Excessive use of chemical fertilizers in agriculture has become a major global source of pollution, leading to issues such as soil compaction, reduced fertility, eutrophication of water bodies, and air pollution. To address these challenges, the application of biofertilizers, such as rhizobial inoculants, has gradually become an effective, low-cost, and sustainable solution. In this study, the variety Trifolium ambiguum Bieb. (Mengnong clover No. 1) was used as the test material, and two rhizobial strains (R1 and R2) were employed for field inoculation trials. In April 2022, Caucasian clover was planted in an experimental field at Inner Mongolia Agricultural University. Each plot measured 3 m × 4 m and was arranged in a completely randomized design with three replications. In the regreening stage of 2023, rhizobial inoculation treatments were applied, with a control group included for comparison. This research examined the effects of rhizobial inoculation on the growth indicators of Caucasian clover, soil physicochemical properties, soil enzyme activities, and soil microbial communities. The results showed that rhizobial treatment increased the plant height and yield of Caucasian clover, improved soil physicochemical properties and enzyme activities, and positively affected soil microbial diversity and abundance. These changes enhanced soil fertility and optimized microbial community structure, promoting plant growth. The inoculation effect of strain R1 was superior to R2. In conclusion, rhizobial inoculants R1 and R2 can serve as effective biofertilizers for agricultural production.

Caucasian clover (Trifolium ambiguum M.Bieb.) is a perennial legume known for its exceptional cold tolerance, commonly used in agriculture and ecosystems in cold climates. Given the impact of climate change, enhancing the cold adaptation of Caucasian clover is crucial for sustaining agricultural productivity. This study employs metabolomics, transcriptomics, and Weighted Gene Co-expression Network Analysis (WGCNA) to investigate the molecular mechanisms of Caucasian clover’s response to low-temperature stress. Metabolomic analysis showed that low-temperature stress triggered the accumulation of fatty acids, amino acids, and antioxidants, which are critical for maintaining membrane stability and antioxidant capacity, thus protecting the plant from oxidative damage. Transcriptomic analysis revealed significant upregulation of genes involved in cold adaptation, particularly those related to antioxidant defense, membrane lipid repair, and signal transduction, including genes in the ABA signaling pathway and antioxidant enzymes, thereby improving cold tolerance. WGCNA identified gene modules closely linked to cold adaptation, especially those involved in antioxidant defense, fatty acid metabolism, signal transduction, and membrane repair. These modules function synergistically, with coordinated gene expression enhancing cold resistance. This study also investigated the isoflavonoid biosynthesis pathway under low-temperature stress, highlighting its role in enhancing antioxidant capacity and cold tolerance. Low-temperature stress induced upregulation of key enzyme genes, such as Isoflavone Synthase (IFS) and Isoflavone-7-O-Glucosyltransferase (IF7GT), promoting antioxidant metabolite accumulation and further enhancing the plant’s cold adaptation. Overall, this study offers novel molecular insights into the cold tolerance mechanisms of Caucasian clover and provides valuable theoretical support for breeding cold-resistant crops in cold climates.

The rapid increase in fertilizer use has led to the degradation of soil quality, nutrient imbalances, reduced biodiversity, and soil compaction. To address these challenges, hybrid soybeans with efficient biological nitrogen fixation capabilities and broad environmental adaptability were selected as green manure to reduce fertilizer application, thereby improving soil fertility and structure. This study utilized the varieties “Forage Soybean S001” (S001), “Neinong S002 Forage Soybean” (S002), “Mengnong S003 Forage Soybean” (S003), “Mengnong S004 Forage Soybean” (S004), “Mengnong S005 Forage Soybean” (S005), and “Mengnong S006 Forage Soybean” (S006) as green manure materials. The clean tillage (CK) treatment served as the control, ensuring a residue-free soil surface while maintaining consistent practices in soil preparation, irrigation, and field management across all treatments. Field planting of green manure and subsequent crops was conducted at the M-Grass Ecology and Environment (Group) Company’s experimental site in Hohhot in early May of 2023 and 2024. The plots each measured 20 m², with three replications arranged in a randomized block design. A combination of field experiments and laboratory analyses was utilized to investigate the effects of incorporating various hybrid soybean varieties as green manure on soil nutrient levels, soil enzyme activity, soil microbial communities, and the subsequent growth of oats. The results indicated that incorporating various hybrid soybean varieties as green manure into the soil significantly improved soil nutrient levels and enzyme activity. The diversity and richness of soil bacterial communities increased significantly, accompanied by alterations in community structure and composition. These changes enhanced soil fertility and optimized the microbial community structure, promoting the growth of subsequent crops. Among all the treatments, S001 and S004 were particularly effective in enhancing the soil environment, indicating their potential as superior green manure resources for broader application.

Isoflavones are important secondary metabolites in leguminous plants with significant physiological functions and economic value. However, the genetic variation, transcriptional regulation, and metabolic pathways governing isoflavone biosynthesis in Trifolium pratense remain largely unexplored. In this study, we systematically analyzed 500 accessions of T. pratense for isoflavone content and performed RNA-seq-based transcriptomic profiling to investigate the molecular mechanisms underlying isoflavone biosynthesis. Cluster analysis revealed significant genetic variation, with distinct transcriptional profiles between high- (H1, H2, H3) and low-isoflavone (L1, L2, L3) groups. GO and KEGG pathway enrichment analyses identified key metabolic pathways, including phenylpropanoid metabolism, flavonoid biosynthesis, carbohydrate metabolism, and hormone signaling, which play crucial roles in isoflavone regulation. Weighted gene co-expression network analysis (WGCNA) identified three key gene modules—MEblue, MEturquoise, and MEyellow—strongly correlated with isoflavone content. The MEturquoise and MEyellow modules were upregulated in high-isoflavone groups and were enriched in phenylpropanoid biosynthesis, lipid metabolism, and transcriptional regulation, suggesting that these pathways actively promote isoflavone accumulation. Conversely, the MEblue module, highly expressed in low-isoflavone groups, was enriched in sugar metabolism and MAPK signaling, indicating a potential metabolic flux shift away from secondary metabolism. Moreover, key rate-limiting enzymes (PAL, C4H, 4CL, CHS, and IFS) exhibited higher expression in high-isoflavone groups, highlighting their importance in precursor supply and enzymatic catalysis. Additionally, transcription factors such as MYB, WRKY, and NAC were identified as potential regulators of isoflavone biosynthesis, indicating a complex interplay between hormonal, circadian, and environmental signals. This study provides a comprehensive molecular framework for understanding isoflavone biosynthesis in T. pratense and identifies key regulatory genes and metabolic pathways that could be targeted for genetic improvement, metabolic engineering, and molecular breeding. The findings offer valuable insights into enhancing isoflavone production in legumes for agricultural, nutritional, and pharmaceutical applications.

Background: Saline-alkali soil seriously restricts plant growth. Caucasian clover (Trifolium ambiguum Bieb.) is a high-quality leguminous forage with strong adaptability and stress resistance. However, current research on Caucasian clover under saline-alkali stress remains limited. The purpose of this study is to use NaHCO3 solution to carry out alkali stress on Caucasian clover plantsand to understand the changes of plant growth and physiological characteristics under stress, which is of great significance for its popularization and cultivation in alkaline soil. Methods: To study the effects of alkaline stress on the growth and physiological characteristics of Caucasian clover, plants were subjected to varying concentrations (0, 50, 100, 150, 200 mmol/L) of NaHCO3 in pot experiments. Post-stress, parameters such as dry weight, root-to-shoot ratio, main root length, chlorophyll, malondialdehyde (MDA) contentand the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were measured. Result: This study demonstrated that alkaline stress significantly affected the growth and physiological characteristics of Caucasian clover. With increasing alkaline stress, the aboveground and underground dry weights, as well as the main root length, decreased significantly. Photosynthesis was inhibited, with chlorophyll content increasing under low alkaline stress but decreasing sharply at higher concentrations. The activities of antioxidant enzymes (SOD, POD, CAT) increased significantly under low stress but gradually decreased under higher stress levels. Meanwhile, MDA content increased significantly under high alkaline stress. Correlation analysis showed a strong positive correlation between SOD activity and CAT activity, as well as between aboveground dry weight and main root length (P less than 0.01), while chlorophyll content was negatively correlated with MDA content (P less than 0.05).