Acetylene Reduction Assay Research Articles

Biological soil crust (BSC) is an effective biofertilizer on Vigna mungo (L.).

Biological soil crusts (BSC) (Cyanobacteria) play an important role in the soil nitrogen fixation and soil stabilization. However, limited researches were carried out about the diversity and distribution of Bio crust in sacred forests. The study aims to identify the distribution of cyanobacteria in biological soil crusts from different sacred groves of Ariyalur and Pudukottai, Tamil Nadu. We identified following microbes of Microcoleus, Scytonema, Anabaena and Nostoc in biological soil crust. A surface experiment was conducted for the efficacy of biological soil crusts on crops seedling growths. The efficacy was assessed by measuring the root & shoot length, dry & fresh weight of the plant, total chlorophyll, protein & carbohydrate content, with reducing sugar. The plant growth was higher in biocrust inoculated pot than the control. In corresponding nitrogenase activity was determined by the acetylene reduction assay, and phytohormones documentation was executed by the high-performance liquid chromatography. The results indicating that the biological soil crusts are the promising factors influencing the plant growth by plant growth-stimulating auxins and nitrogenase activity.

Multiphase characterization of wild Vigna associated root nodule bacteria from Japanese subtropical islands unveiled novel high temperature resistant Bradyrhizobium strains having high symbiotic compatibility with soybean and mungbean

ABSTRACT Vigna riukiuensis plant – a rare type of vigna, found only in Taiwan and the islands of Okinawa prefecture, Japan – possesses intrinsic property of high level of salt and heat tolerance. To understand the diversity and identify suitable rhizobia, multiphase characterization of root nodule bacteria associated with V. riukiuensis grown in Ishigaki and Iriomote Islands of Okinawa prefecture was performed. Multigene phylogenetic analysis of housekeeping genes based on 16S rRNA gene sequences, 16S-23S rRNA gene internal transcribed spacer (ITS) and 23S rRNA gene sequences identified three main groups closely similar to Bradyrhizobium japonicum, B. elkanii and B. jicamae family. However, analysis of symbiotic nifH and nodD1 genes and their phylogenetic trees showed similar topology, having only few discrepancies in comparison to the housekeeping gene phylogeny. Interestingly, for some of the isolates having similarity with B. elkanii, growth was observed at 40°C, which exceed the highest record for B. elkanii to the best of our knowledge. All the isolates were observed to have the capability of forming root nodules and fix nitrogen in their original host plant V. riukiuensis and two other crops: soybean and mungbean. Most of the isolates showed similar or higher nitrogen-fixing capability in comparison with B. diazoefficiens USDA110 in V. riukiuensis and V. radiata (mungbean), and Iri 5/6 in V. riukiuensis, Iri 5/12 in soybean and Ishi 7/2 in mungbean showed highest acetylene reduction assay (in µmol/h/gm nodule dry weight) activity, which was significantly higher than B. diazoefficiens USDA110. In addition, six isolates attained higher soybean biomass production compared with B. diazoefficiens USDA110, suggesting high symbiotic compatibility with soybean. Among them, Iri 5/7 of B. elkanii group contributed 29% higher soybean biomass production than B. diazoefficiens USDA110 and could grow at 40°C, hence it could be a promising soybean inoculant in the tropics. Abbreviations ARA: acetylene reduction assay, ISR: intergenic spacer regions, ITS: internal transcribed spacer

Algal associates and the evidence of cyanobacterial nitrogen fixation in the velamen roots of epiphytic orchids

The hanging velamen roots in epiphytic orchids are unique kind of aerial roots in the plant world. The diverse kinds of microbial associates in it suggest its ecological and functional complexities. In this context, diversity and population characteristics of the entire algal flora in the velamen roots of four tropical epiphytic orchids Acampe praemorsa, Rhynchostylis retusa, Epidendrum radicans and Oncidium sphacelatum from different geographic locations of Kerala state, India were explored and compared. The potential nitrogen-fixing activity (NFA) of cyanobacteria in the velamen roots was analysed by acetylene reduction assay to understand the nitrogen-fixing role of cyanobacterial species in the same as a source of nitrogen to the host. The total nitrogen content of the velamen roots and leaves was compared to generate indirect evidence of accumulation of nitrogen in roots by primary addition through nitrogen fixation. Altogether 24 species of algal associates were isolated from the velamen roots of four epiphytic orchids. A majority of algae were found as cyanobacterial species; many of them are well-known nitrogen-fixing categories. The total tissue nitrogen content of the velamen roots was found significantly higher than that of leaves. The NFA of cyanobacteria in the velamen roots revealed its potential role in nitrogen fixation. This preliminary level empirical evidence is sufficient to consider the velamen roots of epiphytic orchids as an ecologically complex aerial ‘organ ecosystem’ with microbial partnerships for nutritional support, especially nitrogen availability in epiphytic orchids.

Interaction between graphene oxide and nitrogen-fixing bacterium Azotobacter chroococcum: Transformation, toxicity and nitrogen fixation

Nitrogen is an essential element and 80% of nitrogen fixation from atmosphere is achieved by biological nitrogen fixation. When graphene enters the environment, it would inevitably interact with nitrogen-fixing bacteria and might disturb the nitrogen cycle. Herein, the interaction between graphene oxide (GO) and nitrogen-fixing bacterium Azotobacter chroococcum was studied to reveal the potential impact of graphene materials on biological nitrogen fixation. After incubation with A. chroococcum, GO was separated, washed and characterized by different techniques. The toxicity of GO to A. chroococcum was monitored by colony-forming unit (CFU) counting, growth curve, live/dead staining and ultrastructural observations. The nitrogen fixation activity of A. chroococcum was measured by acetylene reduction assay and the soil nitrogen contents were measured. GO was immediately reduced by cell secretions and kept stable thereafter. GO stimulated A. chroococcum growth at low concentrations and showed inhibitive effect at high concentrations. GO induced cell death and cell wall break at high concentration. The toxicological mechanism was assigned to membrane damage and oxidative stress. In the presence of soil, GO showed similar concentration-dependent but alleviated toxicity, while the soil nitrogen contents slightly increased at high concentrations. Our results collectively indicated that GO was bio-reduced and toxic to nitrogen-fixation bacteria.

Divulging diazotrophic bacterial community structure in Kuwait desert ecosystems and their N2-fixation potential.

Kuwait is a semi-arid region with soils that are relatively nitrogen-poor. Thus, biological nitrogen fixation is an important natural process in which N2-fixing bacteria (diazotrophs) convert atmospheric nitrogen into plant-usable forms such as ammonium and nitrate. Currently, there is limited information on free-living and root-associated nitrogen-fixing bacteria and their potential to fix nitrogen and aid natural plant communities in the Kuwait desert. In this study, free living N2-fixing diazotrophs were enriched and isolated from the rhizosphere soil associated with three native keystone plant species; Rhanterium epapposum, Farsetia aegyptia, and Haloxylon salicornicum. Root-associated bacteria were isolated from the root nodules of Vachellia pachyceras. The result showed that the strains were clustered in five groups represented by class: γ-proteobacteria, and α-proteobacteria; phyla: Actinobacteria being the most dominant, followed by phyla: Firmicutes, and class: β-proteobacteria. This study initially identified 50 nitrogen-fixers by16S rRNA gene sequencing, of which 78% were confirmed to be nitrogen-fixers using the acetylene reduction assay. Among the nitrogen fixers identified, the genus Rhizobium was predominant in the rhizosphere soil of R. epapposum and H. salicornicum, whereas Pseudomonas was predominant in the rhizosphere soil of F. aegyptia, The species Agrobacterium tumefaciens was mainly found to be dominant among the root nodules of V. pachyceras and followed by Cellulomonas, Bacillus, and Pseudomonas genera as root-associated bacteria. The variety of diazotrophs revealed in this study, signifying the enormous importance of free-living and root-associated bacteria in extreme conditions and suggesting potential ecological importance of diazotrophs in arid ecosystem. To our knowledge, this study is the first to use culture-based isolation, molecular identification, and evaluation of N2-fixing ability to detail diazotroph diversity in Kuwaiti desert soils.

Characterization of endophytic bacterial communities within greenhouse and field-grown rhizomes of three rare pitcher plant species (Sarracenia oreophila, S. leucophylla, and S. purpurea spp. venosa) with an emphasis on nitrogen-fixing bacteria

Several Sarracenia species are endangered due to habitat destruction. Knowledge of their microbiomes may better inform reintroduction efforts. We characterized bacterial communities within rhizomes of three species with an emphasis on diazotrophs. Microbiomes of wild and greenhouse-grown Sarracenia were characterized using cultivation-independent and cultivation-dependent methods, including Illumina MiSeq, 16S rRNA and nifH qPCR, colony identification via 16S rRNA sequencing, and nifH colony PCR. Rhizomes were tested for biological nitrogen fixation using acetylene reduction assays. Diazotrophs make up a notable proportion of the S. oreophila microbiome according to qPCR. Microbiomes of greenhouse S. oreophila varied from wild ones by having greater proportions of diazotrophs as indicated by qPCR. Wild and greenhouse plant microbiomes also differed with respect to several phylogenetic groups. Acetylene reduction assays indicated that nitrogen fixation occurred in some rhizomes. Multiple colonies from S. leucophylla and S. purpurea were nifH+, and 16S rRNA sequencing likewise revealed dissimilarities between wild and greenhouse-grown plant microbiomes. Our study is the first to characterize bacterial communities within Sarracenia rhizomes and demonstrates that substantial differences exist between wild and greenhouse plants. We provide evidence that Sarracenia can contain large populations of diazotrophs that may help explain how these plants survive in oligotrophic environments.

Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and coastal environments of Southeastern Arabian Sea (SEAS)

Nitrogen fixation by diazotrophic bacteria serve as an important source of fixed nitrogen in an aquatic ecosystem and thereby directly influence the carbon flux and primary production. Currently there is little information about the cultivable heterotrophic diazotrophs and the eco-physiological roles of nitrogen fixing bacteria in aquatic environments. The focus of the present study was to understand the diversity of cultivable heterotrophic diazotrophs and to evaluate their nitrogen fixation capability in the estuarine and coastal environments of the Southeastern Arabian Sea. The heterotrophic diazotrophic bacteria were isolated on nitrogen-free media and the potential activity of nitrogen fixation was estimated by acetylene reduction assay. The molecular basis of the nitrogen fixation capability among the above isolates was analyzed testing for the presence of dinitrogenase reductase (nifH) gene. The 16S rRNA gene based identity revealed that the cultivable heterotrophic diazotrophs belonged to α, β, γ-Proteobacteria and Firmicutes. The results also revealed that α-Proteobacterium Nitratireducter kimnyeongensis was found to be a potential diazotroph (190.3 ± 4.55 nmol C2H4/mg protein/day), which was isolated from the coastal ecosystem. Various strains of γ-Proteobacteria such as Klebsiella pneumonia, Klebsiella quasipneumonia and Klebsiella variicola also exhibited relatively high nitrogen-fixing activity compared to that of Pseudomonas flavescens and Halomonas meridiana Other cultivable diazotrophs encountered in the study area were Oceanobacillus iheyencis, Bacillus aerius, Exiguobacterium profundum, Staphylococcus warneri, Bacillus amyloliquefaciens, Staphylococcus arlettae and Staphylococcus caprae. The results revealed that Gram-negative bacterial strains possessed relatively superior nitrogen fixation activity over Gram-positive isolates (p<0.01). In the light of our observations, we hypothesize that heterotrophic diazotrophs play key role in nitrogen fixation process and contribute to primary production in estuarine and coastal environments of Southeastern Arabian Sea.

Chinese Black Truffle-Associated Bacterial Communities of Tuber indicum From Different Geographical Regions With Nitrogen Fixing Bioactivity

It is well known that the microbes associated with truffle fruiting bodies play a very important role during the truffle lifecycle. Tuber indicum, commonly called Chinese black truffle, is a species endemic to Eastern Asia and in the genus of Tuber. Here, we reported the bacterial communities of T. indicum from different geographical regions and described the bacterial diversity from three compartments (soil, ectomycorrhizae and ascocarps) of T. indicum using high-throughput sequencing combined tissue culture. The results revealed that Bradyrhizobium was the dominant genus in fruiting bodies of T. indicum from nine geographical sites in China, and the microbes in T. indicum ascocarps were influenced by geological locations and soil characteristics. More specific bacterial taxa were enriched in the fruiting bodies than in the ectomycorrhizae and soil. In addition, 60 cultural bacteria were isolated from T. indicum fruiting bodies (4 families, 24 genera), and Pseudomonas, Alcaligenes faecalis, Microbacterium, and Arthrobacter were dominant. One of 13 strains that have potential nitrogen-fixation activities was further verified by an acetylene reduction assay (ARA). Together, this research provides new and important data for better understanding of the interaction between truffle and associated microbe and the biology of truffle itself.

Mangrove-diazotroph relationships at the root, tree and forest scales: diazotrophic communities create high soil nitrogenase activities in Rhizophora stylosa rhizospheres.

The tidal flats on which mangrove plants grow tend to have low soil nitrogen contents because nitrogen-containing litter is repeatedly washed offshore by ebb tides. Under such circumstances, it is unclear how mangrove plants acquire the nitrogen required to support their vigorous growth. In the present work, chemical and biological characteristics of diazotrophy around mangrove plant roots were surveyed under natural conditions to elucidate mangrove-diazotroph relationships. Soil nitrogenase activity of a representative mangrove plant, Rhizophora stylosa, which has a broad geographical distribution, was measured using the acetylene reduction assay at forest, tree and prop root scales. In addition, diazotrophic community composition was compared between rhizosphere and bulk soil based on sequencing of nifH genes. Soil nitrogenase activity was high near prop roots, and this pattern was enhanced as soil live root content increased. At the forest scale, we observed high soil nitrogenase activity (acetylene-reducing activity) inside the forest (the highest value was 90.9 µmol C2H2 min-1 cm-3, average 46.8 ± 18.2 µmol C2H2 min-1 cm-3). Rates decreased sharply from the forest to the tidal flat (range 1.2-22.2 µmol C2H2 min-1 cm-3, average 7.9 ± 4.5 µmol C2H2 min-1 cm-3). The nifH operational taxonomic unit composition differed significantly among forest and tree rhizospheres and the bulk soil (P < 0.0001). Our results suggest that the accumulation of diazotrophs around R. stylosa mangrove trees enhances the supply of biologically fixed nitrogen to the mangrove roots. This supply is especially important when the soil naturally contains little nitrogen. This nitrogen acquisition system may be a key process that explains the high productivity of mangrove ecosystems.

Allelic Variants for Candidate Nitrogen Fixation Genes Revealed by Sequencing in Red Clover (Trifolium pratense L.).

Plant–rhizobia symbiosis can activate key genes involved in regulating nodulation associated with biological nitrogen fixation (BNF). Although the general molecular basis of the BNF process is frequently studied, little is known about its intraspecific variability and the characteristics of its allelic variants. This study’s main goals were to describe phenotypic and genotypic variation in the context of nitrogen fixation in red clover (Trifolium pretense L.) and identify variants in BNF candidate genes associated with BNF efficiency. Acetylene reduction assay validation was the criterion for selecting individual plants with particular BNF rates. Sequences in 86 key candidate genes were obtained by hybridization-based sequence capture target enrichment of plants with alternative phenotypes for nitrogen fixation. Two genes associated with BNF were identified: ethylene response factor required for nodule differentiation (EFD) and molybdate transporter 1 (MOT1). In addition, whole-genome population genotyping by double-digest restriction-site-associated sequencing (ddRADseq) was performed, and BNF was evaluated by the natural 15N abundance method. Polymorphisms associated with BNF and reflecting phenotype variability were identified. The genetic structure of plant accessions was not linked to BNF rate of measured plants. Knowledge of the genetic variation within BNF candidate genes and the characteristics of genetic variants will be beneficial in molecular diagnostics and breeding of red clover.

Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation

To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for use with soil/rhizosphere samples. We tested the impact of different carbon (C) sources, oxygen concentrations (O2), and incubation times on FLNF rates of two low-fertility Michigan soils via ARA and 15N2. FLNF rates were greatest with addition of a C cocktail, at low O2, and with 7-day incubations for both methods. FLNF via ARA was 1700x greater with a C cocktail versus glucose only and via 15N2 was 17x greater with a C cocktail compared to other C sources and no-C controls. Specific O2 optimum varied by method and site. A 7-day incubation was needed for the ARA, but a 3-day incubation was suitable for 15N2. Lastly, we confirm previously identified issues with the ARA of acetylene-independent ethylene production/consumption resulting in potential FLNF measurement error of 1.3–52.3 μg N g−1 day−1. We present an optimized method for measuring potential FLNF in soil/rhizosphere samples which will allow for consistent and comparable FLNF rate measurements. Researchers should account for C source, O2, and incubation time when assessing FLNF and use the ARA method with caution.

Multiple factors drive the abundance and diversity of the diazotrophic community in typical farmland soils of China.

Biological nitrogen fixation plays an important role in nitrogen cycling by transferring atmospheric N2 to plant-available N in the soil. However, the diazotrophic activity and distribution in different types of soils remain to be further explored. In this study, 152 upland soils were sampled to examine the diazotrophic abundance, nitrogenase activity, diversity and community composition by quantitative polymerase chain reaction, acetylene reduction assay and the MiSeq sequencing of nifH genes, respectively. The results showed that diazotrophic abundance and nitrogenase activity varied among the three soil types. The diazotrophic community was mainly dominated by Bradyrhizobium, Azospirillum, Myxobacter, Desulfovibrio and Methylobacterium. The symbiotic diazotroph Bradyrhizobium was widely distributed among soils, while the distribution of free-living diazotrophs showed large variation and was greatly affected by multiple factors. Crop type and soil properties directly affected the diazotrophic ɑ-diversity, while soil properties, climatic factors and spatial distance together influenced the diazotrophic community. Network structures were completely different among all three types of soils, with most complex interactions observed in the Red soil. These findings suggest that diazotrophs have various activities and distributions in the three soil types, which played different roles in nitrogen input in agricultural soil in China, being driven by multiple environmental factors.

Bioaugmentation of soil with Enterobacter cloacae AKS7 enhances soil nitrogen content and boosts soil microbial functional-diversity.

Biofertilizer happens to be a promising alternative of chemical fertilizer in the establishment of sustainable agricultural practices. Following this observation, several nitrogen-fixing bacteria were isolated from the soil in which an isolate (AKS7) was selected for further studies as AKS7 showed considerable competence in growth on nitrogen-free growth medium. Acetylene reduction assay confirmed that AKS7 can fix atmospheric nitrogen efficiently. The result of Kjeldahl assay revealed that the organism (AKS7) could fix nitrogen up to 12mg in 8days. A strong positive correlation (r = 0.987) was observed between microbial cell biomass and the amount of nitrogen fixed by AKS7 over a period of 8days. The organism was identified as Enterobacter cloacae through molecular and biochemical tests. To investigate the in situ nitrogen fixation by E. cloacae AKS7, naturally attenuated (AKS7 not-inoculated) and bioaugmented (AKS7-inoculated) soil microcosms were prepared. The bioaugmented microcosm showed higher level of soil nitrogen content than naturally attenuated microcosm. A large number of heterotrophic as well as nitrogen-fixing microorganisms were counted in bioaugmented microcosm than naturally attenuated microcosm. Results of the carbon source utilization patterns of BiOLOG ECO plate revealed that bioaugmented microcosm exhibited higher level of functional richness and evenness that lead to the exhibition of higher level of microbial functional-diversity in bioaugmented microcosm than the naturally attenuated microcosm. Taken together, the results indicated that augmentation of E. cloacae AKS7 into soil enhanced the nitrogen content and soil microbial functional-diversity considerably.

Repeatable, continuous and real‐time estimates of coupled nitrogenase activity and carbon exchange at the whole‐plant scale

Abstract Symbiotic nitrogen fixation (SNF) by higher plants and their bacterial symbionts is a globally important input of nitrogen. Our understanding of the mechanisms that control SNF and the time‐scales over which they operate has been constrained by the limitations of the existing methods for measuring SNF. One method, Acetylene Reduction Assays by Cavity ring‐down laser Absorption Spectroscopy (ARACAS), seems promising, as it is highly sensitive and gives rapid, continuous, repeatable and real‐time measurements of nitrogenase activity. ARACAS has been used to study nitrogen fixation in lichens, mosses and asymbiotic bacteria, but adapting it to higher plants poses challenges because acetylene and ethylene can influence plant function. Here, we report modifications to ARACAS that allow it to be used on higher plants in an environmentally controlled incubation chamber. The modifications include lower concentrations of acetylene (2%) and ethylene and concurrent measurements of whole‐chamber CO2 exchange, H2O exchange and nitrogenase activity, linking nitrogenase activity to whole‐plant rates of photosynthesis and respiration. After propagating the error terms from all sources, we establish the following parameters of the method: (a) The detection limit of our method was 2–3 ppbv C2H4 per hour, although it rose substantially when we used tank‐derived acetylene, which has much higher ethylene contamination; (b) Repeated measures at a frequency of 3 days or longer did not diminish nitrogenase activity or photosynthesis, although daily measurements diminished nitrogenase activity; (c) This method can detect changes at time‐scales as short as seconds; (d) Continuous measurement of nitrogenase activity is maintained above 90% of the maximum rate for 7.0 ± 1.3 (M ± SD) hours. This method has the potential to improve our understanding of the controls over SNF, and therefore, how SNF and global nitrogen and carbon cycling are likely to be affected by global change.

Regulation of nitrogen fixation from free-living organisms in soil and leaf litter of two tropical forests of the Guiana shield

Background and aimsBiological fixation of atmospheric nitrogen (N2) is the main pathway for introducing N into unmanaged ecosystems. While recent estimates suggest that free-living N fixation (FLNF) accounts for the majority of N fixed in mature tropical forests, the controls governing this process are not completely understood. The aim of this study was to quantify FLNF rates and determine its drivers in two tropical pristine forests of French Guiana.MethodsWe used the acetylene reduction assay to measure FLNF rates at two sites, in two seasons and along three topographical positions, and used regression analyses to identify which edaphic explanatory variables, including carbon (C), nitrogen (N), phosphorus (P) and molybdenum (Mo) content, pH, water and available N and P, explained most of the variation in FLNF rates.ResultsOverall, FLNF rates were lower than measured in tropical systems elsewhere. In soils seasonal variability was small and FLNF rates differed among topographies at only one site. Water, P and pH explained 24% of the variation. In leaf litter, FLNF rates differed seasonally, without site or topographical differences. Water, C, N and P explained 46% of the observed variation. We found no regulatory role of Mo at our sites.ConclusionsRates of FLNF were low in primary rainforest on poor soils on the Guiana shield. Water was the most important rate-regulating factor and FLNF increased with increasing P, but decreased with increasing N. Our results support the general assumption that N fixation in tropical lowland forests is limited by P availability.

Biofertilizer Production for Agronomic Application and Evaluation of Its Symbiotic Effectiveness in Soybeans

This study was conducted to evaluate the effects of Bradyrhizobium japonicum SAY3-7, Bradyrhizobium elkanii BLY3-8, and Streptomyces griseoflavus P4 on the symbiotic effectiveness of soybeans before biofertilizer production, to produce biofertilizer containing the studied three strains (SAY3-7, BLY3-8, and P4), to test the effectiveness of the biofertilizer on soybean varieties, and to assess the varietal effects and interaction effects between variety and biofertilizer on plant growth, nodulation, nitrogen fixation, nutrient absorption, and seed yield. Nitrogen fixation was measured using the acetylene reduction assay and ureide methods. Contents of nutrients (N, P, K, Ca, and Mg) were also measured to calculate their uptakes. In this study, synergistic effects of nitrogen fixation were induced by combined inoculation with SAY3-7, BLY3-8 and P4 in all tested soybean varieties. Therefore, we assumed that an effective biofertilizer could be produced using these effective bacteria (SAY3-7, BLY3-8, and P4). After making biofertilizer using these effective bacteria, packages were stored at 30 °C. The populations of the bacteria in the biofertilizer were maintained at a density of 1 × 108 colony forming units (cfu) g−1 for P4 and 7 × 109 cells g−1 for Bradyrhizobium. Diluting biofertilizer by 10−3 proved more effective for nodulation and nitrogen fixation than other dilution treatments. Moreover, this biofertilizer significantly promoted plant growth, nodulation, nitrogen fixation, nutrient uptakes, and seed yield in Yezin-3 and Yezin-6 soybean varieties. Yezin-6 is a more efficient variety than Yezin-3 for improved plant growth, nodulation, nitrogen fixation, nutrient absorption, and seed yield. Taken together, the application of an effective biofertilizer and the use of an efficient soybean variety can play important roles in promoting plant growth, nodulation, nitrogen fixation, and higher seed yield.

Identification of Nitrogen-Fixing Bradyrhizobium Associated With Roots of Field-Grown Sorghum by Metagenome and Proteome Analyses.

Sorghum (Sorghum bicolor) is cultivated worldwide for food, bioethanol, and fodder production. Although nitrogen fixation in sorghum has been studied since the 1970s, N2-fixing bacteria have not been widely examined in field-grown sorghum plants because the identification of functional diazotrophs depends on the culture method used. The aim of this study was to identify functional N2-fixing bacteria associated with field-grown sorghum by using “omics” approaches. Four lines of sorghum (KM1, KM2, KM4, and KM5) were grown in a field in Fukushima, Japan. The nitrogen-fixing activities of the roots, leaves, and stems were evaluated by acetylene reduction and 15N2-feeding assays. The highest nitrogen-fixing activities were detected in the roots of lines KM1 and KM2 at the late growth stage. Bacterial cells extracted from KM1 and KM2 roots were analyzed by metagenome, proteome, and isolation approaches and their DNA was isolated and sequenced. Nitrogenase structural gene sequences in the metagenome sequences were retrieved using two nitrogenase databases. Most sequences were assigned to nifHDK of Bradyrhizobium species, including non-nodulating Bradyrhizobium sp. S23321 and photosynthetic B. oligotrophicum S58T. Amplicon sequence and metagenome analysis revealed a relatively higher abundance (2.9–3.6%) of Bradyrhizobium in the roots. Proteome analysis indicated that three NifHDK proteins of Bradyrhizobium species were consistently detected across sample replicates. By using oligotrophic media, we purified eight bradyrhizobial isolates. Among them, two bradyrhizobial isolates possessed 16S rRNA and nif genes similar to those in S23321 and S58T which were predicted as functional diazotrophs by omics approaches. Both free-living cells of the isolates expressed N2-fixing activity in a semi-solid medium according to an acetylene reduction assay. These results suggest that major functional N2-fixing bacteria in sorghum roots are unique bradyrhizobia that resemble photosynthetic B. oligotrophicum S58T and non-nodulating Bradyrhizobium sp. S23321. Based on our findings, we discuss the N2-fixing activity level of sorghum plants, phylogenetic and genomic comparison with diazotrophic bacteria in other crops, and Bradyrhizobium diversity in N2 fixation and nodulation.

Azospirillum ramasamyi sp. nov., a novel diazotrophic bacterium isolated from fermented bovine products.

A Gram-stain-negative, rod-shaped, aerobic bacterium, designated M2T2B2T, was isolated from fermented bovine products in Suwon, Republic of Korea. The strain displayed growth at 15-45 °C (optimum, 28-30 °C), pH 6.0-10.0 (pH 7.0) and 0-2 % (w/v) NaCl (0 %). Colonies were light pink-coloured, round and convex. The cells were positive for oxidase and weakly positive for catalase. The major fatty acids in whole cells of strain M2T2B2T were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), followed by summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), summed feature 2 (C12 : 0 aldehyde/unidentified 10.928/C14 : 0 3-OH/iso-C16 : 1 I), C16 : 0, C18 : 1 2-OH, C16 : 0 3-OH and C17 : 1ω6c. The polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, and three unidentified aminolipids. Ubiquinone 10 was the predominant ubiquinone. The DNA G+C content was 68.0 mol%. The strain could fix atmospheric nitrogen, which was evaluated by the acetylene reduction assay. Further, whole genome sequence analysis revealed the presence of a nif gene cluster. Strain M2T2B2T showed the highest 16S rRNA, rpoD and nifH gene sequence similarity to members of the genus Azospirillum, and showed 97.6 % 16S rRNA gene sequence similarity to Azospirillum oryzae COC8T. The phenotypic, phylogenetic and genomic analyses support the proposal of strain M2T2B2T as being a novel species of the genus Azospirillum, for which the name Azospirillumramasamyi sp. nov. is proposed. The type strain is M2T2B2T (=KACC 14063T=NBRC 106460T).

Effects of Biofertilizer Produced from Bradyrhizobium and Streptomyces griseoflavus on Plant Growth, Nodulation, Nitrogen Fixation, Nutrient Uptake, and Seed Yield of Mung Bean, Cowpea, and Soybean

The use of biofertilizers is important for sustainable agriculture, and the use of nodule bacteria and endophytic actinomycetes is an attractive way to enhance plant growth and yield. This study tested the effects of a biofertilizer produced from Bradyrhizobium strains and Streptomyces griseoflavus on leguminous, cereal, and vegetable crops. Nitrogen fixation was measured using the acetylene reduction assay. Under N-limited or N-supplemented conditions, the biofertilizer significantly promoted the shoot and root growth of mung bean, cowpea, and soybean compared with the control. Therefore, the biofertilizer used in this study was effective in mung bean, cowpea, and soybean regardless of N application. In this study, significant increments in plant growth, nodulation, nitrogen fixation, nitrogen, phosphorus, and potassium (NPK) uptake, and seed yield were found in mung beans and soybeans. Therefore, Bradyrhizobium japonicum SAY3-7 plus Bradyrhizobium elkanii BLY3-8 and Streptomyces griseoflavus are effective bacteria that can be used together as biofertilizer for the production of economically important leguminous crops, especially soybean and mung bean. The biofertilizer produced from Bradyrhizobium and S. griseoflavus P4 will be useful for both soybean and mung bean production.

Soil nitrogen response to shrub encroachment in a degrading semi-arid grassland

Abstract. Transitions from grass- to shrub-dominated states in drylands by woody plant encroachment represent significant forms of land cover change with the potential to alter the spatial distribution and cycling of soil resources. Yet an understanding of how this phenomenon impacts the soil nitrogen pool, which is essential to primary production in arid and semi-arid systems, is poorly resolved. In this study, we quantified how the distribution and speciation of soil nitrogen, as well as rates of free-living biological nitrogen fixation, changed along a gradient of increasing mesquite (Prosopis velutina Woot.) cover in a semi-arid grassland of the southwestern US. Our results show that site-level concentrations of total nitrogen remain unchanged with increasing shrub cover as losses from inter-shrub areas (sum of grass and bare-soil cover) are proportional to increases in soils under shrub canopies. However, despite the similar carbon-to-nitrogen ratio and microbial biomass of soil from inter-shrub and shrub areas at each site, site-level concentrations of inorganic nitrogen increase with shrub cover due to the accumulation of ammonium and nitrate in soils beneath shrub canopies. Using the acetylene reduction assay technique, we found increasing ratios of inorganic nitrogen to bioavailable phosphorus inhibit rates of biological nitrogen fixation by free-living soil bacteria. Overall, these results provide a greater insight into how grassland-to-shrubland transitions influence the soil N pool through associated impacts on the soil microbial biomass.