To investigate the precise and efficient symbiosis between Sinorhizobium meliloti LL2 and different alfalfa varieties, we conducted experiments using eight alfalfa varieties along with the S. meliloti LL2. Our objective was to identify highly effective symbiotic combinations by analyzing differences in nodulation, nitrogen fixation, and biomass accumulation. The results revealed that Gannong NO.9 had higher values for single effective root nodule weight (1.30 mg) and the number of infected cells in root nodules (2795) compared to other varieties (p < 0.05). Additionally, Gannong NO.9 exhibited the highest nitrogenase activity (0.91 μmol·g⁻¹·h⁻¹), nitrogen fixation percentage (67.16%), and amount of nitrogen fixation (18.80 mg/pot). Moreover, there was a significant 26.50% increase in aboveground tissue nitrogen accumulation compared to the control check (CK) (p < 0.05). Furthermore, underground tissue showed excellent values for nitrogen accumulation (35.68 mg/plant) and crude protein content (17.75%) when compared with other treatments. The growth of plants was demonstrated by the combined impact of nodulation and nitrogen fixation. The distribution of biomass after nitrogen fixation was compared to the control group (p < 0.05) to investigate accumulation. The eight combinations of symbiotic nitrogen fixation (SNF) were classified into six distinct types based on their significantly different biomass growth rates compared to CK. ① Aboveground accumulation type: Gannong NO.9 (there was a 24.31% increase in aboveground dry weight); ② aboveground and underground accumulation type: Qingshui (the aboveground dry weight increased by 135.94%, while the underground dry weight grew by 35.26%); ③ aboveground accumulation, underground depletion type: Gannong NO.5 ( ); ④ zero-growth type (there was no significant difference in dry weights, both above and below ground, compared to CK): WL168HQ, WL319HQ and Longzhong; ⑤ aboveground and underground depletion type: WL298HQ (the aboveground dry weight decreased by 29.29%, while the underground dry weight decreased by 20.23%); ⑥ underground depletion type: Gannong NO.3 (the underground dry weight showed a decrease of 34.49%); no type with aboveground consumption and underground accumulation was found. The study clarified the optimal combination of LL2 and Gannong NO.9, finding that biomass accumulation after symbiotic nitrogen fixation is variety-dependent.

Evaluating key traits of male sterile mutant accessions in rhizomatous alfalfa (Medicago sativa L.) is crucial for selecting plants for artificial hybrid breeding of rhizomatous maternal lines. In this study, branch cuttings from four male sterile mutant accessions of ‘Qingshui’ alfalfa were used as experimental samples. We evaluated phenotypic traits, which included pollen viability and stigma receptivity, as well as nutritional quality, using difference analysis, correlation analysis, and principal component analysis. Prioritizing pollen viability and stigma receptivity, while considering phenotypic traits and nutritional quality as supplementary factors, allowed us to comprehensively evaluate 24 rhizomatous alfalfa individuals. This evaluation led to the identification of four male sterile mutant accessions with superior traits. The pollen from accession 4-4 was found to be partially fertile, whereas the remaining 23 alfalfa individuals were entirely male sterile. All 24 individuals exhibited stigma receptivity levels suitable for effective pollination. Principal component analysis revealed that among the assessed traits, the leaf–stem ratio contributed most significantly, followed by crude protein content, while neutral detergent fiber content had the least impact on overall quality. Additionally, the number of branches showed a strong positive correlation with individual plant yield (p < 0.01). No significant correlations were detected among plant height, stem diameter, forage grading index, crude protein, neutral detergent fiber, acid detergent fiber content, and yield. Overall, our comprehensive evaluation suggests that accessions 1-2, 2-2, 3-1, and 4-3 are most suitable for use as parental lines in artificial hybrid breeding.

Different rhizobial strains can lead to distinct symbiotic phenotypes in alfalfa, yet molecular differences at the mature nodule stage remain unclear. Here, we analyzed 21-day post-inoculation (dpi) nodules induced by strains WE2 and WWL2. We measured nitrogenase activity (acetylene reduction assay, ARA) and performed dual RNA-seq to compare gene expression in both the alfalfa host and the rhizobia. On the host side, WE2-induced nodules showed higher expression of mature nodule marker genes (ENOD93 and leghemoglobin (Lb) genes) and higher expression of genes encoding SWEET transporters and amino acid and peptide transporters. Host differentially expressed genes were enriched in pathways related to transmembrane transport, redox and heme-related functions, and processes linked to maintaining microaerobic conditions. On the rhizobial side, WE2 nodules showed higher expression of genes involved in microaerobic respiration and nitrogen fixation (e.g., nif/fix and key respiratory chain genes), whereas WWL2 nodules showed higher expression of genes linked to transport, chemotaxis/motility, and environmental information processing. Together, these host and rhizobia expression patterns suggest coordinated differences between host pathways related to resource supply and microaerobic conditions and rhizobial expression programs for respiration and nitrogen fixation. Based on these associations, we propose a working model and provide candidate genes and pathways for functional validation and inoculant screening.