A database for the taxonomic and phylogenetic identification of the genus Bradyrhizobium using multilocus sequence analysis.

Soil is living medium and it acts as a precarious reserve in agriculture and food production. To enhance crop yields for ever-increasing human population, chemical fertilizers are being applied in the soil. But, the haphazard usage of fertilizers, predominantly nitrogenous and phosphorus, headed to considerable contamination of soil, air and water. Moreover, unwarranted consumption of these agrochemicals also cause lethal effects on soil microorganisms and disturbs the soil fertility. Due to current public apprehensions about the side effects of these agrochemicals, understanding plant and rhizospheric microbial interactions is gaining momentum. It is considered to be important to effectively manage level of nitrogen in soil through biological nitrogen fixation (BNF) to maintain agricultural sustainability. The fixed N is directly taken up in the plants and is less vulnerable to volatilization, denitrification and leaching. Thus, mutualistic symbiosis amongst legume plant and nodulating rhizobia plays a key role in ecological environments. Legume-rhizobia symbioses provide approximately 45% of N used in agriculture and contributions of BNF from the symbiotic association accounts for at least 70 million metric tons per year into terrestrial ecosystems. In agricultural systems, about 80% of BNF contributed by symbiotic association made between leguminous plants and species of Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, Mesorhizobium and Allorhizobium. The populations of these root-nodule forming bacteria can be changed ecologically, agronomically, edaphically and genetically to increase legume production and soil productivity. Moreover, legume-rhizobia symbioses also provide non-polluting and economical ways to augment N2-fixing potential under stress conditions. Scientists have identified numerous symbiotic systems tolerant in harsh situations of salinity, alkalinity, acidity, drought, toxic metals have been recognized and alteration in rhizobial population under stressed environments can be an indicator of soil fertility. Moreover, interactions among rhizobia, plant growth-promoting rhizobacteria (PGPR) and mycorrhiza as well show significant part in increasing soil fertility and crop yields. In this chapter, significance of biological nitrogen fixation in persistent food supply, influence of extreme environments on legume-rhizobia symbiosis as well as interaction of rhizobia with belowground microbial species are discussed. The eco-friendly approach to increase crop production and soil health by inoculation of symbiotic bacteria as biofertilizers is described for sustainable agriculture.

The 12th International Symposium on biological nitrogen fixation (BNF) with Non-Legumes took place in Buzios, Rio de Janeiro, Brazil from October 3 to 8, 2010. It was the latest in a series of meetings that, fittingly, first began in Brazil, in 1977 in the city of Piracicaba. This Special Issue of Plant and Soil comprises 21 papers from the Buzios Symposium, as well as a commentary and three obituaries. Given the volatility (and general upward trend) in oil prices and global attempts to alleviate greenhouse gas (GHG) emissions associated with the agricultural use of Ncontaining mineral fertilizers produced by the energyintensive Haber-Bosch process, it was inevitable that the main focus of the 2010 Symposium would be very much on the substitution of fertilizers in favour of the increased use of BNF in non-legume cropping systems. In the intervening years since the last time the symposium was held in Brazil (August 1987 in Rio de Janeiro), knowledge about non-legume BNF has increased enormously, encompassing everything from more accurate N-based quantification studies in the field through to the molecular identification of their associated diazotrophs (both culturable and unculturable) and the localization of these bacteria via high resolution microscopy. The completely sequenced genomes of many diazotrophic bacteria are also now available, and more are deposited in databases every year. Indeed, such technologies, which only a few years ago were considered remarkable, are now becoming standard tools in this field. In the case of sugarcane and other biofuel crops, the location of the 2010 meeting in Brazil was especially pertinent, as the highly advanced Brazilian bioethanol programme, which produces over 27 billion litres of ethanol per year, is based upon the cultivation of sugarcane, and Brazilian cane has long been known to be able to derive much of its N-requirements via BNF. The consequent low fertilizer use by Brazilian cane producers not only makes for enormous economic savings, but also mitigates against GHG emissions from the production of N-containing fertilizers, as well as their transport and application and nitrous oxide emissions. However, in spite of the strong likelihood that cane can benefit significantly from BNF, little is known about how much N it can really fix, and as field-based BNF quantification studies may take several years to complete, we are thus fortunate to include two such studies in this Special Issue (both utilizing the N natural abundance technique): one on sugarcane by Urquiaga et al., and one on the C-4 “energy crop” Plant Soil (2012) 356:1–3 DOI 10.1007/s11104-012-1317-1

The symbioses between legumes and nitrogen-fixing rhizobia make the greatest contribution to the global nitrogen input via the process of biological nitrogen fixation (BNF). Bradyrhizobium stands out as the main genus nodulating basal Caesalpinioideae. We performed a polyphasic study with 11 strains isolated from root nodules of Chamaecristafasciculata, an annual multi-functional native legume of the USA. In the 16S rRNA gene phylogeny the strains were clustered in the Bradyrhizobium japonicumsuperclade. The results of analysis of the intergenic transcribed spacer (ITS) indicated less than 89.9 % similarity to other Bradyrhizobium species. Multilocus sequence analysis (MLSA) with four housekeeping genes (glnII, gyrB, recA and rpoB) confirmed the new group, sharing less than 95.2 % nucleotide identity with other species. The MLSA with 10 housekeeping genes (atpD, dnaK, gap, glnII, gltA, gyrB, pnp, recA, rpoB and thrC) indicated Bradyrhizobium daqingense as the closest species. Noteworthy, high genetic diversity among the strains was confirmed in the analyses of ITS, MLSA and BOX-PCR. Average nucleotide identity and digital DNA-DNA hybridization values were below the threshold of described Bradyrhizobium species, of 89.7 and 40 %, respectively. In the nifH and nodC phylogenies, the strains were grouped together, but with an indication of horizontal gene transfer, showing higher similarity to Bradyrhizobium arachidis and Bradyrhizobium forestalis. Other phenotypic, genotypic and symbiotic properties were evaluated, and the results altogether support the description of the CNPSo strains as representatives of the new species Bradyrhizobiumfrederickii sp. nov., with CNPSo 3426T (=USDA 10052T=U686T=CL 20T) as the type strain.

Biological nitrogen fixation in secondary regrowth and mature rainforest of central Amazonia

Whole genome sequence comparisons have become essential for establishing a robust scheme in bacterial taxonomy. To generalize this genome-based taxonomy, fast, reliable, and cost-effective genome sequencing methodologies are required. MinION, the palm-sized sequencer from Oxford Nanopore Technologies, enables rapid sequencing of bacterial genomes using minimal laboratory resources. Here we tested the ability of Nanopore sequences for the genome-based taxonomy of Vibrionaceae and compared Nanopore-only assemblies to complete genomes of five Rumoiensis clade species: Vibrio aphrogenes, V. algivorus, V. casei, V. litoralis, and V. rumoiensis. Comparison of overall genome relatedness indices (OGRI) and multilocus sequence analysis (MLSA) based on Nanopore-only assembly and Illumina or hybrid assemblies revealed that errors in Nanopore-only assembly do not influence average nucleotide identity (ANI), in silico DNA-DNA hybridization (DDH), G+C content, or MLSA tree topology in Vibrionaceae. Our results show that the genome sequences from Nanopore-based approach can be used for rapid species identification based on the OGRI and MLSA.

Nowadays application of biological nitrogen fixation (BNF) through rhizobia inoculums is highly promoted as a solution to solve the problem of poor soil fertility in areas where legumes are cultivated. This is due to the fact that, rhizobia enhance nitrogen fixation, induce disease resistance, reduce heavy metal in the soil, facilitate bioavailabity of iron in soil and is environmental friendly. To get rhizobia strains which are suitable for inoculants production, isolation and molecular characterization of elite rhizobia are highly needed. Molecular characterization acts as a spark plug for discovery of many microbes including Rhizobia. Multi Locus Sequence Analysis (MLSA), 16S rRNA gene sequence analysis, DNA-DNA hybridization and SDS-PAGE analysis of the whole-cell proteins are the molecular techniques mostly used in characterizing rhizobia. But before deciding to use or not to use rhizobia inoculants in certain areas, knowing the population size of indigenous rhizobia found in that area is very important, because this is a major factor which determines inoculums responses as well gives clues on which areas need or do not need inoculation. The Most Probable Number (MPN) method is mostly used in enumerating rhizobia population of the soil. Given that, in most of the developing countries, including Tanzania, Biological Nitrogen Fixation (BNF) technology is not fully flourished; more efforts in isolation, molecular characterization of elite rhizobia and estimation of indigenous rhizobia population in various areas are required.

Biological nitrogen fixation (BNF) is dramatically affected by environmental constraints such as water stress or heavy metals. It has been reported that these stresses induce the over-production of reactive oxygen species (ROS) and, in turn, oxidative stress that may be responsible for the above-mentioned BNF decline at the molecular level. Oxidative stress, occurring under different environmental stresses, has been widely related to physiological damage. However, a direct relationship between oxidative stress and the decline of BNF, independently from any other cellular damage resulting from adverse environmental situations, has yet to be demonstrated. In order to study the likely in vivo relationship between ROS and BNF inhibition in the legume-Rhizobium symbiosis, two paraquat (PQ) doses, 1 (LPQ) and 10 (HPQ) mmol m(-3), were applied to pea roots for 96 h in order to exacerbate ROS production. Whole-plant physiology and nodule metabolism parameters were determined every 24 h to monitor the evolution of plant responses to ROS. LPQ provoked BNF decline, which was preceded by a prior decrease in sucrose synthase (SS) activity. However, HPQ gave rise to a faster and more pronounced BNF inhibition, which coincided with a decline in SS and also with a reduction in leghaemoglobin (Lb) content. These results indicate a likely involvement of ROS in the effects of environmental stresses on BNF. Furthermore, these results support the occurrence of two regulation pathways for BNF under oxidative stress, one of these involving carbon shortage and the other involving Lb/oxygen flux.

Biological N(2) fixation represents the major source of N input in agricultural soils including those in arid regions. The major N(2)-fixing systems are the symbiotic systems, which can play a significant role in improving the fertility and productivity of low-N soils. The Rhizobium-legume symbioses have received most attention and have been examined extensively. The behavior of some N(2)-fixing systems under severe environmental conditions such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, and pesticides is reviewed. These major stress factors suppress the growth and symbiotic characteristics of most rhizobia; however, several strains, distributed among various species of rhizobia, are tolerant to stress effects. Some strains of rhizobia form effective (N(2)-fixing) symbioses with their host legumes under salt, heat, and acid stresses, and can sometimes do so under the effect of heavy metals. Reclamation and improvement of the fertility of arid lands by application of organic (manure and sewage sludge) and inorganic (synthetic) fertilizers are expensive and can be a source of pollution. The Rhizobium-legume (herb or tree) symbiosis is suggested to be the ideal solution to the improvement of soil fertility and the rehabilitation of arid lands and is an important direction for future research.

Climate change poses unprecedented challenges to global agriculture, necessitating transformative strategies to enhance nitrogen sustainability while ensuring food security. Conventional nitrogen management, heavily reliant on synthetic fertilizers, exacerbates environmental degradation via greenhouse gas emissions, nutrient leaching, and soil degradation. This review synthesizes advances in sustainable nitrogen fixation (SNF) strategies, emphasizing biological, technological, and systemic innovations to reconcile productivity with ecological resilience. We explore the potential of classical biological nitrogen fixation (BNF) via legume-rhizobia symbiosis and asymbiotic diazotrophs, alongside emerging synthetic biology tools such as CRISPR-edited crops and engineered microbial consortia, to reduce dependency on synthetic inputs. Cutting-edge interventions—including nanotechnology-enabled fertilizers, AI-driven nutrient modeling, and real-time nanosensors—are highlighted for their role in optimizing nitrogen-use efficiency and precision agriculture. Furthermore, systems-level approaches integrating circular economy principles, landscape-scale nitrogen cycling, and climate-smart practices underscore the importance of holistic agroecosystem management. Despite these advancements, challenges persist in scalability, environmental stress adaptation, and interdisciplinary implementation. Ethical considerations, equitable technology access, and policy frameworks are critical to ensuring safe and inclusive deployment. The review concludes that a paradigm shift toward integrated, data-driven nitrogen management—bridging microbial ecology, synthetic biology, and digital innovations—is essential for building climate-resilient agricultural systems. Prioritizing transdisciplinary research, stakeholder engagement, and context-specific solutions will be pivotal in realizing sustainable nitrogen fixation as a cornerstone of global food security and environmental stewardship.

About 60 % of the earth’s available nitrogen is fixed via biological nitrogen fixation (BNF). Being a major contributor to BNF, Rhizobium-legume symbiosis can provide well over half of the biological source of fixed nitrogen. Actually, Rhizobium-legume symbiosis results in the formation of nodules on legume roots where rhizobia fix nitrogen from the atmosphere. But nodulation and nitrogen fixation is a complex process and is dependent on the compatibility and potential of both partners of Rhizobium-legume symbiosis under variable soil and environmental conditions. Although, some selected efficient and effective traits of rhizobia and legumes have shown encouraging results, there is a need of consistent positive influence on nodulation and nitrogen fixation to maximize the growth and yield of legumes under variable conditions. Hence, the use of means capable of improving both the legume growth and the growth and function of symbiotic rhizobia is essential. Co-inoculation of Rhizobium species with favorably interacting traits of plant growth-promoting rhizobacteria (PGPR) is considered an applied, cost-effective, efficient, and environment-friendly approach to further improve legume growth and productivity under variable conditions because they can provide broad spectrum mechanisms of actions and improve reliability of inocula without genetic engineering. In addition, these PGPR when used in combination with rhizobia have also shown the strategies for dealing with stressful conditions like salinity, pH, temperature, drought, heavy metal, and pathogens which could further impose limitations on the capacity of Rhizobium-legume symbiosis. This chapter highlights various PGPR traits compatible with specific legume rhizobia and their phytostimulatory mechanisms contributing to augmentation in rhizobial growth and function for growth and yield enhancement of legumes under variable conditions.

A total of 11 rhizobial strains from five woody legumes (Acacia confusa, Dalbergia odorifera, Dalbergia fusca, Erythrophleum fordii and Pterocarpus macarocarpus) in Southern China were identified, using 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA) of three housekeeping genes (recA, glnII and dnaK). Among the 11 isolated rhizobia, seven were classified as Agrobacterium tumefaciens,Bradyrhizobium elkanii, Ensifer adhaerens and Rhizobium multihospitium and four were within the genus Rhizobium that might be novel species when considering their clustering independently in topology tree and relatively low recA gene sequence similarities (<94%) to the recognized species. In each gene phylogeny, the 16S rRNA gene was found to be incongruent with each house-keeping gene, indicated that this gene should not be used as a single marker in rhizobial taxonomy. In addition, more robust topology trees were produced through the analysis of a concatenation of three housekeeping genes than through each housekeeping gene and 16S rRNA gene trees, with high bootstrap support (≥55%). Overall, our results support that the MLSA has higher discrimination potential in rhizobial taxonomy than the 16S rRNA gene and is suitable to estimate phylogenetic relationships among rhizobia species. Key words: Multilocus sequence analysis, 16S rRNA, rhizobia and woody legume.

In some leguminous plants, associations with nitrogen-fixing microorganisms allow their nutrition with nitrogen (N) from the atmosphere. This process is known as Biological Nitrogen Fixation (BNF), where through nitrogenase enzymes, N2 is converted to an available form. This process can replace in part, or in total, nitrogen fertilizers. Cowpea bean is a legume species that is recognized for its high capacity to carry out BNF. In the last decades, studies have encouraged small farmers from north and northeast Brazil to use inoculants with rhizobia species since the results of researches have demonstrated that inoculation is an interesting strategy to improve cowpea production. Considering the specific function of molybdenum (Mo) in the N assimilation, different doses of Mo were tested in this study in order to find doses that could improve and enhance BNF. Therefore, this study aimed to compare nitrogen fertilization and BNF in the N assimilation by plants with different Mo doses. Inoculation was performed with the strains UFLA 03-84 and INPA 03-11B. Doses of Mo were applied in seeds and each pot contained five seeds. Thirty-five days after germination, the plants were analyzed for shoot dry matter and fresh matter, N contents and accumulation, as well as the Soil-Plant Analysis Development (SPAD) Index and nodulation in inoculated plants. The different doses of Mo and also the nodulation treatments did not show significant differences in the contents of N. Plants with N fertilization had significant higher shoot dry matter and root dry matter production, in addition to higher N foliar contents and N accumulation. Therefore, BNF was not as efficient as nitrogen fertilization in the evaluated experimental conditions using cowpea beans.

Multilocus sequence analysis of Brazilian Rhizobium microsymbionts of common bean ( Phaseolus vulgaris L.) reveals unexpected taxonomic diversity

Pseudomonas syringae pv. porri causes bacterial leaf spot and blight of leek (Allium porrum) and is in wet crop seasons responsible for substantial losses. The local diversity within this pathogen in Flanders, Belgium, was investigated to obtain insights into its epidemiology. Therefore, symptomatic leek leaves were collected from 112 fields and bacteria were isolated. An oxidase negative, HR positive, fluorescent Pseudomonas was consistently recovered from the diseased tissues. Isolates were identified as P. syringae pv. porri by rpoD gene sequencing and by confirmation of pathogenicity in leek. Genomic profiles generated with BOX-PCR subdivided them into two groups, with one group containing 5 of the 37 analyzed strains. Those five isolates were all obtained in 2012 and the plant origins indicated seed transmitted infection. Draft genome sequences were produced for a P. syringae pv. porri strain from each BOX group and sequences of seven housekeeping genes were extracted for multi locus sequence analysis (MLSA). This resulted in the clustering of both P. syringae pv. porri strains with the P. syringae pv. oryzae strain 1_6 as did the whole genome sequence comparisons by ANI analysis. The P. syringae pv. porri isolates, designated LMG 28495 and LMG 28496, differed in a type III effector gene, HrpW, and in the number of mobile elements in the genome. Overall, the data demonstrate that two P. syringae pv. porri variants are present in symptomatic leek in Flanders which can be discriminated and possibly traced using a genomic profiling method such as BOX-PCR . Furthermore, the draft genome sequences of both strains will facilitate the development of sensitive and specific methods for early detection.

The tree Senegalia (Acacia) senegal is well adapted to soils of arid regions having a vital role in reforestation and increasing soil fertility through biological nitrogen fixation in addition to its utility as edible seeds and gum. Fifty-nine root nodule bacterial (RNB) strains were isolated; characterized at phenotypic and genotypic level; and host range was studied. The Senegalia-RNB strains were able to grow up to 3% NaCl and on a wide range of pH (5–11) with optimum growth at alkaline pH showing their adaptation to alkaline desert soil. The carbon utilization pattern of Senegalia-RNB strains was significantly different from existing closely related type strains of Ensifer. The PCR-RFLP (ARDRA) as well as RAPD patterns revealed considerable genetic diversity among the strains forming distinct genetic groups. The 16S rRNA phylogeny of selected six (AS11, AS18, AS27, AS34, AS50 and AS53) strains showed that they belong to fast growing species of Ensifer with few being similar to the previously described Ensifer strains from Vachellia jacquemontii and Vachellia leucophloea from Thar Desert of India. The symbiotic (nifH and nodA) gene phylogeny showed incongruence with that of 16S rRNA gene (species) phylogeny suggesting HGT incidences under hot-arid and alkaline environment. The Senegalia-Ensifer strains possessing novel symbiotic genes could effectively cross-nodulate species of Vachellia (V. jacquemontii, V. leucophloea and V. nilotica) but failed to nodulate the crop Vigna radiata. Multi locus sequence analysis (MLSA) is required to determine novelty of these Ensifer strains and more cross-inoculation studies will help in understanding their potential use as inoculums in nursery practices for reforestation program in arid and semi-arid regions.