia inoculation increased relative abundances of potentially beneficial bacteria in peanut rhizosphere, and also altered rhizobacterial co-occurrence association networks and important network hub taxa. Similarly, plant development also significantly influenced the structure, composition and co-occurrence association networks of rhizobacterial communities. CONCLUSIONS: Bradyrhizobial inoculants increased peanut growth and yields, they and plant development affected the assembly of peanut rhizobacterial communities. SIGNIFICANCE AND IMPACT OF THE STUDY: Rhizobial inoculants improved the host plant performance that might also be associated with the dynamic changes in rhizobacterial community except enhancing the biological nitrogen fixation and helps to profoundly understand the mechanism how rhizobia inoculants improve plant growth and yields.

Empowering rice seedling growth by endophytic Bradyrhizobium sp. SUTN9-2.

Bradyrhizobium sp. strain SUTN9-2 was confirmed as rice endophytic bacteria and also as rice growth promotion agent. SUTN9-2 showed the capability of plant growth promotion characteristics, such as indole-3-acetic acid (IAA) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase productions and nitrogen fixation. In this study, the ability of SUTN9-2 to stimulate rice growth was investigated at different stages with N-free and NH4 NO3 under in vivo condition. The rice dry weight and chlorophyll content could be enhanced when SUTN9-2 was inoculated in N-free, especially at seedling stage (7 and 14 dai). The rice dry weight was also increased when SUTN9-2 was inoculated with NH4 NO3 at 7 and14 dai. The results of quantitative analysis of IAA and ACC deaminase were inconsistent with the expression of genes involved in IAA (nit) and ACC deaminase (acdS) productions. This inconsistently could implied that IAA and ACC deaminase produced from SUTN9-2 do not directly affect rice growth, but other factors resulting from the production of IAA and ACC deaminase could be involved. Moreover, the expression of genes involved in nitrogen fixation (nifH and nifV) of SUTN9-2 was also induced in rice tissues. This finding suggested that rice growth promotion may be supported by NH4 NO3 together with nitrogen fixation by SUTN9-2. SIGNIFICANCE AND IMPACT OF THE STUDY: Indole-3-acetic acid, 1-amino-cyclopropane-1-carboxylic acid deaminase productions and nitrogen fixation may play important roles in rice growth promotion by endophytic SUTN9-2, especially at early rice seedling growth stage, which has the potential to be used as rice seedling growth promoter in the system of rice intensification.

Potential of Rice Stubble as a Reservoir of Bradyrhizobial Inoculum in Rice-Legume Crop Rotation.

Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with leguminous and nonleguminous plants, such as rice. Therefore, it can be expected that rice endophytic bradyrhizobia can be applied in the rice-legume crop rotation system. Some endophytic bradyrhizobial strains were isolated from rice (Oryza sativa L.) tissues. The rice biomass could be enhanced when supplementing bradyrhizobial strain inoculation with KNO3, NH4NO3, or urea, especially in Bradyrhizobium sp. strain SUTN9-2. In contrast, the strains which suppressed rice growth were photosynthetic bradyrhizobia and were found to produce nitric oxide (NO) in the rice root. The expression of genes involved in NO production was conducted using a quantitative reverse transcription-PCR (qRT-PCR) technique. The nirK gene expression level in Bradyrhizobium sp. strain SUT-PR48 with nitrate was higher than that of the norB gene. In contrast, the inoculation of SUTN9-2 resulted in a lower expression of the nirK gene than that of the norB gene. These results suggest that SUT-PR48 may accumulate NO more than SUTN9-2 does. Furthermore, the nifH expression of SUTN9-2 was induced in treatment without nitrogen supplementation in an endophytic association with rice. The indole-3-acetic acid (IAA) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase produced in planta by SUTN9-2 were also detected. Enumeration of rice endophytic bradyrhizobia from rice tissues revealed that SUTN9-2 persisted in rice tissues until rice-harvesting season. The mung bean (Vigna radiata) can be nodulated after rice stubbles were decomposed. Therefore, it is possible that rice stubbles can be used as an inoculum in the rice-legume crop rotation system under both low- and high-organic-matter soil conditions.IMPORTANCE This study shows that some rice endophytic bradyrhizobia could produce IAA and ACC deaminase and have a nitrogen fixation ability during symbiosis inside rice tissues. These characteristics may play an important role in rice growth promotion by endophytic bradyrhizobia. However, the NO-producing strains should be of concern due to a possible deleterious effect of NO on rice growth. In addition, this study reports the application of endophytic bradyrhizobia in rice stubbles, and the rice stubbles were used directly as an inoculum for a leguminous plant (mung bean). The degradation of rice stubbles leads to an increased number of SUTN9-2 in the soil and may result in increased mung bean nodulation. Therefore, the persistence of endophytic bradyrhizobia in rice tissues can be developed to use rice stubbles as an inoculum for mung bean in a rice-legume crop rotation system.

Predominant populations of indigenous soybean-nodulating Bradyrhizobium japonicum strains obtained from organic farming systems in Minnesota.

AIMS: Bradyrhizobium from organic fields in Minnesota were isolated and genotyped to assess diversity of soybean-bradyrhizobia in organic farming systems that can be used to improve soybean productivity. METHODS AND RESULTS: Soil samples were collected from 25 organic fields in Minnesota during May to July 2012. Soybean (cv. Lambert) was used as a host to trap indigenous bradyrhizobia in each sample. Genetic diversity of Bradyrhizobium strains (n=733) was determined using the horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (HFERP) DNA fingerprinting technique and the soybean-bradyrhizobia were classified into 79 different genotypes. Of these, 15 dominant genotypes were found and were highly similar (>92% fingerprint similarity) to serotypes USDA 127 (40.4%), USDA 4 (31.8%) and USDA 123 (15.5%), which were the three main populations of soybean-bradyrhizobia in organic fields. CONCLUSIONS: Bradyrhizobium japonicum serogroup USDA 4 strains were found to make up a previously unrecognized, predominant rhizobial population in the organic farming soils examined. The relative abundance of strain USDA 4 was negatively correlated with that of USDA 127 and this relationship may be influenced by the levels of NO3 -N and other soil edaphic factors. SIGNIFICANCE AND IMPACT OF THE STUDY: The local community of bradyrhizobia can be affected by applying inoculant bacteria to organic fields. Based on these results, soybean production in organic farms may be improved by displacing strains similar to USDA 4 with those better at nitrogen fixation and competitive ability than indigenous strains.

Soybean (Glycine max (L.) Merr.) response to application of mineral nitrogen and bradyrhizobia on Nitisols of Teppi, Southwest Ethiopia.

Soybean (Glycine max (L.) Merr.), an important crop grown for its protein source for humans and livestock, is widely introduced in different parts of Ethiopia. However, the productivity of the crop is far below its potential in the country due to different factors, among which low soil fertility is a major contributor. Hence, this field experiment was conducted with the objective of determining the optimum rate of starter nitrogen (N) and bradyrhizobium inoculation on yield and yield components of soybean in the 2019 and 2020 cropping seasons. Two levels of bradyrhizobia (inoculated and uninoculated) and six levels of starter nitrogen (0, 9, 18, 27, 36, and 54 kg N ha-1) were arranged in a factorial design. The result showed that soybean grain yield increased by about 60 % with inoculation of bradyrhizobia applied with low rates of starter nitrogen fertilizer, regardless of cropping seasons. Application of a nitrogen rate above 18 kg N ha-1 leads to yield decline and has no significant variation from bradyrhizobia inoculation only. Regardless of the cropping seasons, elevated levels of starter nitrogen beyond 27 kg ha-1 suppressed nodulation and nodule dry matter. Starter N at a rate of 9 and 18 kg N ha-1 improved soybean nodulation by 125-130 % over control and 95 % over bradyrhizobia inoculation alone. Thus, it was recommended to apply bradyrhizobia strains with 9 or 18 kg N ha-1 starter nitrogen for better yield of soybean as well as adequate nitrogen fixation in Nitisols having moderate soil nitrogen levels similar to the Teppi areas.

Effect of graphene on soybean root colonization by Bradyrhizobium strains.

Legume crops such as soybean obtain a large portion of their nitrogen nutrition through symbiotic nitrogen fixation by diazotrophic rhizobia bacteria in root nodules. However, nodule occupancy by low-capacity nitrogen-fixing rhizobia can lead to lower-than-optimal levels of nitrogen fixation. Seed/root coating with engineered materials such as graphene-carrying biomolecules that may promote specific attraction/attachment of desirable bacterial strains is a potential strategy that can help overcome this rhizobia competition problem. As a first step towards this goal, we assessed the impact of graphene on soybean and Bradyrhizobium using a set of growth, biochemical, and physiological assays. Three different concentrations of graphene were tested for toxicity in soybean (50, 250, and 1,000 mg/l) and Bradyrhizobia (25, 50, and 100 mg/l). Higher graphene concentrations (250 mg/l and 1,000 mg/l) promoted seed germination but slightly delayed plant development. Spectrometric and microscopy assays for hydrogen peroxide and superoxide anion suggested that specific concentrations of graphene led to higher levels of reactive oxygen species in the roots. In agreement, these roots also showed higher activities of antioxidant enzymes, catalase, and ascorbate peroxidase. Conversely, no toxic effects were detected on Bradyrhizobia treated with graphene, and neither did they have higher levels of reactive oxygen species. Graphene treatments at 250 mg/l and 1,000 mg/l significantly reduced the number of nodules, but rhizobia infection and the overall nitrogenase activity were not affected. Our results show that graphene can be used as a potential vehicle for seed/root treatment.

Scrutiny of NolA and NodD1 Regulatory Roles in Symbiotic Compatibility Unveils New Insights into Bradyrhizobium guangxiense CCBAU53363 Interacting with Peanut (Arachis hypogaea) and Mung Bean (Vigna radiata).

Bradyrhizobium guangxiense CCBAU53363 efficiently nodulates peanut but exhibits incompatible interaction with mung bean. By comparing the common nod region with those of other peanut bradyrhizobia efficiently nodulating these two hosts, distinctive characteristics with a single nodD isoform (nodD1) and a truncated nolA were identified. However, the regulatory roles of NodD1 and NolA and their coordination in legume-bradyrhizobial interactions remain largely unknown in terms of explaining the contrasting symbiotic compatibility. Here, we report that nolA was important for CCBAU53363 symbiosis with peanut but restricted nodulation on mung bean, while nodD1 was dispensable for CCBAU53363 symbiosis with peanut but essential for nodulation on mung bean. Moreover, nolA exerted a cumulative contribution with nodD1 to efficient symbiosis with peanut. Additionally, mutants lacking nolA delayed nodulation on peanut, and both nolA and nodD1 were required for competitive nodule colonization. It is noteworth that most of the nodulation genes and type III secretion system (T3SS)-related genes were significantly downregulated in a strain 53ΔnodD1nolA mutant compared to wild-type strain CCBAU53363, and the downregulated nodulation genes also had a greater impact than T3SS-related genes on the symbiotic defect of 53ΔnodD1nolA on peanut, which was supported by a more severe symbiotic defect induced by 53ΔnodC than that with the 53ΔnodD1nopP, 53ΔnodD1rhcJ, and 53ΔnodD1ttsI mutants. NolA did not regulate nod gene expression but did regulate the T3SS effector gene nopP in an indirect way. Meanwhile, nolA, nodW, and some T3SS-related genes besides nopP were also demonstrated as new "repressors" that seriously impaired CCBAU53363 symbiosis with mung bean. Taken together, the roles and essentiality of nolA and nodD1 in modulating symbiotic compatibility are sophisticated and host dependent. IMPORTANCE The main findings of this study were that we clarified that the roles and essentiality of nodD1 and nolA are host dependent. Importantly, for the first time, NolA was found to positively regulate T3SS effector gene nopP to mediate incompatibility on mung bean. Additionally, NolA does not regulate nod genes, which are activated by NodD1. nolA exerts a cumulative effect with nodD1 on CCBAU53363 symbiosis with peanut. These findings shed new light on our understanding of coordinated regulation of NodD1 and NolA in peanut bradyrhizobia with different hosts.

Spontaneously Produced Lysogenic Phages Are an Important Component of the Soybean Bradyrhizobium Mobilome.

The ability of Bradyrhizobium spp. to nodulate and fix atmospheric nitrogen in soybean root nodules is critical to meeting humanity's nutritional needs. The intricacies of soybean bradyrhizobia-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention, even though these interactions may significantly impact soybean yield. In batch culture, four soybean bradyrhizobia strains, Bradyrhizobium japonicum S06B (S06B-Bj), B. japonicum S10J (S10J-Bj), Bradyrhizobium diazoefficiens USDA 122 (USDA 122-Bd), and Bradyrhizobium elkanii USDA 76T (USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced tailed phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ~3-fold after 48 h of incubation. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each soybean bradyrhizobia genome, preventing accurate identification of spontaneously produced prophage (SPP) genomes. A DNA sequencing and mapping approach accurately delineated the boundaries of four SPP genomes within three of the soybean bradyrhizobia chromosomes and suggested that the SPPs were capable of transduction. In addition to the phages, S06B-Bj and USDA 76-Be contained three to four times more insertion sequences (IS) and large, conjugable, broad host range plasmids, both of which are known drivers of horizontal gene transfer (HGT) in soybean bradyrhizobia. These factors indicate that SPP along with IS and plasmids participate in HGT, drive bradyrhizobia evolution, and play an outsized role in bradyrhizobia ecology. IMPORTANCE Previous studies have shown that IS and plasmids mediate HGT of symbiotic nodulation (nod) genes in soybean bradyrhizobia; however, these events require close cell-to-cell contact, which could be limited in soil environments. Bacteriophage-assisted gene transduction through spontaneously produced prophages provides a stable means of HGT not limited by the constraints of proximal cell-to-cell contact. These phage-mediated HGT events may shape soybean bradyrhizobia population ecology, with concomitant impacts on soybean agriculture.

High Diversity of Bradyrhizobial Species Fix Nitrogen with Woody Legume Spartocytisus supranubius in a High Mountain Ecosystem.

The symbiosis between rhizobia and legumes is of pivotal importance in nitrogen-poor ecosystems. Furthermore, as it is a specific process (most legumes only establish a symbiosis with certain rhizobia), it is of great interest to know which rhizobia are able to nodulate key legumes in a specific habitat. This study describes the diversity of the rhizobia that are able to nodulate the shrub legume Spartocytisus supranubius in the harsh environmental conditions of the high mountain ecosystem of Teide National Park (Tenerife). The diversity of microsymbionts nodulating S. supranubius was estimated from a phylogenetic analysis of root nodule bacteria isolated from soils at three selected locations in the park. The results showed that a high diversity of species of Bradyrhizobium and two symbiovars can nodulate this legume. Phylogenies of ribosomal and housekeeping genes showed these strains distributed into three main clusters and a few isolates on separate branches. These clusters consist of strains representing three new phylogenetic lineages of the genus Bradyrhizobium. Two of these lineages belong to the B. japonicum superclade, which we refer to as B. canariense-like and B. hipponense-like, as the type strains of these species are the closest species to our isolates. The third main group was clustered within the B. elkanii superclade and is referred to as B. algeriense-like as B. algeriense is its closest species. This is the first time that bradyrhizobia of the B. elkanii superclade have been reported for the canarian genista. Furthermore, our results suggest that these three main groups might belong to potential new species of the genus Bradyrhizobium. Analysis of the soil physicochemical properties of the three study sites showed some significant differences in several parameters, which, however, did not have a major influence on the distribution of bradyrhizobial genotypes at the different locations. The B. algeriense-like group had a more restrictive distribution pattern, while the other two lineages were detected in all of the soils. This suggests that the microsymbionts are well adapted to the harsh environmental conditions of Teide National Park.

Coordinated regulation of symbiotic adaptation by NodD proteins and NolA in the type I peanut bradyrhizobial strain Bradyrhizobium zhanjiangense CCBAU51778.

Type I peanut bradyrhizobial strains can establish efficient symbiosis in contrast to symbiotic incompatibility induced by type II strains with mung bean. The notable distinction in the two kinds of key symbiosis-related regulators nolA and nodD close to the nodABCSUIJ operon region between these two types of peanut bradyrhizobia was found. Therefore, we determined whether NolA and NodD proteins regulate the symbiotic adaptations of type I strains to different hosts. We found that NodD1-NolA synergistically regulated the symbiosis between the type I strain Bradyrhizobium zhanjiangense CCBAU51778 and mung bean, and NodD1-NodD2 jointly regulated nodulation ability. In contrast, NodD1-NolA coordinately regulated nodulation ability in the CCBAU51778-peanut symbiosis. Meanwhile, NodD1 and NolA collectively contributes to competitive nodule colonization of CCBAU51778 on both hosts. The Fucosylated Nod factors and intact type 3 secretion system (T3SS), rather than extra nodD2 and full-length nolA, were critical for effective symbiosis with mung bean. Unexpectedly, T3SS-related genes were activated by NodD2 but not NodD1. Compared to NodD1 and NodD2, NolA predominantly inhibits exopolysaccharide production by promoting exoR expression. Importantly, this is the first report that NolA regulates rhizobial T3SS-related genes. The coordinated regulation and integration of different gene networks to fine-tune the expression of symbiosis-related genes and other accessory genes by NodD1-NolA might be required for CCBAU51778 to efficiently nodulate peanut. This study shed new light on our understanding of the regulatory roles of NolA and NodD proteins in symbiotic adaptation, highlighting the sophisticated gene networks dominated by NodD1-NolA.

Genetic and phenotypic diversity of microsymbionts nodulating promiscuous soybeans from different agro-climatic conditions.

BACKGROUND: Global food supply is highly dependent on field crop production that is currently severely threatened by changing climate, poor soil quality, abiotic, and biotic stresses. For instance, one of the major challenges to sustainable crop production in most developing countries is limited nitrogen in the soil. Symbiotic nitrogen fixation of legumes such as soybean (Glycine max (L.) Merril) with rhizobia plays a crucial role in supplying nitrogen sufficient to maintain good crop productivity. Characterization of indigenous bradyrhizobia is a prerequisite in the selection and development of effective bioinoculants. In view of this, bradyrhizobia were isolated from soybean nodules in four agro-climatic zones of eastern Kenya (Embu Upper Midland Zone, Embu Lower Midland Zone, Tharaka Upper Midland Zone, and Tharaka Lower Midland Zone) using two soybean varieties (SB8 and SB126). The isolates were characterized using biochemical, morphological, and genotypic approaches. DNA fingerprinting was carried out using 16S rRNA gene and restricted by enzymes HaeIII, Msp1, and EcoRI.  RESULTS: Thirty-eight (38) bradyrhizobia isolates obtained from the trapping experiments were placed into nine groups based on their morphological and biochemical characteristics. Most (77%) of the isolates had characteristics of fast-grower bradyrhizobia while 23% were slow-growers. Restriction digest revealed significant (p < 0.015) variation within populations and not among the agro-climatic zones based on analysis of molecular variance. Principal coordinate analysis demonstrated sympatric speciation of indigenous bradyrhizobia isolates. Embu Upper Midland Zone bradyrhizobia isolates had the highest polymorphic loci (80%) and highest genetic diversity estimates (H' = 0.419) compared to other agro-climatic zones. CONCLUSION: The high diversity of bradyrhizobia isolates depicts a valuable genetic resource for selecting more effective and competitive strains to improve promiscuous soybean production at a low cost through biological nitrogen fixation.

Soybean Nodulation Response to Cropping Interval and Inoculation in European Cropping Systems.

To support the adaption of soybean [Glycine max (L) Merrill] cultivation across Central Europe, the availability of compatible soybean nodulating Bradyrhizobia (SNB) is essential. Little is known about the symbiotic potential of indigenous SNB in Central Europe and the interaction with an SNB inoculum from commercial products. The objective of this study was to quantify the capacity of indigenous and inoculated SNB strains on the symbiotic performance of soybean in a pot experiment, using soils with and without soybean history. Under controlled conditions in a growth chamber, the study focused on two main factors: a soybean cropping interval (time since the last soybean cultivation; SCI) and inoculation with commercial Bradyrhizobia strains. Comparing the two types of soil, without soybean history and with 1-4 years SCI, we found out that plants grown in soil with soybean history and without inoculation had significantly more root nodules and higher nitrogen content in the plant tissue. These parameters, along with the leghemoglobin content, were found to be a variable among soils with 1-4 years SCI and did not show a trend over the years. Inoculation in soil without soybean history showed a significant increase in a nodulation rate, leghemoglobin content, and soybean tissue nitrogen concentration. The study found that response to inoculation varied significantly as per locations in soil with previous soybean cultivation history. An inoculated soybean grown on loamy sandy soils from the location Müncheberg had significantly more nodules as well as higher green tissue nitrogen concentration compared with non-inoculated plants. No significant improvement in a nodulation rate and tissue nitrogen concentration was observed for an inoculated soybean grown on loamy sandy soils from the location Fehrow. These results suggest that introduced SNB strains remained viable in the soil and were still symbiotically competent for up to 4 years after soybean cultivation. However, the symbiotic performance of the SNB remaining in the soils was not sufficient in all cases and makes inoculation with commercial products necessary. The SNB strains found in the soil of Central Europe could also be promising candidates for the development of inoculants and already represent a contribution to the successful cultivation of soybeans in Central Europe.

Late-Season Nitrogen Applications Increase Soybean Yield and Seed Protein Concentration.

Low seed and meal protein concentration in modern high-yielding soybean [Glycine max L. (Merr.)] cultivars is a major concern but there is limited information on effective cultural practices to address this issue. In the objective of dealing with this problem, this study conducted field experiments in 2019 and 2020 to evaluate the response of seed and meal protein concentrations to the interactive effects of late-season inputs [control, a liquid Bradyrhizobium japonicum inoculation at R3, and 202 kg ha-1 nitrogen (N) fertilizer applied after R5], previous cover crop (fallow or cereal cover crop with residue removed), and short- and full-season maturity group cultivars at three U.S. locations (Fayetteville, Arkansas; Lexington, Kentucky; and St. Paul, Minnesota). The results showed that cover crops had a negative effect on yield in two out of six site-years and decreased seed protein concentration by 8.2 mg g-1 on average in Minnesota. Inoculant applications at R3 did not affect seed protein concentration or yield. The applications of N fertilizer after R5 increased seed protein concentration by 6 to 15 mg g-1, and increased yield in Arkansas by 13% and in Minnesota by 11% relative to the unfertilized control. This study showed that late-season N applications can be an effective cultural practice to increase soybean meal protein concentration in modern high-yielding cultivars above the minimum threshold required by the industry. New research is necessary to investigate sustainable management practices that increase N availability to soybeans late in the season.

Polyphasic analysis reveals correlation between phenotypic and genotypic analysis in soybean bradyrhizobia (Bradyrhizobium spp.).

Soybean bradyrhizobia (Bradyrhizobium spp.) are bacteria that fix atmospheric nitrogen within the root nodules of soybean, a crop critical for meeting global nutritional protein demand. Members of this group differ in symbiotic effectiveness, and historically both phenotypic and genotypic approaches have been used to assess bradyrhizobial diversity. However, agreement between various approaches of assessment is poorly known. A collection (n=382) of soybean bradyrhizobia (Bradyrhizobium japonicum, B. diazoefficiens, and B. elkanii) were characterized by Internal Transcribed Spacer - Restriction Fragment Length Polymorphism (ITS-RFLP), cellular fatty acid composition (fatty acid methyl esters, FAME), and serological reactions to assess agreement between phenotypic and genotypic methods. Overall, 76% of the accessions demonstrated identical clustering with each of these techniques. FAME was able to identify all 382 accessions, whereas 14% were non-reactive serologically. One ITS-RFLP group, containing 36 Delaware isolates, produced multiple ITS amplicons indicating they possess multiple ribosomal RNA (rrn) operons. Cloning and sequencing revealed that these strains contained as many as three heterogenous rrn operons, a trait previously unknown in bradyrhizobia. A representative subset of 96 isolates was further characterized using 16S rRNA and Internal Transcribed Spacer (ITS) amplicon sequencing. ITS sequences showed better inter- and intra-species discrimination (65-99% identity) than 16S sequences (96-99% identity). This study shows that phenotypic and genotypic approaches are strongly correlated at the species level but should be approached with caution. We also suggest using combined 16S and ITS genotyping data to obtain better inter- and intra-species resolution in bradyrhizobia classification.

Mutualistic co-evolution of T3SSs during the establishment of symbiotic relationships between Vigna radiata and Bradyrhizobia.

This study supports the idea that the evolution of type III secretion system (T3SS) is one of the factors that controls Vigna radiata-bradyrhizobia symbiosis. Based on phylogenetic tree data and gene arrangements, it seems that the T3SSs of the Thai bradyrhizobial strains SUTN9-2, DOA1, and DOA9 and the Senegalese strain ORS3257 may share the same origin. Therefore, strains SUTN9-2, DOA1, DOA9, and ORS3257 may have evolved their T3SSs independently from other bradyrhizobia, depending on biological and/or geological events. For functional analyses, the rhcJ genes of ORS3257, SUTN9-2, DOA9, and USDA110 were disrupted. These mutations had cultivar-specific effects on nodulation properties. The T3SSs of ORS3257 and DOA9 showed negative effects on V. radiata nodulation, while the T3SS of SUTN9-2 showed no effect on V. radiata symbiosis. In the roots of V. radiata CN72, the expression levels of the PR1 gene after inoculation with ORS3257 and DOA9 were significantly higher than those after inoculation with ORS3257 ΩT3SS, DOA9 ΩT3SS, and SUTN9-2. The T3Es from ORS3257 and DOA9 could trigger PR1 expression, which ultimately leads to abort nodulation. In contrast, the T3E from SUTN9-2 reduced PR1 expression. It seems that the mutualistic relationship between SUTN9-2 and V. radiata may have led to the selection of the most well-adapted combination of T3SS and symbiotic bradyrhizobial genotype.

Detection of the type III secretion system and its phylogenetic and symbiotic characterization in peanut bradyrhizobia isolated from Guangdong Province, China.

The distribution of rhcRST and rhcJ-C1 fragments located in different loci of the type III secretion system (T3SS) gene cluster in the peanut-nodulating bradyrhizobia isolated from Guangdong Province, China was investigated by PCR-based sequencing. T3SS was detected in approximately one-third of the peanut bradyrhizobial strains and the T3SS-harboring strains belonging to different Bradyrhizobium genomic species. Diverse T3SS groups corresponding to different symbiotic gene types were defined among the 23 T3SS-harboring strains. The same or similar T3SS genes were detected in different genospecies, indicating that interspecies horizontal transfer of symbiotic genes had occurred in the Bradyrhizobium genus.

Eco-geographical diversity of cowpea bradyrhizobia in Senegal is marked by dominance of two genetic types.

The genetic diversity of native cowpea rhizobia originating from 60 sites across four eco-geographic zones in Senegal was studied. More than 300 cowpea nodules were analyzed by PCR-RFLP of the 16S-23S rDNA InterGenic Spacer region (IGS). Alignments of IGS sequences indicated that all genotypes were grouping within the Bradyrhizobium genus. The geographical distribution showed that apart from five IGS types, the others were specifically found in only one region. The diversity was significantly higher in the Senegal River valley zone, which presents lower mean annual rainfalls and slightly alkaline soils. Interestingly, two IGS types dominated the Senegalese rhizobial collection, one IGS type (VI) was found on more than half of the nodules collected in the northern Senegal River valley while another IGS type (I) was recovered from the great majority of nodules in the three other regions sampled. Two representative strains from each of these two dominant types were isolated and further analyzed. Multi Locus Sequence Analyses using 6 housekeeping genes indicate that they belong to a new Bradyrhizobium species closely related to B. yuanmingense. Phylogenetic analyses of 2 symbiotic genes nodC and nifH show that they are clustered with B. arachidis. Physiological tests on these strains have shown that under laboratory conditions, the growth of the IGS type VI strains was slightly less affected by a higher osmotic strength in the medium and to alkaline pH, which corroborates the soil physico-chemical parameters.