Soybean Root Nodules Research Articles

Activity of nitrogen fixation and antioxidant enzymes in symbiotic systems Glycine max – Bradyrhizobium japonicum for complex treatment with lectin and fungicides

The dynamics of the nitrogen fixation activity of the root nodules, the growth of the vegetative mass of plants and the change in the activity of antioxidant enzymes (superoxide dismutase, ascorbate and guaiacol peroxidase) in different soybean organs for treatment of seeds by rhizobia incubated with lectin, in combination with fungicides have been studied. The objects of the study were symbiotic systems formed with the participation of soybean (Glycine max (L.) Merr.) Almaz and Bradyrhizobium japonicum (standard strain 634b) incubated with lectin. As disinfectants of soybean seeds, the following preparations with fungicidal activity were used – Maxim XL 035 PS, Fever, Standak Top according to one rate of active substance consumption of each preparation specified by the manufacturer. One part of the seeds treated with fungicides was inoculated with pure culture of suspension of rhizobia for one hour (titre of suspension concentration was 108 cells/ml). Another part of the seeds treated with fungicides was inoculated with rhizobia suspension, which was previously incubated with a solution of commercial lectin soybean at a concentration of 100 μg/ml. The research was conducted in strictly controlled conditions of a model vegetative experiment using microbiological, physiological, biochemical methods, gas chromatography, spectrophotometry. It was found that processing of soybean seeds with fungicides (Fever and Maxim XL) together with rhizobium inoculation contributed to the preservation of the nitrogen fixation activity of the root nodules and the growth of vegetative mass of plants. Under these conditions, the intensification of the activity of superoxide dismutase and ascorbate peroxidase was observed, as well as inhibition of the activity of guaiacol peroxidase in soybean root nodules in the phase of three true leaves and increased activity of all investigated enzymes in the phase of mass flowering. It has been established that the use of complex treatment of seeds by soybean rhizobia incubated with lectin and fungicides leads to an increase in the activity of superoxide dismutase and guaiacol peroxidase in root nodules in the phase of three true leaves and the growth of the activity of ascorbate peroxidase in the phase of mass flowering. At the same time, the inhibition of the growth of vegetative mass of plants and their symbiotic properties occurred, as evidenced by the decrease in the nitrogen fixation activity of the root nodules for the joint treatment of seeds with fungicides and lectin. A specific reaction of investigated enzymes in the roots and leaves of soybean was shown, which was more pronounced in the phase of three true leaves, indicating the development of a typical antioxidant reaction to a complex treatment, as a kind of stress that is leveled to the phase of mass flowering. The degree of reaction of antioxidant enzymes in the studied symbiotic systems Glycine max – Bradyrhizobium japonicum depends on the nature of the active substance fungicides and the manifestation of their joint effect in a complex with rhizobia incubated with lectin.

Nitrogen Fertilization of Soybean Affects Root Growth and Nodulation on Two Soil Types in Mississippi

ABSTRACTGreenhouse studies were conducted to evaluate the influence of nitrogen (N) sources [urea + ​N-(n-butyl) thiophosphoric triamide, NBPT (urease inhibitor) and polymer-coated urea (PCU)] and rates on soybean root characteristics, nodule formation, and biomass production on two soil types (silt loam and clay) commonly cropped to soybean in Mississippi. About 15% less belowground biomass was produced in clay soil than in silt loam soil directly corresponding to all other root parameters including root length, root area, root diameter, and nodule number. Pooled across N rates, N additions resulted in 19% and 52% decrease in belowground biomass and number of nodules, respectively, across soils compared to soybean receiving no N. The N rate was the most critical factor as it influenced all root growth parameters. Number of nodules were 24% greater with PCU than urea + NBPT. Nitrogen additions and clay soil negatively impacted soybean root growth, nodulation, and belowground biomass production.Abbreviations: Polymer-coated urea, PCU; N-(n-butyl) thiophosphoric triamide, NBPT

Effects of bamboo biochar on soybean root nodulation in multi-elements contaminated soils

Improvements in plant physiological performance by means of biochar application in soils contaminated by multi-elements are determinants of agroecosystem functioning. This study analyzed the effects of bamboo-derived biochar on root nodulation and plant growth in a moderately acidic Andosol (pH = 5.56) contaminated with multi-elements during a 70-day investigation of soybean growth.Bamboo biochar that had been pyrolyzed at a temperature below 500°C was applied to soils at three different and moderately high rates (5%, 10%, and 15%, w/w). Biochar amendment beyond 5% stimulated root nodulation as well as soybean growth. The nodule weight per root system was significantly enhanced by 186% and 243% over the control at the 10% and 15% addition rates, respectively. The primary explanation for these stimulatory effects was attributed to an increase in the K and Mo supplies for plant uptake that was induced by the biochar application, whereas the increased availability of P contributed to a lesser extent. Leaf CO2 assimilation rate was slightly enhanced at the highest application rate, but this enhancement was not associated with an increase in biomass. The incorporation of biochar into the soil reduced extractable-NH4NO3 Cd, Cu, Mn, Ni, and Zn, but not Pb, regardless of the application dose. This change was accompanied by a significant (P < 0.05) suppression of the uptake od trace elements in soybean shoots at the optimum application rate (10%); the degree of reduction followed this order: Pb>Mn>Cd>Zn>Cu>Ni. The increase in soil pH and the diffusion/adsorption of trace elements onto the biochar may have contributed to the lowering of the concentration of trace elements in the soil as well as in soybean shoots.

Identification and changes of the drought-induced cysteine protease transcriptome in soybean (Glycine max) root nodules

The formation and lifespan of soybean root nodules is affected by drought-stress, compromising the nitrogen supply of soybean plants. Expression of nodule cysteine proteases, involved in the regulation of the bacterial symbiosis and leghemoglobin degradation, increases during nodule senescence. Changes in the root nodule transcriptome were studied under varying drought-stress conditions using RNA-seq to identify nodule cysteine proteases induced following exposure to drought-stress conditions. Eight cysteine proteases belonging to the C1 family (papain-like cysteine proteases) and three cysteine proteases belonging to the C13 family (legumains or vacuole processing enzymes (VPE)) were induced by drought-stress. Expression of one C1 cysteine protease, Glyma.10G207100, was highly induced during drought but not under natural nodule senescence. A possible function of one C13 cysteine protease (<alpha>1-VPE-deficient), which was highly expressed in nodules under drought, was further investigated in Arabidopsis mutant plants. C13 mutant plants showed decreased C1 cysteine protease activity, had higher biomass and protein levels under stress conditions compared to wild-type plants. From our study we conclude that the C1 cysteine protease, Glyma.14G085800 will be a useful indicator for drought-induced premature senescence of soybean root nodules and that silencing of C13 cysteine proteases (VPEs) might be a strategy to reduce the consequences C1 cysteine protease activity during drought-stress.

Involvement of programmed cell death in suppression of the number of root nodules formed in soybean induced by Bradyrhizobium infection

ABSTRACTLegumes establish symbiosis with nitrogen-fixing rhizobia through root nodules to acquire nitrogen. Legumes control nodule number through systemic (autoregulation of nodulation) as well as local regulation. Moreover, plants defend themselves against bacteria and other pathogens through the induction of localized (localized acquired resistance) and systemic (SAR, systemic acquired resistance; ISR, induced systemic resistance) responses. Herein, we show that the number of root nodules is suppressed by programmed cell death (PCD), and is simultaneously controlled by SAR and ISR in soybean (Glycine max [L.] Merr.). The wild-type soybean cultivar Williams 82 showed markedly fewer root nodule primordia and PCD symptoms, including accelerated DNA degradation, enhanced generation of reactive oxygen species (visualized by 3,3′-diaminobenzidine staining), and excessive cell death (detected on staining with trypan blue) compared to the hypernodulation mutant NOD1-3. These results suggest that PCD suppresses the formation of root nodules in wild-type soybean. In addition, microarray and gene ontology analyses showed that essential components of hypersensitive response (HR) or disease resistance, such as resistance (R) genes, mitogen-activated protein kinase cascade, SAR, salicylic acid, jasmonic acid, ethylene, etc., were activated in wild-type plants. These analyses corroborate the above findings, demonstrating that the suppression of root nodule formation by PCD is accompanied by HR, and is simultaneously controlled by SAR and ISR in soybean. These findings provide new insight into the control of nodulation to balance nutritional requirements and energy status in legumes.

Antagonistic endophytic bacteria associated with nodules of soybean (Glycine max L.) and plant growth-promoting properties.

A total of 276 endophytic bacteria were isolated from the root nodules of soybean (Glycine max L.) grown in 14 sites in Henan Province, China. The inhibitory activity of these bacteria against pathogenic fungus Phytophthora sojae 01 was screened in vitro. Six strains with more than 63% inhibitory activities were further characterized through optical epifluorescence microscopic observation, sequencing, and phylogenetic analysis of 16S rRNA gene, potential plant growth-promoting properties analysis, and plant inoculation assay. On the basis of the phylogeny of 16S rRNA genes, the six endophytic antagonists were identified as belonging to five genera: Enterobacter, Acinetobacter, Pseudomonas, Ochrobactrum, and Bacillus. The strain Acinetobacter calcoaceticus DD161 had the strongest inhibitory activity (71.14%) against the P. sojae 01, which caused morphological abnormal changes of fungal mycelia; such changes include fracture, lysis, formation of a protoplast ball at the end of hyphae, and split ends. Except for Ochrobactrum haematophilum DD234, other antagonistic strains showed the capacity to produce siderophore, indole acetic acid, and nitrogen fixation activity. Regression analysis suggested a significant positive correlation between siderophore production and inhibition ratio against P. sojae 01. This study demonstrated that nodule endophytic bacteria are important resources for searching for inhibitors specific to the fungi and for promoting effects for soybean seedlings.

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.

Impact of pyrochar and hydrochar on soybean (Glycine max L.) root nodulation and biological nitrogen fixation

AbstractThe aim of this study was to identify effects of carbonized organic material (“biochar”) on soybean growth, root nodulation and biological nitrogen fixation, and to elucidate possible underlying mechanisms.Soybean (Glycine max L.) was grown in four arable soils amended with carbonized organic material produced from wood or maize as feedstocks, by pyrolysis (“pyrochar”) or hydrothermal carbonization (“hydrochar”). Nodulation by Bradyrhizobium, biological nitrogen fixation (BNF) assessed by 15N techniques, plant growth, nutrient uptake and changes in chemical soil properties after soil amendment were determined. Data were analyzed by means of a three way ANOVA on the factors soil, carbonization technique and feedstock. It turned out that soybean root nodulation and BNF was influenced by the carbonization technique used to prepare the soil amendment. Hydrochar, in average and across all soils, increased nodule dry matter and BNF by factors of 3.4 and 2.3, respectively, considerably more than pyrochar, which led to 1.8 and 1.2 fold increases, respectively. Nodule dry matter and BNF correlated positively with available soil sulfur and negatively with available soil nitrogen. Hydrochars provided more available sulfur than pyrochars, and hydrochars caused a decrease in nitrogen availability in the soil solution, thereby exerting a positive influence on nodulation and BNF. Pyrochar amendment increased soil pH but had no effect on nodulation and BNF. Plant growth was affected by the soil and by the feedstock used for the “biochar”, and increased slightly more in treatments with pyrochar and hydrochar made from maize, which was richer in nitrogen and potassium.The results show that carbonized organic materials, and specifically hydrochar, have the capacity to increase BNF in soils. We suggest that this enhancement in BNF in response to soil amendments with carbonized organic materials is due to an increase in available sulfur and a reduction of available soil nitrogen.

Spatial distribution of organelles in leaf cells and soybean root nodules revealed by focused ion beam-scanning electron microscopy.

Analysis of cellular ultrastructure has been dominated by transmission electron microscopy (TEM), so images collected by this technique have shaped our current understanding of cellular structure. More recently, three-dimensional (3D) analysis of organelle structures has typically been conducted using TEM tomography. However, TEM tomography application is limited by sample thickness. Focused ion beam-scanning electron microscopy (FIB-SEM) uses a dual beam system to perform serial sectioning and imaging of a sample. Thus FIB-SEM is an excellent alternative to TEM tomography and serial section TEM tomography. Animal tissue samples have been more intensively investigated by this technique than plant tissues. Here, we show that FIB-SEM can be used to study the 3D ultrastructure of plant tissues in samples previously prepared for TEM via commonly used fixation and embedding protocols. Reconstruction of FIB-SEM sections revealed ultra-structural details of the plant tissues examined. We observed that organelles packed tightly together in Nicotiana benthamiana Domin leaf cells may form membrane contacts. 3D models of soybean nodule cells suggest that the bacteroids in infected cells are contained within one large membrane-bound structure and not the many individual symbiosomes that TEM thin-sections suggest. We consider the implications of these organelle arrangements for intercellular signalling.

Ensifer shofinae sp. nov., a novel rhizobial species isolated from root nodules of soybean (Glycine max)

Two bacterial strains isolated from root nodules of soybean were characterized phylogenetically as members of a distinct group in the genus Ensifer based on 16S rRNA gene comparisons. They were also verified as a separated group by the concatenated sequence analyses of recA, atpD and glnII (with similarities ≤93.9% to the type strains for defined species), and by the average nucleotide identities (ANI) between the whole genome sequence of the representative strain CCBAU 251167T and those of the closely related strains in Ensifer glycinis and Ensifer fredii (90.5% and 90.3%, respectively). Phylogeny of symbiotic genes (nodC and nifH) grouped these two strains together with some soybean-nodulating strains of E. fredii, E. glycinis and Ensifer sojae. Nodulation tests indicated that the representative strain CCBAU 251167T could form root nodules with capability of nitrogen fixing on its host plant and Glycine soja, Cajanus cajan, Vigna unguiculata, Phaseolus vulgaris and Astragalus membranaceus, and it formed ineffective nodules on Leucaena leucocephala. Strain CCBAU 251167T contained fatty acids 18:1 ω9c, 18:0 iso and 20:0, differing from other related strains. Utilization of l-threonine and d-serine as carbon source, growth at pH 6.0 and intolerance of 1% (w/v) NaCl distinguished strain CCBAU 251167T from other type strains of the related species. The genome size of CCBAU 251167T was 6.2Mbp, comprising 7,581 predicted genes with DNA G+C content of 59.9mol% and 970 unique genes. Therefore, a novel species, Ensifer shofinae sp. nov., is proposed, with CCBAU 251167T (=ACCC 19939T=LMG 29645T) as type strain.

Molecular identification of Bradyrhizobium japonicum strains isolated from root nodules of soybean (Glycine max L.)

The aim of this study was to isolate and identify Bradyrhizobium japonicum strains on the basis of molecular characteristics. From root nodules of different soybean cultivars were obtained 56 isolates, characterized according to morphological, cultural, and biochemical properties. Among these isolates, 33 isolates showing resemblance with Bradyrhizobium sp. were further subjected to molecular identification. Following DNA extraction, a partial 16S rDNA gene sequence from the isolates was amplified by PCR using universal primers fD1 (27F) and rP3 (1492R). Purification and sequencing of the amplified fragments were done in the bio?technology company Macrogen, Seoul, South Korea. Sequences were analyzed using the pro?gram FinchTV and BLAST (Basic Local Alignment Search Tool) and compared to sequences in GenBank and the Bradyrhizobium ID-database for identification. Comparison of the se?quences with the Bradyrhizobium ID-database showed that all tested isolates were identified as Bradyrhizobium japonicum. Each isolate was deposited in the NCBI GenBank database under a unique accession number. Identification of Bradyrhizobium species from root nodules of soybean is of great importance because the symbiosis between rhizobia and legumes are a cheaper and usually more effective agronomic practice for ensuring an adequate supply of nitrogen for legumes, while preserving and improving fertility and productivity of soils.

Genetic Characterization of Soybean Rhizobia Isolated from Different Ecological Zones in North-Eastern Afghanistan.

Seventy rhizobial isolates were obtained from the root nodules of two soybean (Glycine max) cultivars: Japanese cultivar Enrei and USA cultivar Stine3300, which were inoculated with different soil samples from Afghanistan. In order to study the genetic properties of the isolates, the DNA sequences of the 16S rRNA gene and symbiotic genes (nodD1 and nifD) were elucidated. Furthermore, the isolates were inoculated into the roots of two soybean cultivars, and root nodule numbers and nitrogen fixation abilities were subsequently evaluated in order to assess symbiotic performance. Based on 16S rRNA gene sequences, the Afghanistan isolates obtained from soybean root nodules were classified into two genera, Bradyrhizobium and Ensifer. Bradyrhizobium isolates accounted for 54.3% (38) of the isolates, and these isolates had a close relationship with Bradyrhizobium liaoningense and B. yuanmingense. Five out of the 38 Bradyrhizobium isolates showed a novel lineage for B. liaoningense and B. yuanmingense. Thirty-two out of the 70 isolates were identified as Ensifer fredii. An Ensifer isolate had identical nodD1 and nifD sequences to those in B. yuanmingense. This result indicated that the horizontal gene transfer of symbiotic genes occurred from Bradyrhizobium to Ensifer in Afghanistan soil. The symbiotic performance of the 14 tested isolates from the root nodules of the two soybean cultivars indicated that Bradyrhizobium isolates exhibited stronger acetylene reduction activities than Ensifer isolates. This is the first study to genetically characterize soybean-nodulating rhizobia in Afghanistan soil.

Distinct properties of two glutamine synthetase isoforms in soybean root nodules

Glutamine synthetase (GS) in nodule cytosol plays a major role in the assimilation of the ammonium produced by biological nitrogen fixation. To characterize the GS protein family in Glycine max root nodules, the catalytical properties of 2 GS1 isoenzymes (GS1β1 and GS1γ1) were compared in this study. Although, GmGS1β1 and GmGS1γ1 have very similar kinetic characteristics, they also exhibit distinct enzymatic properties in terms of thermal stability and the transferase to synthetase activity (GSt/GSs) ratios. The results demonstrated that GmGS1γ1, which displayed lower thermal stability and GSt/GSs ratios than GmGS1β1, might be considered as superior isoform to participate in the efficient assimilation of ammonia only when it is needed. Also, it is proposed that the difference of enzymatic properties between isoforms contribute to their differential roles in ammonia assimilation under variable internal and external environments.

Effect of previous cropping of rapeseed (Brassica napus L.) on soybean (Glycine max) root mycorrhization, nodulation, and plant growth

Arbuscular mycorrhizal fungi (AMF) improve the uptake of immobile mineral nutrients such as phosphate, thereby enhancing the growth of most plants. However, plants belonging to the Brassicaceae family such as rapeseed (Brassica napus L.) do not associate with AMF. In Argentina, one of the crops frequently used in rotation with rapeseed is soybean (Glycine max L.). The aim of this study was to evaluate the impact of the non-mycorrhizal rapeseed (inoculated with the phosphorus solubilizing bacteria Bacillus sp. LRCP-4 or Arthrobacter sp. LRCP-11) as the preceding crop, on soybean plants growth, nodulation and AMF colonization. Green house experiments were done using soil samples from rapeseed cultivated fields to growth soybean plants. Results indicated that the soybean interaction with the microsymbiont Bradyrhizobium japonicum E109 was not affected and that the growth of plants in soil previously planted with rapeseed inoculated with Bacillus sp. LRCP-4 was increased (31% and 29% for shoot and root fresh weight respectively). However, it was evident from this study that inclusion of rapeseed in the soybean-based system decrease by a 30% the AMF soybean root colonization.

Drought Stress Responses in Soybean Roots and Nodules.

Drought is considered to be a major threat to soybean production worldwide and yet our current understanding of the effects of drought on soybean productively is largely based on studies on above-ground traits. Although the roots and root nodules are important sensors of drought, the responses of these crucial organs and their drought tolerance features remain poorly characterized. The symbiotic interaction between soybean and rhizobia facilitates atmospheric nitrogen fixation, a process that provides essential nitrogen to support plant growth and development. Symbiotic nitrogen fixation is important for sustainable agriculture, as it sustains plant growth on nitrogen-poor soils and limits fertilizer use for crop nitrogen nutrition. Recent developments have been made in our understanding of the drought impact on soybean root architecture and nodule traits, as well as underpinning transcriptome, proteome and also emerging metabolome information, with a view to improve the selection of more drought-tolerant soybean cultivars and rhizobia in the future. We conclude that the direct screening of root and nodule traits in the field as well as identification of genes, proteins and also metabolites involved in such traits will be essential in order to gain a better understanding of the regulation of root architecture, bacteroid development and lifespan in relation to drought tolerance in soybean.

The Bradyrhizobium japonicum Ferrous Iron Transporter FeoAB Is Required for Ferric Iron Utilization in Free Living Aerobic Cells and for Symbiosis

The bacterium Bradyrhizobium japonicum USDA110 does not synthesize siderophores for iron utilization in aerobic environments, and the mechanism of iron uptake within symbiotic soybean root nodules is unknown. An mbfA bfr double mutant defective in iron export and storage activities cannot grow aerobically in very high iron medium. Here, we found that this phenotype was suppressed by loss of function mutations in the feoAB operon encoding ferrous (Fe(2+)) iron uptake proteins. Expression of the feoAB operon genes was elevated under iron limitation, but mutants defective in either gene were unable to grow aerobically over a wide external ferric (Fe(3+)) iron (FeCl3) concentration range. Thus, FeoAB accommodates iron acquisition under iron limited and iron replete conditions. Incorporation of radiolabel from either (55)Fe(2+) or (59)Fe(3+) into cells was severely defective in the feoA and feoB strains, suggesting Fe(3+) reduction to Fe(2+) prior to traversal across the cytoplasmic membrane by FeoAB. The feoA or feoB deletion strains elicited small, ineffective nodules on soybean roots, containing few bacteria and lacking nitrogen fixation activity. A feoA(E40K) mutant contained partial iron uptake activity in culture that supported normal growth and established an effective symbiosis. The feoA(E40K) strain had partial iron uptake activity in situ within nodules and in isolated cells, indicating that FeoAB is the iron transporter in symbiosis. We conclude that FeoAB supports iron acquisition under limited conditions of soil and in the iron-rich environment of a symbiotic nodule.

Sulfur Fertilization Influence on Growth and Yield Traits of Three Korean Soybean Varieties

Field experiment was carried out at the Research Farm of Korea University, Republic of Korea to study effects of sulfur (S) fertilization on growth traits, yield and yield components of three Korean soybean varieties (Poongsunnamulkong, Saedanbaekkong and Daewonkong). Seeds were sown on black polythene mulched soil bed at the spacing of 15 X 60 cm both at control and sulfur fertilized (gypsum with 16% sulfur content at the rate of 200kg ha-1) plots following a Randomized Complete Block Design of the experiment applying three doses of sulfur together at the time of seed sowing and two doses together at V4 vegetative growth stage (4 nodes on the main stem beginning with the unifoliate node). The results showed that sulfur fertilization significantly increased all studied growth traits of three Soybean varieties except plant height when they all reached physiological maturity stage (120 DAS). It was also evident that sulfur fertilization in the form of gypsum can also increase the number and weight of soybean root nodules and enhance the ability of nitrogen fixation, and so reduce the input of nitrogen fertilizer. Similarly, all the studied yield and yield component related parameters were also significantly influenced by sulfur fertilization except seed number per pod and 100 seed weight. Varieties and sulfur fertilization also interacted significantly in all studied growth traits but the notable sole interaction effect was in plant height. Leaf area of Poongsunnamulkong and Saedanbaekkong variety increased significantly with sulfur fertilization while it was decreased significantly for Daewonkong. Similar trend was observed in their production of number of pods per plant and grain yield per plant indicating leaf area increment by sulfur fertilization played an important role in increasing yield of Poongsunnamulkong and Saedanbaekkong varieties though their performance was reverse in other growth traits. The improvement in growth and yield attributes after sulfur fertilization led to higher biological yield and enhanced seed yield.

Microsymbiont diversity and phylogeny of native bradyrhizobia associated with soybean (Glycine max L. Merr.) nodulation in South African soils

The genetic diversity and identification of slow- and fast-growing soybean root nodule bacterial isolates from different agro-climatic regions in Mpumalanga, Limpopo and Gauteng Provinces of South Africa were evaluated. The 16S-rDNA-RFLP analysis of 100 rhizobial isolates and eight reference type strains placed the isolates into six major clusters, and revealed their site-dependent genomic diversity. Sequence analysis of single and concatenated housekeeping genes (atpD, glnII and gyrB), as well as the symbiotic gene nifH captured a considerably higher level of genetic diversity and indicated the dominance of Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum in Mpumalanga, Limpopo and Gauteng Provinces. Gene sequence similarities of isolates with type strains of Bradyrhizobium ranged from 97.3 to 100% for the 16S rDNA, and 83.4 to 100% for the housekeeping genes. The glnII gene phylogeny showed discordance with the other genes, suggesting lateral gene transfer or recombination events. Concatenated gene sequence analysis showed that most of the isolates did not align with known type strains and might represent new species from South Africa. This underscores the high genetic variability associated with soybean Bradyrhizobium in South African soils, and the presence of an important reservoir of novel soybean-nodulating bradyrhizobia in the country. In this study, the grouping of isolates was influenced by site origin, with Group I isolates originating from Limpopo Province and Groups II and III from Mpumlanga Province in the 16S rDNA-RFLP analysis.

Formononetin stimulates mycorrhizal fungi colonization on the surface of active root nodules in soybean

The symbiosis of Leguminosae with arbuscular mycorrhizal fungi (AMF) and N2-fixing nodulating bacteria (NFNB) can occur simultaneously, forming a tripartite symbiosis. In particular, AMF can colonize root nodules, although this interaction is not yet well elucidated, especially with regard to nodule activity and to the influence of external factors, such as biostimulants. In this study, we hypothesized that the application of the flavonoid formononetin, used to stimulate root colonization by native AMF, increases the AMF colonization of soybean (Glycine max) root nodules, especially under low availability of phosphorus (P). To test this hypothesis, we performed a field experiment in randomized blocks in a 4 × 3 factorial design, with 4 treatments of formononetin (0, 0.46, 0.92 and 1.84 g per kg seed) and 3 of P (0, 60 and 120 kg ha−1) with 5 replicates. Nodules and roots were collected during the R2 stage (full flowering) and evaluated with respect to AMF colonization. Formononetin stimulated mycorrhizal fungi colonization of active nodules, especially when no P was applied, as also observed for AMF root colonization; however, it had no effect with 60 and 120 kg P ha−1. Thus, the application of formononetin increases surface AMF colonization of active nodules and roots, but its effect disappears with an increase in P and the inactivity of the nodule.

Ensifer glycinis sp. nov., a rhizobial species associated with species of the genus Glycine.

Rhizobial strains from root nodules of Astragalus mongholicus and soybean (Glycine max) were characterized phylogenetically as members of the genus Ensifer (formerly named Sinorhizobium), based on 16S rRNA gene sequence comparisons. Results based upon concatenated sequence analysis of three housekeeping genes (recA, atpD and glnII, ≤ 93.8 % similarities to known species) and average nucleotide identity (ANI) values of whole genome sequence comparisons (ranging from 89.6 % to 83.4 % to Ensifer fredii and Ensifer saheli, respectively) indicated the distinct positions of these novel strains within the genus Ensifer. Phylogeny of symbiotic genes (nodC and nifH) of three novel strains clustered them with rhizobial species Ensifer fredii and Ensifer sojae, both isolated from nodules of Glycine max. Cross-nodulation tests showed that the representative strain CCBAU 23380T could form root nodules with nitrogen fixation capability on Glycine soja, Albizia julibrissin, Vigna unguiculata and Cajanus cajan, but failed to nodulate Astragalus mongholicus, its original host legume. Strain CCBAU 23380T formed inefficient nodules on G. max, and it did not contain 18 : 0, 18 : 1ω7c 11-methyl or summed feature 1 fatty acids, which differed from other related strains. Failure to utilize malonic acid as a carbon source distinguished strain CCBAU 23380T from the type strains of related species. The genome size of CCBAU 23380T was 6.0 Mbp, comprising 5624 predicted genes with DNA G+C content of 62.4 mol%. Based on the results above, a novel species, Ensifer glycinis sp. nov., is proposed, with CCBAU 23380T (=LMG 29231T =HAMBI 3645T) as the type strain.