Biological nitrogen fixation by Rhizobium-legume symbioses represents an environmentally friendly and inexpensive alternative to the use of chemical nitrogen fertilizers in legume crops. Rhizobial inoculants, applied frequently as biofertilizers, play an important role in sustainable agriculture. However, inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields. Strains with excellent performance under controlled conditions are typically selected as inoculants, but the rates of nodule occupancy compared to native strains are rarely investigated. Lack of persistence in the field after agricultural cycles, usually due to the transfer of symbiotic genes from the inoculant strain to naturalized populations, also limits the suitability of commercial inoculants. When rhizobial inoculants are based on native strains with a high nitrogen fixation ability, they often have superior performance in the field due to their genetic adaptations to the local environment. Therefore, knowledge from laboratory studies assessing competition and understanding how diverse strains of rhizobia behave, together with assays done under field conditions, may allow us to exploit the effectiveness of native populations selected as elite strains and to breed specific host cultivar-rhizobial strain combinations. Here, we review current knowledge at the molecular level on competition for nodulation and the advances in molecular tools for assessing competitiveness. We then describe ongoing approaches for inoculant development based on native strains and emphasize future perspectives and applications using a multidisciplinary approach to ensure optimal performance of both symbiotic partners.

Nodulation regulation: Legumes convert atmospheric nitrogen into biologically useful ammonium with the help of symbiotic bacteria housed in root nodules. Much of nodule development is controlled by the transcription factor NODULE INCEPTION (NIN). Feng et al . show that NIN is proteolytically processed to release a fragment that regulates the later stages of nodulation when the nodules acquire nitrogen-fixing capability. In related work, Jiang et al . identified members of the NIN-like protein (NLP) transcription factor family as being regulators of leghemoglobin expression acting through an unusual promotor motif shared across legumes. —PJH

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N ₂ ) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N ₂ fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N ₂ fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different Rhizobium leguminosarum strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple Rhizobium -legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.

Bacteria navigate their way often as individual cells through their chemical and biological environment in aqueous medium or across solid surfaces. They swim when starved or in response to physical and chemical stimuli. Flagella-driven chemotaxis in bacteria has emerged as a paradigm for both signal transduction and cellular decision-making. By altering motility, bacteria swim toward nutrient-rich environments, movement modulated by their chemotaxis systems with the addition of pili for surface movement. The numbers and types of chemoreceptors reflect the bacterial niche and lifestyle, with those adapted to complex environments having diverse metabolic capabilities, encoding far more chemoreceptors in their genomes. The Alpha-proteobacteria typify the latter case, with soil bacteria such as rhizobia, endosymbionts of legume plants, where motility and chemotaxis are essential for competitive symbiosis initiation, among other processes. This review describes the current knowledge of motility and chemotaxis in six model soil bacteria: Sinorhizobium meliloti, Agrobacterium fabacearum, Rhizobium leguminosarum, Azorhizobium caulinodans, Azospirillum brasilense, and Bradyrhizobium diazoefficiens. Although motility and chemotaxis systems have a conserved core, rhizobia possess several modifications that optimize their movements in soil and root surface environments. The soil provides a unique challenge for microbial mobility, since water pathways through particles are not always continuous, especially in drier conditions. The effectiveness of symbiont inoculants in a field context relies on their mobility and dispersal through the soil, often assisted by water percolation or macroorganism movement or networks. Thus, this review summarizes the factors that make it essential to consider and test rhizobial motility and chemotaxis for any potential inoculant.

Common bean is a poor symbiotic N-fixer, with a low response to inoculation owing to its promiscuous nodulation with competitive but inefficient resident rhizobia. Consequently, farmers prefer to fertilize them rather than rely on their capacity for Biological Nitrogen Fixation (BNF). However, when rhizobial inoculants are based on autochthonous strains, they often have superior BNF performance in the field due to their genetic adaptations to the local environment. Nevertheless, there is scant information at the genomic level explaining their superiority or on how their genomes may influence the inoculant performance. This information is especially important in technologically advanced agri-systems like Europe, where environmental concerns and increasingly stringent fertilizer regulations are encouraging a return to the use of rhizobial inoculants, but based upon strains that have been thoroughly characterized in terms of their symbiotic performance and their genetics. The aim of this study was to design an inoculant formulation based on a superior autochthonous strain, Rhizobium leguminosarum bv. phaseoli LCS0306, to assess its performance in the field, and to determine the genomic features contributing to the high effectiveness of its symbiosis with common bean. Plants inoculated with the autochthonous strain LCS0306 fixed significantly more nitrogen than those with the allochthonous strains R. phaseoli ATCC 14482^T and R. etli CFN42^T, and had grain yield similar to the nitrogen-fertilized controls. Inoculation with LCS0306 was particularly efficacious when formulated with a carrier based upon a mixture of perlite and biochar. Whole genome comparisons revealed no differences in the classical symbiotic genes of strain LCS0306 within the symbiovar phaseoli. However, its symbiotic superior performance might be due to its genomic versatility, as it harbors a large assortment of genes contributing to fitness and competitiveness. It is concluded that inoculation with elite rhizobia formulated with perlite-biochar carriers might constitute a step-change in the sustainable cultivation of common bean in Spanish soils.