Legume-nodulating Bacteria Research Articles

Rapid Identification of Rhizobia Nodulating Soybean by a High-Resolution Melting Analysis

Soybean [Glycine max (L.) Merr.] is one of the most important and oldest crops. Due to its ability to form symbiotic interactions with nitrogen-fixing bacteria, it is a valuable source of nitrogen for agriculture and proteins for humans and livestock. In Europe, for instance, in Poland, the soybean cultivation area is still not large but is gradually increasing due to climate change. The lack of indigenous soybean microsymbionts in Polish soils forces the application of commercial strains to establish effective symbioses. Fast and reliable identification methods are needed to study the persistence, competitiveness, and dispersal of bradyrhizobia introduced as inocula. Our study aimed to apply real-time PCR coupled with high-resolution melting curve (HRM) analysis to detect and differentiate bacterial strains occupying soybean nodules. HRM-PCR was performed on crude extracts from nodules using primers specific for recA, a highly conserved nonsymbiotic gene. By comparing them with the reference strains, we were able to identify and assign Bradyrhiobium strains that had been introduced into field locations in Poland. In conclusion, HRM analysis was proven to be a fast and accurate method for identifying soybean microsymbionts and might be successfully used for identifying other legume-nodulating bacteria.

Isolation of Rhizobia From the Nodules of Bambara Groundnuts for Inoculant Production

Rhizobia symbiotic interactions with legumes fix atmospheric nitrogen into the soil, which is essential in amending the characteristically low-nitrogen soils in most farming communities in northern Ghana. A high potential for improvement of Bambara groundnuts production in low-nitrogen soils is by the exploitation of colonization of the plant roots with rhizobial inoculation. This experiment sought to isolate Legume Nodulating Bacteria (LNB) obtained from root nodules of Bambara groundnut (Vigna subterranea) plants and to identify effective strains for improved production of the crop. Roots nodules of Bambara plants used in this study were obtained from preserved plants and the isolates were authenticated for their symbiotic effectiveness under screen house conditions. Nodulation of the isolates was examined in plastic pots containing sterile river sand and test crop (Bambara seeds). The experiment included reference strains, a positive nitrogen control and an un-inoculated control. The results were obtained after two months of data collection. The difference in results was explained via nodulation capacity. Out of the two isolates obtained, 2CL showed a high nodulation capability, rating it as highly effective. The outcome of this study provides stakeholders with the prospect for the use of effective isolates as inoculants to improve Bambara groundnut yield in general and in northern Ghana in particular.

Members of Ensifer and Rhizobium genera are new bacterial endosymbionts nodulating Pisum sativum (L.).

A total of 84 Pisum sativum legume nodulating bacteria (LNB) were isolated from seven geographical sites from southern Tunisia. Phylogenetic analyses based on partial sequences of 16S rRNA gene and the housekeeping genes glnII, and recA grouped strains into six clusters, four of which belonged to the genus Rhizobium and two to the Ensifer genus. Among Rhizobium clusters, 41 strains were affiliated to Rhizobium leguminosarum, two strains to R. pisi, two strains to R. etli, and interestingly two strains belonged to previously undescribed Rhizobium species. The remaining two strains were closely related to Ensifer medicae (two strains) and Ensifer meliloti (two strains). A symbiotic nodC gene-based phylogeny and host specificity test showed that all Rhizobium strains nodulating pea belonged to the symbiovar viciae, whereas the Ensifer strains were associated with the symbiovar meliloti never described to date. All strains under investigation differed in the number of induced root nodules and the effectiveness of atmospheric nitrogen fixation. The R. leguminosarum PsZA23, R. leguminosarum PsGBL42, and E. medicae PsTA22a, forming the most effective symbiosis with the plant host, are potential candidates for inoculation programs.

Diversity of Indigenous Symbiotic Nitrogen Fixing Bacteria from Blackgram [Vigna mungo (L.) Hepper] Cultivated in Rice Fallows

Background: Legume nodulating bacteria (LNB), also known as rhizobia produce root nodules on leguminous plants where in nitrogen fixation takes place. In agriculture, this symbiotic relationship is significant as it provides nitrogenous fertilizers to the leguminous crops. Traditionally rhizobia were exclusively members of the Rhizobiaceae family in the Alphaproteobacteria class of bacteria. Several reports revealed that large number of diazotrophs have the ability to nodulate and fix atmospheric N2 in legumes in addition to rhizobia which are outside the Alphaproteobacteria class. Relatively less information is available on the genetic diversity of indigenous rhizobia nodulating blackgram cultivated in rice fallows. Hence the present study was carried out to know the diversity of symbiotic native nitrogen fixing bacteria isolated from the root nodules of blackgram grown in rice fallows using partial 16S rRNA sequencing.Methods: Nitrogen fixing microsymbionts (Rhizobium strains) from root nodules of blackgram were isolated on yeast extract mannitol agar (YEMA) medium. The pure cultures were maintained at 28±2°C for 48 h. Identification of Rhizobium isolates was done by using various tests and Morphological characterization of isolates was also done by using standard microbiological methods. The PCR amplification and sequencing of 16S rRNA gene of isolates were carried out to identify the isolates.Result: In the present study four strains of Rhizobium designated as VM-2, VM-8, VM-9 and VM-15 were isolated. Morphologically colonies of all strains are round, transparent, entire, convex and smooth. They are Gram-ve and rod shaped. Mucilage production is noticed in VM-2, VM-8 and VM-9. The 16S rRNA gene sequences revealed that the strain VM-2 showed a close relation with Rhizobium, VM-8 and VM-9 with Bradyrhizobium where as VM-15 with Achromobater. It was concluded that the symbiotic nitrogen fixing bacteria isolated from the root nodules of blackgram cultivated in rice fallows exhibited high genetic diversity.

Methods for Isolation and Characterization of Nitrogen-Fixing Legume-Nodulating Bacteria.

Symbiotic nitrogen fixation (SNF) is a characteristic feature of nodulating legumes. The wild legumes are comparatively less explored for their SNF ability; hence, it is essential to study nodulation and identify the microsymbiont diversity associated with them. This chapter aims to describe the methodology for nodule hunting; trapping, isolation, and characterization of root nodule bacteria (RNB) at phenotypic, genotypic, and symbiotic levels. The documentation of nodulating native legume species and the rhizobial diversity associated with them in various parts of world has gained attention as this symbiotic association provides fixed nitrogen, improves productivity of plants in an ecofriendly manner. Before field-based applications the symbiotic bacteria need to be assessed for their N fixing ability as well as characterized at molecular level. The phylogeny based on symbiosis-essential genes supplemented with the host-range studies helps in better understanding of the symbiotaxonomy of rhizobia. More efficient symbiotic couples need to be screened by cross-nodulation studies for their application in agricultural practices.

Recent changes to the classification of symbiotic, nitrogen-fixing, legume-associating bacteria: a review

The Rhizobia are collectively comprised of gram negative soil bacteria that have the ability to form symbiotic nitrogen-fixing root and/or stem nodules in association with leguminous plants. The taxonomy of these bacteria is continually in a state of flux, in large part due to rapid development of refined molecular biology techniques. The isolation and characterization of new, and often different, legumes-nodulating bacteria on a variety of plant hosts has resulted in the naming of many new rhizobial species. Here we update the taxonomy of the legume-nodulating bacteria and describe newly identified rhizobia capable of nodulating edible legumes and legume trees. In 1990, there was only one bacterial species that was known to nodulate common bean worldwide (Rhizobium leguminosarum sv. phaseoli), one species that nodulated faba bean (Rhizobium leguminosarum sv. viciae), and two species that nodulated soybean (Bradyrhizobium japonicum and Rhizobium fredii). Today, nearly 14, 11, 6, 5, 5, 4, 3 and 2 species have been defined that are capable of nodulating common bean, soybean, cowpea, chickpea, peanut, lentils, faba bean and pea, respectively. The recent use of whole genome based taxonomy (genomotaxonomy) will surely change how we define this important group of bacteria. The identification of several rhizobial species that are able to nodulate and fix nitrogen with edible legumes may enhance the production of these crops and can compensate for worldwide deficiencies in human nutritional needs in the future.

The Diversity of Legume-Nodulating Bacteria from Several Agroecosystems in Sumberjaya, Lampung

Bacteria that capable of forming root nodules on legumes are known as Rhizobia. They have also known as Legume- Nodulating Bacteria (LNB). They can fi x nitrogen from the atmosphere. Diversity of Legume-Nodulating Bacteria is affected by biotic factors (such as their genetic factors, plants, and competition with the other soil microbes) and abiotic factors (such as land use, soil’s temperature, pH, chemistry and soil’s properties). The aim of this experiment is to know the diversity of eleven Legume- Nodulating Bacteria based on their phenotypic and genotypic characters. The eleven LNB used in this experiments were isolated from several agroecosystems in Sumberjaya, Lampung. The analysis of these LNB diversity were carried out by characterizing both phenotypic and genotypic properties. The diversity analysis showed that the eleven LNB isolates had high diversity, based on nodule formation, and classifi ed into two groups of cross inoculation group. Key words: Rhizobia, phenotypic diversity, genotypic diversity

Legume Nodulating Bacterium, Achromobacter xylosoxidans Found in Tropical Shrub Agroecosystem, Sumatera, Indonesia

Legume nodulating bacteria (LNB), known also as rhizobia, are soil bacteria, which are able to form rootnodules and fi x nitrogen in the leguminous plants. The LNB availability in the soil depends on the type ofagroecosystem, where plant grows. In this study, we isolated LNB from the shrub agroecosystem in Sumatera,Indonesia, and obtained four selected bacterial strains. Among them, the isolate UGM48a formed root nodulein Macroptilium atropurpureum and showed highest number of nitrogenase activity. UGM48a also contains nifHand nodA genes. An analysis of the PCR-amplifi ed 16S rDNA and BLASTn analysis showed that UGM48adisplayed 96% similarity with Achromobacter xylosoxidans. In addition, UGM48a were successfully nodulatedGlycine max (L.) merr var. wilis. This is the fi rst report detecting A. xylosoxidans as nodule-forming species forGlycine max possesing the positive copy of nodA gene. Keywords : Legume Nodulating Bacteria, shrub agroecosystem, Achromobacter xylosoxidans, nodA, Glycine max

Abundance of Legume Nodulating Bacteria in Soils of Diverse Land Use Systems in Cameroon

Improving biological nitrogen fixation through legume nodulating bacteria (LNB) inoculation requires knowledge on the abundance and effectiveness of indigenous population in the ferralsols. The Most Probable Number method (MPN) was used to determine the naturally occurring LNB population in 64 representative soils of four sites in the humid forest zone of Cameroon. Nodulation Macroptilium atropurpureum was examined under tubes experiment in four location sites: Bertoua, Ebolowa, Bokito and Yaounde, and within each of the locations, in four land use systems (LUSs) of different levels of disturbance: mixed farm; fallows; cocoa plantation and forest. The LNB population size varied from 0.78 to 5.25 log units of soil depending on the land use. The most disturbed LUSs (farms and fallows) exhibiting the highest number of LNB on average. Undisturbed ones (cocoa plantation and forest) showed the lowest number of LNB. The percentage of the need for inoculation was more frequent in the forests (94%) and plantations (75 %). It is low in the in the fallows (25%) and the farms (44 %). In total 60 % of the soils analysed need inoculation. These results are important for future investigations on alternative inoculant strains for improving legume production in Cameroon.

High diversity of Bradyrhizobium strains isolated from several legume species and land uses in Brazilian tropical ecosystems

The genus Bradyrhizobium stands out among nitrogen-fixing legume-nodulating bacteria because it predominates among the efficient microsymbionts of forest, forage, and green manure legume species, as well as important species of grain legumes, such as soybean, cowpea, and peanut. Therefore, the diversity of Bradyrhizobium strains is a relevant resource from environmental and economic perspectives, and strains isolated from diverse legume species and land uses in Brazilian tropical ecosystems were assessed in this study. To accomplish this, sequences of four housekeeping genes (atpD, dnaK, gyrB, and recA) were individually analysed, with the first three also being considered using multilocus sequence analysis (MLSA). The sensitivity of the strains to different antibiotics, their tolerance to different levels of salinity, and their ability to nodulate soybean plants were also measured. The phylogenetic trees based on each individual gene, and on the concatenated housekeeping genes, revealed several strain clusters separated from any currently described species. The Bradyrhizobium strains studied were generally resistant to antibiotics. All strains were able to grow at salinity levels of up to 0.5% NaCl, whereas only strains UFLA03-142, UFLA03-143, UFLA03-145, and UFLA03-146 grew in the presence of 1% NaCl. Together, the results indicated that some of the strains studied were potential novel species, indicating that the various soils and ecosystems in Brazil may harbour an as yet unknown diversity of rhizobia.

Isolation and Screening of Indigenous Bambara Groundnut (Vigna Subterranea) Nodulating Bacteria for their Tolerance to Some Environmental Stresses

Environmental stresses are important limiting factors for crops production. The aim of this experiment is to isolate Legume Nodulating Bacteria (LNB) obtained from root nodules of bambara groundnut (Vigna subterranea L.) plants and evaluate their performance under some environmental constraints. Samples were collected in Cameroon from three location sites of the Humid-forest zone: Logbessou in the Littoral region; Mfoua in the South and Boga in the Centre region. Nodulation of bambara groundnut was examined in plastic bags and root nodules were collected from seedling. After their isolation, the bacteria were confirmed as LNB by re-nodulating Macroptilium atropurpureum. The morphological, cultural and phenotypic characteristics (utilization of carbon, tolerance to salt, pH, aluminium) of isolates were determined. The results obtained were analyzed statistically by ANOVA using the software SPSS analysis version 11.5. Duncan test was used to measure the difference among the means at a level of p<0.05. A collection of 18 isolates was obtained on Yeast Extract Mannitol Agar medium. Authentication experiments, confirmed that the majority of the isolates (66.67%) were LNB due to their ability to infect the host plant. Bambara groundnut isolates are different morphologically. Dendrogram of the phenotypic characteristics showed that, below the boundary level of 50% average similarity, isolates fell into at least three distinct groups. All isolates showed fast-growing capacity. Most isolates (66.67%) were able to grow in a medium with pH as low as and Al concentration of 50 µM (58.33 %). Some isolates (50%) showed weak growth capacity at 4% NaCl. The bambara groundnut isolates tested were able to use a broad range of carbohydrates as sole source of carbon. The isolates from the present study may be useful to increase the symbiotic nitrogen fixation in legume.

The use of local substrates in the production of legume nodulating bacteria inoculants for Sub- Saharan Africa: Preliminary results

Objective: The aim is to assess cheaper organic local products as an alternative for legume nodulating bacteria (LNB) inoculants production.Methodology and results: The study was done using two selected LNB strains (VUID1 and AHYP21), 16 local media (C and N sources), and groundnuts. The LNB biomass obtained from the local media was greater than the one obtained from the standard YEM media. VUID1 strain had a better yield in sucrose/soybean water, cow milk/yeast extract, cow milk/soybean water than the mannitol/yeast water media; while AHYP21 strain yielded better in the glycerol/yeast water, sucrose/yeast extract, cow milk/yeast extract and cow milk/soybean water media. Local carriers (Forest humus, horticultural compost, palm tree compost, and household organic compost) increased groundnuts growth and nitrogen uptake. These preliminary results showed the potential of local media and carriers in the production of inoculants for leguminous plants.Conclusions and application of findings: All the local substrates showed an important potential as local culture media and carriers for the production of LNB inoculants. Instead of mannitol and yeast extract, other products can be used for the culture of LNB. The production of LNB inoculants is easy, using organic local matter as carriers of inoculants. LNB inoculants will be more accessible for farmers.Key-words: Carrier, groundnuts, inoculants, legume nodulating bacteria, local media

Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria

Bacterial strains from Zn–Pb mine tailings were isolated by trapping with Anthyllis vulneraria, a legume-host suitable for mine substratum phytostabilisation. Sequence analysis of the 16S rRNA gene and three housekeeping genes (atpD, dnaK and recA) showed that they were related to those of the genus Aminobacter. DNA–DNA relatedness of representative isolates supported the placement of novel strains in Aminobacter as a new species. Phenotypic data emphasize their differentiation from the other related species of Aminobacter and Mesorhizobium. Aminobacter isolates exhibited nodA sequences tightly related with M. loti as the closest nodA relative. By contrast, their nodA sequences were highly divergent from those of M. metallidurans, another species associated with A. vulneraria that carries two complete copies of nodA. Therefore, the novel bacterial strains efficient on A. vulneraria represented the first occurrence of legume symbionts in the genus Aminobacter. They represent a new species for which the name Aminobacter anthyllidis sp. nov. is proposed (type strain STM4645T=LMG26462T=CFBP7437T).

Solubilisation of inorganic phosphates by inoculant strains from tropical legumes

Microbial solubilisation of low soluble inorganic phosphates is an important process contributing for the phosphorus available to plants in tropical soils. This study evaluates the ability of inoculant strains for tropical legumes to solubilise inorganic phosphates of low solubility that are found in tropical soils. Seven strains of Leguminosae nodulating bacteria (LNB) were compared with one another and with a non-nodulating positive control, Burkholderia cepacia (LMG 1222T). Four of the strains are used as inoculants for cowpeas (Vigna unguiculata) (Bradyrhizobium sp. UFLA 03-84; Bradyrhizobium elkani INPA 03-11B and Bradyrhizobium japonicum BR3267) or for common beans (Phaseolus vulgaris) (Rhizobium tropici CIAT 899T). Rhizobium etli UFLA 02-100 and Rhizobium leguminosarum 316C10a are also efficient nodulators of beans and Cupriavidus taiwanensis LMG 19424T nodulates on Mimosa pudica. Two experiments, with solid and liquid media, were performed to determine whether the strains were able to solubilise CaHPO4, Al(H2PO4)3 or FePO4.2H2O. On solid GELP medium none of the strains dissolved FePO4.2H2O, but LMG 1222, UFLA 03-84 and CIAT 899 solubilised CaHPO4 particularly well. These strains, along with LMG 19424 and BR 3267, were also able to increase the solubility of Al(H2PO4)3. In liquid GELP medium, LMG 1222 solubilised all phosphate sources, but no legume nodulating strain could increase the solubility of Al(H2PO4)3. The strains CIAT 899 and UFLA 02-100 were the only legume nodulating bacteria able to solubilise the other phosphate sources in liquid media, dissolving both CaHPO4 and FePO4.2H2O. There was a negative correlation between the pH of the culture medium and the concentration of soluble phosphate when the phosphorus source was CaHPO4 or FePO4.2H2O. The contribution of these strains to increasing the phosphorus nutrition of legumes and non-legume plant species should be investigated further by in vivo experiments.

A review of work on rhizosphere microbiota in Uganda

AbstractThe rhizosphere is broadly defined as the soil volume under the influence of plant roots, enriched with exudates, secretions and mucilaginous materials. It supports an active microbial population distinctive from the bulk soil. Subsequently, there are many interactions between plant‐microflora, plant‐macro‐fauna, microflora–macrofauna and soil‐plant‐microflora. Some relationships are positive and others parasitic. Significant among the beneficial ones are symbioses between leguminous plant species and legume nodule bacteria and mycorrhizal association occurring widely among plants. The former has been extensively studied and substantial gain made in harnessing biologically fixed nitrogen. Mycorrhizal investigations are still inadequate, especially production of commercial inoculants. Similarly, plant disease and their causative agents raise great concern hence a lot of work has been done. There other benefits that have not been well tapped like production of growth promoting substances and bio‐control agents. Despite the evident importance of the rhizosphere there are no specific citations of deliberate studies on its micro‐biota in Uganda. Therefore there is urgent need for research to determine, the abundance, diversity and distribution of rhizosphere biota, their impact on natural and agro‐ecosystem functioning. The studies should also document indigenous knowledge, impact of current policies and build physical and human capacities to handle current and future challenges.

Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes.

Legume-nodulating bacteria (rhizobia) usually produce N-acyl homoserine lactones, which regulate the induction of gene expression in a quorum-sensing (or population-density)-dependent manner. There is significant diversity in the types of quorum-sensing regulatory systems that are present in different rhizobia and no two independent isolates worked on in detail have the same complement of quorum-sensing genes. The genes regulated by quorum sensing appear to be rather diverse and many are associated with adaptive aspects of physiology that are probably important in the rhizosphere. It is evident that some aspects of rhizobial physiology related to the interaction between rhizobia and legumes are influenced by quorum sensing. However, it also appears that the legumes play an active role, both in terms of interfering with the rhizobial quorum-sensing systems and responding to the signalling molecules made by the bacteria. In this article, we review the diversity of quorum-sensing regulation in rhizobia and the potential role of legumes in influencing and responding to this signalling system.

Molecular diversity of rhizobia nodulating the invasive legume Cytisus scoparius in Australia

To contribute to the understanding of Cytisus scoparius success at invading and establishing itself in Australia. Root-nodule bacteria isolated from C. scoparius, growing on five different sites and originally introduced to Australia, were compared with isolates from indigenous plants growing in France and isolates from native legumes growing on the same Australian sites as C. scoparius. Small-subunit rDNA from 251 isolates were analysed by PCR-RFLP and representatives from different genospecies were selected for sequencing. Phylogenetic analyses revealed a great diversity of lineages belonging to Bradyrhizobium, with one genospecies being specific for Cytisus both in Australia and in France, Rhizobium and Mesorhizobium and one falling outside the described genera of legume-nodulating bacteria. Principal component analysis showed that the Cytisus Australian rhizobial communities are more similar to each other than to their co-occurring native partners. Early established rhizobial symbionts may have an increased probability to contribute inoculum for the development of further nodules. This is a first report comparing rhizobia nodulating C. scoparius in its native and exotic environments. Cytisus scoparius symbionts were identified outside the Bradyrhizobium genus and a new lineage of legume-nodulating bacteria was identified.

Nitrogen‐fixing nodules from rose wood legume trees (Dalbergia spp.) endemic to Madagascar host seven different genera belonging to α‐ and β‐Proteobacteria

Although legume biodiversity is concentrated in tropical regions, the majority of studies on legume nodulating bacteria (LNB) are focused on cultivated leguminous plants from temperate regions. However, recent works on tropical regions tend to indicate that the actual diversity of LNB is largely underestimated. In this study, we report the isolation and characterization of 68 nitrogen-fixing root nodule bacteria collected from eight endemic tree species of Dalbergia in Madagascar. The isolates were characterized by (i) restriction fragment length polymorphism (RFLP) analysis of 16S-IGS rDNA, (ii) 16S rDNA gene sequencing and (iii) nodulation tests. Results revealed a wide diversity of bacteria present in the nodules of Dalbergia. Among the 68 isolated bacteria, 65 belonged to Bradyrhizobium, Mesorhizobium, Rhizobium, Azorhizobium and Phyllobacterium from the alpha-class of Proteobacteria, and three isolates belonged to Burkholderia and Ralstonia from the beta-class of Proteobacteria. Our results also show for the first time that a strain belonging to the Burkholderia cepacia complex is able to induce efficient nodules on a legume plant.

Nodulation of Lupinus albus by Strains of Ochrobactrum lupini sp. nov

The nodulation of legumes has for more than a century been considered an exclusive capacity of a group of microorganisms commonly known as rhizobia and belonging to the alpha-Proteobacteria. However, in the last 3 years four nonrhizobial species, belonging to alpha and beta subclasses of the Proteobacteria, have been described as legume-nodulating bacteria. In the present study, two fast-growing strains, LUP21 and LUP23, were isolated from nodules of Lupinus honoratus. The phylogenetic analysis based on the 16S and 23S rRNA gene sequences showed that the isolates belong to the genus Ochrobactrum. The strains were able to reinfect Lupinus plants. A plasmid profile analysis showed the presence of three plasmids. The nodD and nifH genes were located on these plasmids, and their sequences were obtained. These sequences showed a close resemblance to the nodD and nifH genes of rhizobial species, suggesting that the nodD and nifH genes carried by strain LUP21T were acquired by horizontal gene transfer. A polyphasic study including phenotypic, chemotaxonomic, and molecular features of the strains isolated in this study showed that they belong to a new species of the genus Ochrobactrum for which we propose the name Ochrobactrum lupini sp. nov. Strain LUP21T (LMG 20667T) is the type strain.

Toward More Productive, Efficient, and Competitive Nitrogen-Fixing Symbiotic Bacteria

Abstract The prospects of developing strains of legume nodule bacteria that provide higher productivity of leguminous plants are described. The generic, biochemical, physiological, regulatory, and economic constraints that govern the ability of private and public efforts to construct better inoculants for legume nodulation are discussed. Success in constructing better inoculants requires a two-pronged approach. First, strains need to be improved in order to compete successfully with indigenous strains for root nodulation of legumes. Several loci have been identified to date that affect competitiveness for strain nodule occupancy. Usually mutations in these loci affect the ability of a strain to form nodules rapidly and efficiently. Other loci, such as those that confer antibiotic production, can be added to strains to enhance nodulation competitiveness when co-inoculated with antibiotic-sensitive strains. Second, the inoculum strains must be improved with respect to symbiotic nitrogen fixation. Efforts to...