Medicago, a genus in the Leguminosae or Fabaceae family, includes the most globally significant forage crops, notably alfalfa (Medicago sativa). Its close diploid relative Medicago truncatula serves as an exemplary model plant for investigating legume growth and development, as well as symbiosis with rhizobia. Over the past decade, advances in Medicago genomics have significantly deepened our understanding of the molecular regulatory mechanisms that underlie various traits. In this review, we comprehensively summarize research progress on Medicago genomics, growth and development (including compound leaf development, shoot branching, flowering time regulation, inflorescence development, floral organ development, and seed dormancy), resistance to abiotic and biotic stresses, and symbiotic nitrogen fixation with rhizobia, as well as molecular breeding. We propose avenues for molecular biology research on Medicago in the coming decade, highlighting those areas that have yet to be investigated or that remain ambiguous.

Abstract. Field trials of rhizobial inoculants require simple and reliable methods for identifying the strains used to determine which strain has formed a nitrogen-fixing nodule. This task arises when testing the competitiveness of inoculant strains against local rhizobia strains, to track the fate of inoculant strains over long periods after the introduction of strains, and finally, such methods may be in demand when protecting the rights of strain owners and developers. The essence of the proposed identification method is to search for strain-specific DNA regions that are absent in other genomes of the same species and to construct a primer system for multiplex PCR, allowing simple, reliable and rapid identification of the strain. The advantages of this approach over other identification methods are, firstly, high reproducibility, and secondly, that the method is based on the detection of structural variants, the contribution of which to the evolution of rhizobia genomes is very high, while most genomic fingerprinting methods (AFLP, RAPD, REP, ERIC, etc.) are based on the detection of nucleotide polymorphisms in short fragments of the genome, but miss many events associated with genomic rearrangements and horizontal gene transfer. The use of the proposed method can also serve to monitor the evolutionary dynamics of rhizobial inoculant strains, especially in unique fragments of the genome, which is very important for R. leguminosarum, where the proportion of unique sequences is much higher than in other rhizobia.

Soybean (Glycine max) nitrogen fixation is inhibited by nitrate, which has been linked to a reduction in carbon allocation and metabolism within nodules. However, the underlying mechanisms remain unclear. In this study, we tested the hypothesis that the nitrate-induced suppression of nitrogen fixation is mediated through altered sucrose allocation and catabolism in nodules. Using unilaterally nodulated dual-root soybean plants in sand-based systems, we applied 200 mg·L−1 nitrate exclusively to the non-nodulated roots for 14 days. Nitrate supply enhanced the proportion of dry weight in leaves but reduced it in nodules at 3, 7, and 14 days. Similarly, nodule dry weight, nodule number, acetylene reduction activity (ARA), and specific nodule activity (SNA) all declined significantly during the same intervals. Notably, sucrose content in the nodules decreased significantly by 20.4% after 3 days but recovered at 7 and 14 days. In contrast, sucrose synthase (SuSy) cleavage activity and malate content in nodules decreased significantly following nitrate treatment, with reductions of 27.8% and 30.7% observed at 7 days, and further decreased to 38.5% and 39.2% at 14 days, respectively. These results suggest that transient sucrose scarcity may drive the initial decline in nitrogen fixation capacity, while restricted sucrose catabolism and decreased malate levels may be a consequence rather than a cause.

Plants small secreted peptides (SSPs) regulate root development, immunity and symbiotic relationships in herbaceous plants. These processes are equally important for establishing functional ectomycorrhizal associations in trees. While fungal SSPs involved in ectomycorrhizal establishment have been identified, the role of plant SSPs remains largely unexplored. Although thousands of SSPs were predicted in plant genomes, their small size and high sequence divergence hinder their accurate automated annotations. To address this issue, we combined de novo gene prediction with family-specific motif search to identify 1,053 SSPs from 21 symbiosis-related families in the genomes of two ectomycorrhizal tree species: Populus trichocarpa and Quercus robur. Nearly half of these SSPs are transcriptionally regulated during ectomycorrhizal symbiosis with various fungal partners. Functional assays of selected Populus CLE peptides, a SSP family known to repress arbuscular mycorrhizal and rhizobial symbioses, revealed that five enhanced ectomycorrhizal roots formation. Unlike CLEs involved in the autoregulation of arbuscular mycorrhizal and rhizobial symbioses, these peptides belonged to clades associated with meristem activity. Their activity did not increase lateral root number but inhibited adventitious root growth, suggesting their role in promoting ectomycorrhizal root organogenesis. These findings demonstrate that CLE peptides promote, rather than repress, ectomycorrhizal symbiosis. This functional divergence from their roles in other symbioses suggests that poplar co-opted a distinct set of SSPs to support ectomycorrhizal development. Our results expand the understanding of host tree contributions to ectomycorrhizal development and identify a set of candidate SSPs for future functional studies, thereby highlighting a new layer of regulation in tree-fungal mutualism.

Root hairs anchor the plant in the soil, facilitating nutrient assimilation, water absorption, and interaction of plants with their environment. In legumes, they play a key role in the early infection of rhizobia. This review aimed to summarize the recent progress about the nodulation factor receptors on the root hair surface. It also discussed the importance of downstream signaling pathways of nodulation factor receptors and highlighted Rho of plants signaling pathway that controls infection thread polar growth and nodulation.

In both alkaline and acidic soils, phosphorus, an essential element for plants, reacts with the soil solution and becomes immobile and unavailable to plants. The study aims to use arbuscular mycorrhizal fungi (AMF) as an alternative method for solubilising phosphorus and to evaluate the effect of mycorrhization on the response of three cultivars of Medicago sativa L. to phosphorus deficiency. For this purpose, plants were used in two regimens: with or without mycorrhizal inoculum and with or without phosphorus amendments. The effects of mycorrhizae on the mineral nutrition, growth parameters of plants, and the rhizosphere's chemical properties were evaluated. The results showed that phosphorus deficiency negatively affected growth parameters. For all cultivars, the lowest values were recorded in mycorrhized-free plants regardless of phosphorus supply. In contrast, the mycorrhizal plants that got phosphorus intakes recorded the most significant nutritional metrics and growth parameters values except for root length, where the highest values were recorded in uninoculated control plants that did not receive phosphoric amendment. The shortest roots were recorded in mycorrhized plants growing on P-amended soil. Phosphorus supplementation with mycorrhizal fungi inoculation also improved soil fertility, increasing the amount of available phosphorus and adjusting the pH values.

For agriculture, the symbiosis carried out by rhizobia with legumes stands out as crucial for both economic and environmental reasons. In this process, the bacteria colonize the roots of the plants, inducing the formation of plant organs called nodules. Within these structures, rhizobia fix the environmental nitrogen into ammonia reducing the demand for this essential element required for plant growth. Various bacterial secretion systems (TXSS, Type X Secretion System) are involved in the establishment of this symbiosis, with the T3SS being the most extensively studied. The T6SS is a nanoweapon present in 25% of gram-negative bacteria, commonly used against other gram-negative bacteria, though some of them use it to manipulate eukaryotic cells.

Interestingly, although T6SS is widely distributed among rhizobia, whether it has a specific role in symbiosis with legumes remains elusive. Sinorhizobium fredii USDA257 is a fast-growing rhizobium with the capacity to nodulate a great variety of legume plants. This strain harbors a single T6SS cluster, containing the genes encoding all the structural components of the system and two genes encoding potential effectors that could target the cell wall of the plants and/or be acting as a toxin/antitoxin system. We have demonstrated that this system is active and can be induced in poor culture media. In addition, we have seen by fluorescence microscopy that the T6SS is active in nodules. Competition assays between USDA257 and different preys have shown that USDA257 cannot kill any of them using its T6SS under tested conditions. By constract, nodulation assays demonstrated that USDA257 utilizes this protein secretion system to enhance nodulation and competitiveness with its host Glycine max cv Pekin.

The complex and mutual interactions between plants and their associated microbiota are key for plant survival and fitness. From the myriad of microbes that exist in the soil, plants dynamically engineer their surrounding microbiome in response to varying environmental and nutrient conditions. The notion that the rhizosphere bacterial and fungal community acts in harmony with plants is widely acknowledged, yet little is known about how these microorganisms interact with each other and their host plants. Here, we explored the interaction of two well-studied plant beneficial endophytes, Enterobacter sp. SA187 and the fungus Serendipita indica. We show that these microbes show inhibitory growth in vitro but act in a mutually positive manner in the presence of Arabidopsis as a plant host. Although both microbes can promote plant salinity tolerance, plant resilience is enhanced in the ternary interaction, revealing that the host plant has the ability to positively orchestrate the interactions between microbes to everyone's benefit. In conclusion, this study advances our understanding of plant-microbiome interaction beyond individual plant-microbe relationships, unveiling a new layer of complexity in how plants manage microbial communities for optimal growth and stress resistance.

Through symbiosis with nitrogen-fixing bacteria, cultivated legumes provide themselves and subsequent crops with nitrogen, making genetic improvements of symbiotic efficiency particularly attractive. Here, we identify the symbiosis-specific GBP1 gene which negatively regulates nitrogen fixation by attenuating bacterial nitrogenase activity in Medicago truncatula nodules. GBP1 inactivation increases nitrogen fixation without affecting nodule development and numbers, providing inroads for engineering legumes with increased productivity for sustainable nitrogen provision.

The plant metabolome wields a strong influence on its associated microbiome, with feedback from the microbiome in turn influencing plant resilience and productivity. The root metabolome represents a key element of the chemical signaling that influences microbiome recruitment to plants. We examined how the root metabolome differs between high- and low-performing genotypes of a drought-resilient cereal, Sorghum bicolor L. Moench, and how these differences relate to the root microbiome in field-grown plants. Overall, lower-performing genotypes exhibited a distinct root metabolome from higher-performing genotypes. In particular, lower-performing genotypes exhibited an accumulation of flavonoids, a class of secondary metabolites involved in plant defense and stress response. Network analyses revealed microbes whose abundance covaried with flavonoid content and suggested that higher levels of flavonoids may hinder root colonization by specific microbes. Higher-performing genotypes further exhibited more discriminating metabolites with distinct levels between watering treatments as compared to lower-performing genotypes, pointing to the potential of higher performing genotypes to better cope with stress by potentially modulating their microbiomes via root chemistry. We discuss how insights into crop performance from the lens of metabolomics can improve our knowledge of how crops may more effectively recruit beneficial plant microbiomes.

Nodulation represents a crucial but energy-intensive strategy for legumes to survive in nutrient-poor soils. A recent study by Ren et al (2025)highlights the significance of micronutrients, particularly iron (Fe), in regulating symbiotic nitrogen fixation, which ensures that nodulation occurs only under favourable environmental conditions.

Legumes are vital for sustainable agriculture due to their unique ability to fix atmospheric nitrogen through symbiosis with rhizobia. Recent research has highlighted the significant role of non-rhizobial bacteria (NRB) within root nodules in enhancing this symbiotic relationship, particularly under stress conditions. These NRB exhibit plant growth-promoting (PGP) metabolites by modulating phytohormones and enhancing nutrient availability, thereby improving nodule development and function. Bacteria produce essential hormones, such as auxin (indole-3-acetic acid), cytokinins, gibberellic acids abscisic acid, jasmonic acid, and salicylic acid, and enzymes like 1-aminocyclopropane-1-carboxylate deaminase, which mitigate ethylene's inhibitory effects on nodulation. Furthermore, NRB contribute to nutrient cycling by solubilizing minerals like phosphate, potassium, silicate, zinc, and iron, essential for effective nitrogen fixation. The co-inoculation of legumes with both rhizobia and NRB with multiple PGP metabolites has shown synergistic effects on plant growth, yield, and resilience against environmental stresses. This review emphasizes the need to further explore the diversity and functional roles of nodule-associated non-rhizobial endophytes, aiming to optimize legume productivity through improved nutrient and hormonal management. Understanding these interactions is crucial for developing sustainable agricultural practices that enhance the efficiency of legume-rhizobia symbiosis, ultimately contributing to food security and ecosystem health.

This study evaluated the impact of integrated nutrient management practices and arbuscular mycorrhizal fungi (AMF) on sorghum yield, nutrient uptake, and soil fertility in swell-shrink soils. Eight treatments, including recommended dose of fertilizer (RDF) and various organic amendments (FYM, vermicompost) with and without AMF, were tested in a randomized block design with three replications during the Kharif season 2022-23. Results indicated that the application of 75% RDF combined with vermicompost (2.5 t ha-1) and AMF (5 kg ha-1) significantly enhanced sorghum yield and total nutrient uptake. This particular treatment (T7) demonstrated a marked improvement in yield, achieving a 116.5% increase in grain yield and a 120.2% increase in fodder yield over the control. These results indicate that combining reduced fertilizer inputs with organic amendments and AMF can effectively enhance sorghum productivity while potentially reducing dependence on chemical fertilizers. Further analysis revealed strong, positive correlations between yield and the uptake of essential nutrients, including nitrogen (N), phosphorus (P), potassium (K), and sulfur (S). This indicates that treatments combining AMF and organic amendments not only support higher crop productivity but also optimize nutrient availability in the soil. Enhanced nutrient uptake was particularly evident with the T7 treatment, where AMF likely played a pivotal role in improving phosphorus availability by secreting phosphatase enzymes, supporting both nutrient dynamics and overall crop health. This integrated approach demonstrated its potential for improving soil health and agricultural productivity in swell-shrink soil environments.