Genetic and Physiological Characterization of Soybean-Nodule-Derived Isolates from Bangladeshi Soils Revealed Diverse Array of Bacteria with Potential Bradyrhizobia for Biofertilizers.

Genetic and physiological characterization of bacteria derived from nodules of leguminous plants in the exploration of biofertilizer is of paramount importance from agricultural and environmental perspectives. Phylogenetic analysis of the 16S rRNA gene of 84 isolates derived from Bangladeshi soils revealed an unpredictably diverse array of nodule-forming and endosymbiotic bacteria-mostly belonging to the genus Bradyrhizobium. A sequence analysis of the symbiotic genes (nifH and nodD1) revealed similarities with the 16S rRNA gene tree, with few discrepancies. A phylogenetic analysis of the partial rrn operon (16S-ITS-23S) and multi-locus sequence analysis of atpD, glnII, and gyrB identified that the Bradyrhizobium isolates belonged to Bradyrhizobium diazoefficiens, Bradyrhizobium elkanii, Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense species. In the pot experiment, several isolates showed better activity than B. diazoefficiens USDA110, and the Bho-P2-B2-S1-51 isolate of B. liaoningense showed significantly higher acetylene reduction activity in both Glycine max cv. Enrei and Binasoybean-3 varieties and biomass production increased by 9% in the Binasoybean-3 variety. Tha-P2-B1-S1-68 isolate of B. diazoefficiens significantly enhanced shoot length and induced 10% biomass production in Binasoybean-3. These isolates grew at 1-4% NaCl concentration and pH 4.5-10 and survived at 45 °C, making the isolates potential candidates for eco-friendly soybean biofertilizers in salty and tropical regions.

Characterization of Rhizobia for the Improvement of Soybean Cultivation at Cold Conditions in Central Europe.

In central Europe, soybean cultivation is gaining increasing importance to reduce protein imports from overseas and make cropping systems more sustainable. In the field, despite the inoculation of soybean with commercial rhizobia, its nodulation is low. In many parts of Europe, limited information is currently available on the genetic diversity of rhizobia and, thus, biological resources for selecting high nitrogen-fixing rhizobia are inadequate. These resources are urgently needed to improve soybean production in central Europe. The objective of the present study was to identify strains that have the potential to increase nitrogen fixation by and the yield of soybean in German soils. We isolated and characterized 77 soybean rhizobia from 18 different sampling sites. Based on a multilocus sequence analysis (MLSA), 71% of isolates were identified as Bradyrhizobium and 29% as Rhizobium. A comparative analysis of the nodD and nifH genes showed no significant differences, which indicated that the soybean rhizobia symbiotic genes in the present study belong to only one type. One isolate, GMF14 which was tolerant of a low temperature (4°C), exhibited higher nitrogen fixation in root nodules and a greater plant biomass than USDA 110 under cold conditions. These results strongly suggest that some indigenous rhizobia enhance biological nitrogen fixation and soybean yield due to their adaption to local conditions.

Isolation and Screening of Indigenous Plant Growth-promoting Rhizobacteria from Different Rice Cultivars in Afghanistan Soils.

To develop biofertilizers for rice in Afghanistan, 98 plant growth-promoting rhizobacteria were isolated from rice plants and their morphological and physiological characteristics, such as indole-3-acetic acid production, acetylene reduction, phosphate and potassium solubilization, and siderophore production, were evaluated. The genetic diversity of these bacteria was also analyzed based on 16S rRNA gene sequences. Of 98 bacteria, 89.7% produced IAA, 54.0% exhibited nitrogenase activity, and 40% showed phosphate solubilization and siderophore production. Some isolates assigned to Pseudomonas (brassicacearum, chengduensis, plecoglossicida, resinovorans, and straminea) formed a relationship with rice, and P. resinovorans and P. straminea showed nitrogen fixation. Rhizobium borbori and R. rosettiformans showed a relationship with rice plants and nitrogen fixation. Among the isolates examined, AF134 and AF137 belonging to Enterobacter ludwigii and P. putida produced large amounts of IAA (92.3 µg mL-1) and exhibited high nitrogenase activity (647.4 nmol C2H4 h-1), respectively. In the plant growth test, more than 70% of the inoculated isolates showed significantly increased root and shoot dry weights. Highly diverse bacterial isolates showing promising rice growth-promoting traits were obtained from Afghanistan alkaline soils.

Complete Genome Sequence of Plant Growth-Promoting Bacillus pumilus TUAT1.

Bacillus pumilus TUAT1 was isolated from soil in a university research field. Strain TUAT1 has the ability to promote the growth of plants, including that of rice, and has been commercialized as a biofertilizer. Here, we sequenced and annotated the genome of TUAT1 to understand the molecular mechanisms underlying its plant growth promotion.

Evaluation of Immune Responses Induced by Simultaneous Inoculations of Soybean (Glycine max [L.] Merr.) with Soil Bacteria and Rhizobia.

Legumes form root nodules and fix atmospheric nitrogen by establishing symbiosis with rhizobia. However, excessive root nodules are harmful to plants because of the resulting overconsumption of energy from photosynthates. The delay of an inoculation of the soybean super-nodulation mutant NOD1-3 with Bradyrhizobium diazoefficiens USDA110T by 5 d after an inoculation with several soil bacteria confirmed that one bacterial group significantly decreased root nodules throughout the study period. Moreover, no significant changes were observed in nitrogen fixation by root nodules between an inoculation with USDA 110T only and co-inoculation treatments. To clarify the potential involvement of PR proteins in the restriction of nodule formation in the plants tested, the relative expression levels of PR-1, PR-2, PR-5, and PDF1.2 in NOD1-3 roots were measured using real-time PCR. One group of soil bacteria (Gr.3), which markedly reduced nodule numbers, significantly induced the expression of PR-1, PR-5 and PDF1.2 genes by day 5 after the inoculation. By days 7, 10, and 20 after the inoculation, the expression levels of PR-2 and PR-5 were lower than those with the uninoculated treatment. Inoculations with this group of soil bacteria resulted in lower root nodule numbers than with other tested soil bacteria exerting weak inhibitory effects on nodulation, and were accompanied by the induction of plant defense-related genes. Thus, PR genes appear to play important roles in the mechanisms that suppresses nodule formation on soybean roots.

Transfer of radiocesium from rhizosphere soil to four cruciferous vegetables in association with a Bacillus pumilus strain and root exudation.

This study was carried out to assess the effect of Bacillus pumilus on the roots of four cruciferous vegetables with different root structures in regard to enhancement of 137Cs bioavailability in contaminated rhizosphere soil. Results revealed that B. pumilus inoculation did not enhance the plant biomass of vegetables, although it increased root volume and root surface areas of all vegetables except turnip. The pH changes due to rhizosphere acidification by B. pumilus inoculation and root exudation did not affect the bioavailability of 137Cs. However, concentrations of 137Cs in plant tissues and soil-to-plant transfer values increased as a result of the larger root volume and root surface area of vegetables due to inoculation. Moreover, leafy vegetables, which possessed larger root volume and root surface areas, had a higher 137Cs transfer value than root vegetables.

Growth and (137)Cs uptake and accumulation among 56 Japanese cultivars of Brassica rapa, Brassica juncea and Brassica napus grown in a contaminated field in Fukushima: Effect of inoculation with a Bacillus pumilus strain.

Fifty six local Japanese cultivars of Brassica rapa (40 cultivars), Brassica juncea (10 cultivars) and Brassica napus (6 cultivars) were assessed for variability in growth and (137)Cs uptake and accumulation in association with a Bacillus pumilus strain. Field trial was conducted at a contaminated farmland in Nihonmatsu city, in Fukushima prefecture. Inoculation resulted in different responses of the cultivars in terms of growth and radiocesium uptake and accumulation. B. pumilus induced a significant increase in shoot dry weight in 12 cultivars that reached up to 40% in one B. rapa and three B. juncea cultivars. Differences in radiocesium uptake were observed between the cultivars of each Brassica species. Generally, inoculation resulted in a significant increase in (137)Cs uptake in 22 cultivars, while in seven cultivars it was significantly decreased. Regardless of plant cultivar and bacterial inoculation, the transfer of (137)Cs to the plant shoots (TF) varied by a factor of up to 5 and it ranged from to 0.011 to 0.054. Five inoculated cultivars, showed enhanced shoot dry weights and decreased (137)Cs accumulations, among which two B. rapa cultivars named Bitamina and Nozawana had a significantly decreased (137)Cs accumulation in their shoots. Such cultivars could be utilized to minimize the entry of radiocesium into the food chain; however, verifying the consistency of their radiocesium accumulation in other soils is strongly required. Moreover, the variations in growth and radiocesium accumulation, as influenced by Bacillus inoculation, could help selecting well grown inoculated Brassica cultivars with low radiocesium accumulation in their shoots.

Relations of fine-root morphology on (137)Cs uptake by fourteen Brassica species.

Fourteen Brassica species consisting of seven leafy vegetables and seven root vegetables were examined for (137)Cs uptake differences in relation to their fine-root morphological characters. A pot experiment was conducted from November 2014 to February 2015 in a Phytroton using a contaminated soil of Fukushima prefecture. Leafy vegetables showed bigger root diameters, larger root surface area and larger root volume. Consequently, leafy vegetables had higher (137)Cs uptake compared to root vegetables. Among the three fine-root parameters, only root surface area was observed as a significant contributing factor to higher (137)Cs uptake in terms of transfer factor (TF, dry weight basis). Kakina exhibited higher (137)Cs TF value (0.20) followed by Chinese cabbage (0.18) and mizuna (0.17). Lower TF values were observed in turnip (0.059), rutabaga (Kitanoshou) (0.062) and radish (Ha daikon) (0.064).

Growth and (137)Cs uptake of four Brassica species influenced by inoculation with a plant growth-promoting rhizobacterium Bacillus pumilus in three contaminated farmlands in Fukushima prefecture, Japan.

The effectiveness of the plant growth-promoting rhizobacterium Bacillus pumilus regarding growth promotion and radiocesium ((137)Cs) uptake was evaluated in four Brassica species grown on different (137)Cs contaminated farmlands at Fukushima prefecture in Japan from June to August 2012. B. pumilus inoculation did not enhance growth in any of the plants, although it resulted in a significant increase of (137)Cs concentration and higher (137)Cs transfer from the soil to plants. The Brassica species exhibited different (137)Cs uptake abilities in the order Komatsuna>turnip>mustard>radish. TF values of (137)Cs ranged from 0.018 to 0.069 for all vegetables. Komatsuna possessed the largest root surface area and root volume, and showed a higher (137)Cs concentration in plant tissue and higher (137)Cs TF values (0.060) than the other vegetables. Higher (137)Cs transfer to plants was prominent in soil with a high amount of organic matter and an Al-vermiculite clay mineral type.

Evaluation of the possibility to use the plant-microbe interaction to stimulate radioactive 137Cs accumulation by plants in a contaminated farm field in Fukushima, Japan.

Field experiments in a contaminated farmland in Nihonmatsu city, Fukushima were conducted to assess the effectiveness of the plant-microbe interaction on removal of radiocesium. Before plowing, 93.3% of radiocesium was found in the top 5 cm layer (5,718 Bq kg DW(-1)). After plowing, Cs radioactivity in the 0-15 cm layer ranged from 2,037 to 3,277 Bq kg DW(-1). Based on sequential extraction, the percentage of available radiocesium (water soluble + exchangeable) was fewer than 10% of the total radioactive Cs. The transfer of (137)Cs was investigated in three agricultural crops; komatsuna (four cultivars), Indian mustard and buckwheat, inoculated with a Bacillus or an Azospirillum strains. Except for komatsuna Nikko and Indian mustard, inoculation with both strains resulted in an increase of biomass production by the tested plants. The highest (137)Cs radioactivity concentration in above-ground parts was found in Bacillus-inoculated komatsuna Nikko (121 Bq kg DW(-1)), accompanied with the highest (137)Cs TF (0.092). Furthermore, komatsuna Nikko-Bacillus and Indian mustard-Azospirillum associations gave the highest (137)Cs removal, 131.5 and 113.8 Bq m(-2), respectively. Despite the beneficial effect of inoculation, concentrations of (137)Cs and its transfer to the tested plants were not very high; consequently, removal of (137)Cs from soil would be very slow.

Transfer of radiocesium to four cruciferous vegetables as influenced by organic amendment under different field conditions in Fukushima Prefecture.

Soil-to-plant transfer of radiocesium ((137)Cs) in four cruciferous vegetables as influenced by cattle manure-based compost amendment was investigated. Komatsuna, mustard, radish and turnip were cultivated in three different (137)Cs-contaminated fields at Nihonmatsu City in Fukushima Prefecture from June to August 2012. Results revealed that organic compost amendments stimulated plant biomass production and tended to induce higher (137)Cs concentration in the cruciferous vegetables in most cases. Among the studied sites, Takanishi soil possessing low exchangeable potassium (0.10 cmolc kg(-1)) was associated with an increased concentration of (137)Cs in plants. Radiocesium transfer factor (TF) values of the vegetables ranged from 0.025 to 0.119. The increase in (137)Cs TFs was dependent on larger plant biomass production, high organic matter content, and high sand content in the studied soils. Average (137)Cs TF values for all study sites and compost treatments were higher in Komatsuna (0.072) and radish (0.059), which exhibited a higher biomass production compared to mustard and turnip. The transferability of (137)Cs to vegetables from soils was in the order Komatsuna > radish > mustard > turnip. The highest (137)Cs TF value (0.071) of all vegetables was recorded for a field where the soil had high organic matter content and a high clay proportion of 470 g kg(-1) consisting of Al-vermiculite clay mineral.

Stable cesium uptake and accumulation capacities of five plant species as influenced by bacterial inoculation and cesium distribution in the soil.

The effects of inoculation with Bacillus and Azospirillum strains on growth and cesium accumulation of five plant species, Komatsuna, Amaranth, sorghum, common millet and buckwheat, grown on cesium-spiked soil were assessed for potential use in cesium remediation. Pot experiments were performed using "artificially" Cs-contaminated soil. Three treatments were applied based on Cs location in the soil. For a soil height of 15 cm in the pots, Cs was added as follows: in the top five cm to imitate no ploughing condition; in the bottom five cm simulating inverted ploughing; and uniformly distributed Cs reproducing normal plowing. Generally, inoculation of Cs-exposed plants significantly enhanced growth and tolerance to this element. Transfer factor (ratio of Cs concentration in the plant tissues to that in surrounding soil) was strongly influenced by Cs distribution, with higher values in the top-Cs treatment. Within this treatment, inoculation of Komatsuna with Bacillus and Azospirillum strains resulted in the greatest transfer factors of 6.55 and 6.68, respectively. Cesium content in the shoots was high in the Azospirillum-inoculated Komatsuna, Amaranth, and buckwheat, i.e., 1,830, 1,220, and 1,030 µg per pot, respectively (five plants were grown in each pot). Therefore, inoculation of Komatsuna and Amaranth with the strains tested here could be effective in enhancing Cs accumulation. The decrease of Cs transfer under uniform- and bottom-Cs treatments would suggest that countermeasures aiming at decreasing the transfer of Cs could rely on ploughing practices.

Phylogenetic diversity and symbiotic functioning in mungbean (Vigna radiata L. Wilczek) bradyrhizobia from contrast agro-ecological regions of Nepal.

Nepal consists wide range of climatic and topographical variations. Here, we explored the phylogeny of native mungbean bradyrhizobia isolated from different agro-ecological regions of Nepal and accessed their nodulation and nitrogen fixation characteristics. Soil samples were collected from three agro-ecological regions with contrasting climate and topography. A local mungbean cultivar, Kalyan, was used as a trap plant. We characterized isolates based on the full nucleotide sequence of the 16S rRNA, ITS region, and nodA genes; and partial sequences of nodD1 and nifD genes. We found 50% of isolates phylogenetically related to B. yuanmingense, 13% to B. japonicum, 8% to B. elkanii, and 29% to novel phylogenetic origin. Results of the inoculation test suggested that expression of different symbiotic genes in isolates resulted in different degrees of symbiotic functioning. Our results indicate B. yuanmingense and novel strains are more efficient symbiotic partners than B. elkanii for the local mungbean cv. Kalyan. We also found most mungbean rhizobial genotypes were conserved across agro-ecological regions. All the strains from tropical Terai region belonged to B. yuanmingense or a novel lineage of B. yuanmingense, and dominance of B. japonicum related strains was observed in the Hill region. Higher genetic diversity of Bradyrhizobium strains was observed in temperate and sub-tropical region than in the tropical region.

Phenotypic and genetic characterization of rhizobia associated with alfalfa in the Hokkaido and Ishigaki regions of Japan.

Twenty five rhizobial isolates were obtained from root nodules of Medicago sativa inoculated with soil samples collected from the Sapporo region and Ishigaki Island in Japan. To study their diversity and characterize them in relation to the climatic conditions of their soils of origin, a polyphasic approach analyzing stress tolerance, symbiotic and genetic properties was used. Stress tolerance assays revealed marked variations in salinity, pH and temperature tolerance. Isolates originating from a sub-tropical climate in alkaline soil (Ishigaki Island) tolerated high temperature, salinity and pH levels. Moreover, isolates recovered from a temperate climate in acidic soil (Sapporo) were sensitive to high temperature and salinity, and tolerated acidic pH. Phylogenetic analysis of conserved 16S rRNA and recA genes, and symbiotic nodA and nifDK revealed 25 isolates to be closely related to Ensifer meliloti. Furthermore, the branch patterns of phylogenetic trees constructed from different genes revealed the existence of at least two E. meliloti types in the soils studied. These results may be relevant to programs directed towards improving crop productivity through biofertilization with locally adapted and genetically defined strains.

Stress tolerance and symbiotic and phylogenic features of root nodule bacteria associated with Medicago species in different bioclimatic regions of Tunisia.

Thirty two rhizobial isolates were obtained from different bioclimatic regions of Tunisia using as trap plants, Medicago sativa, Medicago ciliaris, Medicago polymorpha and Medicago minima. To study their diversity and characterize them in relation to Mediterranean conditions, abiotic stress resistance, symbiotic properties and genetic diversity in terms of 16S rRNA and nodA sequences were assessed. Five isolates from M. sativa, three from M. ciliaris and three from M. minima could grow at 45°C. Only two isolates from M. sativa grew at 4% NaCl. The most stress tolerant isolates were obtained from arid soils. A phylogenetic analysis of 16S rRNA genes revealed 29 isolates to be closely related to Ensifer including one (Pl.3-9) that showed a 16S rRNA sequence similar to that of Ensifer meliloti and nodA sequence similar to that of Ensifer medicae. However, three isolates were categorized into Agrobacterium containing the nodA of Ensifer. Furthermore, these isolates developed nodules on original hosts. The results for the four isolates suggest horizontal gene transfer between the species.

Genetic diversity of native soybean bradyrhizobia from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal.

Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, the genetic diversity of native soybean bradyrhizobia isolated from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from three different topographical regions with contrasting climates. A local soybean cultivar, Cobb, was used as a trap plant to isolate bradyrhizobia. A total of 24 isolates selected on the basis of their colony morphology were genetically characterized. For each isolate, the full nucleotide sequence of the 16S rRNA gene and ITS region, and partial sequences of the nifD and nodD1 genes were determined. Two lineages were evident in the conserved gene phylogeny; one representing Bradyrhizobium elkanii (71% of isolates), and the other representing Bradyrhizobium japonicum (21%) and Bradyrhizobium yuanmingense (8%). Phylogenetic analyses revealed three novel lineages in the Bradyrhizobium elkanii clade, indicating high levels of genetic diversity among Bradyrhizobium isolates in Nepal. B. japonicum and B. yuanmingense strains were distributed in areas from 2420 to 2660 m above sea level (asl), which were mountain regions with a temperate climate. The B. elkanii clade was distributed in two regions; hill regions ranging from 1512 to 1935 m asl, and mountain regions ranging from 2420 to 2660 m asl. Ten multi-locus genotypes were detected; seven among B. elkanii, two among B. japonicum, and one among B. yuanmingense-related isolates. The results indicated that there was higher species-level diversity of Bradyrhizobium in the temperate region than in the sub-tropical region along the southern slopes of the Himalayan Mountains in Nepal.