Alfalfa (Medicago sativa) is a perennial forage crop with significant economic and ecological significance. If alfalfa can be planted in saline-alkali land, it will not only improve the utilization rate of marginal land and alleviate the competition between forage and cereal crops for arable land but will also increase the yield of high-quality domestic forage. In this study, we conducted transcriptomic analysis on the saline-alkali-tolerant alfalfa cultivar NQ-1 and compared its metabolite accumulation levels with saline-alkali-sensitive cultivars. The results showed that under saline-alkali stress, the photosynthesis and some secondary metabolic pathways in NQ-1 were activated, such as α-Linolenic acid metabolism, Phenylpropanoid and Flavonoid biosynthesis, and Photosynthesis-related pathways, providing substances and energy for enhancing NQ-1 stress tolerance. Furthermore, some specific flavonoids were detected that may contribute to the saline-alkali tolerance of NQ-1. In addition, transcription factors that may regulate flavonoid biosynthesis in NQ-1 under saline-alkali stress were also identified. This study deepens the understanding of the resistance mechanism of saline-alkali-tolerant cultivars of alfalfa and provides valuable information for molecular design breeding strategies for stress-resistant alfalfa.

Alfalfa (Medicago sativa L.), a perennial leguminous herb, can tolerate cold and saline–alkaline conditions. In this study, alfalfa cultivars LJ and 218TR were exposed to saline–alkaline, cold, and saline–alkaline–cold conditions and compared in terms of phenotypes, physiological indices, key metabolite contents, and stress-responsive gene expression. Malondialdehyde, soluble sugar, proline contents and phenylalanine ammonia-lyase (PAL), superoxide dismutase, catalase, and peroxidase activities initially increased under individual stress conditions, but decreased when stresses were combined. Photosystem II maximum photochemical efficiency and chlorophyll contents decreased under individual and combined stress conditions. Nitroblue tetrazolium-stained leaves revealed that the combined stress treatment significantly increased cell mortality rates and superoxide anion levels. LJ was more tolerant to saline–alkaline, cold, and combined stress treatments than 218TR. Metabolite analyses indicated that for LJ and 218TR, salicylic acid (SA) was the most responsive metabolite to combined stress conditions. Additionally, the expression of isochorismate synthase (ICS) and PAL genes critical for SA biosynthesis was upregulated under single or combined stress conditions, leading to SA accumulation and improved tolerance to saline–alkaline–cold conditions. This study revealed the physiological indices and molecular changes underlying alfalfa responses to saline–alkaline stress combined with cold stress, providing a theoretical basis for breeding stress-tolerant cultivars.

Root-knot nematodes (RKN), especially Meloidogyne incognita, threaten global alfalfa crops because of their broad host range and pathogenic nature. Despite its significance, research on resistance is limited. In this study, 24 varieties from China, the US, Canada, Australia, and France were assessed for resistance using the Disease Index (DI) and Egg Mass Index (EMI). Results identified 19 varieties with varying resistance levels and 5 that were susceptible. Chinese Gannong No. 9 was highly resistant (DI: 10) and achieved the highest composite score (91). The US varieties Dryland and Moste were classified as resistant (DI: 14.3% and 12.5%, respectively) and also ranked highly by composite score (65 and 62.5). A moderate correlation between DI and EMI (r = 0.68) led to some inconsistent classifications, including for 2295, Instict, and WL168HQ, highlighting the importance of using multiple complementary metrics for accurate resistance evaluation. Egg mass production was strongly correlated with galling severity (r = 0.70), while root biomass showed no correlation with galling (r = 0.09), indicating root weight is not a reliable resistance indicator. Preliminary infection dynamics showed similar nematode penetration rates at 2 days post-infection across resistant and susceptible varieties. At 7 days post-infection, both resistant and susceptible varieties retained predominantly J2 larvae (78–89%), with no statistically significant differences in developmental stage distributions. These preliminary observations suggest that resistance-associated effects on nematode development, if present, are not strongly expressed at early stages of infection. The mechanistic basis of resistance in alfalfa remains unresolved and warrants further investigation using additional timepoints, histological analyses of feeding-site development, and molecular characterization. Geographically, American varieties displayed broad performance variation, Chinese varieties showed a bimodal distribution, and Canadian varieties exhibited moderate, consistent resistance. These results offer valuable germplasm for breeding and highlight the importance of multiple resistance metrics. Resistant varieties such as Gannong No. 9 provide important genetic resources for developing durable nematode resistance in alfalfa and can guide variety selection in nematode-infested regions.

Glycyrrhiza pallidiflora Maxim., a perennial legume with high biomass yield and good nutritional value, has potential as a forage resource. This study examined how mixing G. pallidiflora (C) with Leymus chinensis (Y) at varying ratios (C10Y0, C9Y1, C8Y2, C7Y3, C6Y4) affects silage fermentation, chemical composition, and microbial community structure. All treatments were inoculated with Lactiplantibacillus plantarum (1 × 10⁶ CFU/g fresh weight) and ensiled for 120 days. The results indicated that mixed silages markedly improved overall fermentation quality compared to the sole C silage (C10Y0). These mixed silages exhibited superior lactic acid (LA) concentrations, lower pH. Bacterial community profiling revealed that the addition of Y shifted the microbiota from a diverse community to one dominated by Lactobacillus. Although the C6Y4 and C7Y3 groups exhibited lower pH, they showed significantly elevated NH₃-N contents, while their crude protein contents and the relative abundances of Lactobacillus were both lower than those of the C9Y1 and C8Y2 groups. Considering the core requirements of comprehensive quality, the mixing ratios of 9:1 (C9Y1) and 8:2 (C8Y2) demonstrated the optimal effects: at these ratios, the silage maintained a CP content of 12.84–14.48% DM, with NDF and ADF contents stabilized at 47.55–51.09% DM and 33.67–34.14% DM, respectively, and DM content of 28.85–31.32%; in terms of fermentation quality, the pH value decreased from 4.85 in the sole C silage (C10Y0) to 4.04–4.11, the LA content increased from 13.91 g/kg DM to 28.86–30.87 g/kg DM, the LA/AA ratio rose from 1.31 to 3.37–3.97, and the NH₃-N content was reduced by 0.56–0.96% TN compared to the C10Y0 (decreasing to 4.16–4.45% TN), effectively inhibiting protein degradation; at the microbial level, the LAB count reached 9.03–9.05 log₁₀ CFU/g FM, an increase of 2.12–2.14 compared to the C10Y0, with a relative abundance exceeding 80%, successfully suppressing the proliferation of undesirable bacteria such as Raoultella and Weissella and ensuring fermentation stability. This provides technical support for utilizing this plant as a viable alternative forage resource.