Alfalfa (Medicago sativa L.) is an important legume forage, and phosphorus is a significant nutrient element of alfalfa with high quality and high yield. However, the effect mechanism of different phosphorus application rates on soil bacteria, and the phosphorus efficiency of alfalfa are still unclear. Therefore, we present the results of a study in which alfalfa was inoculated with Funneliformis mosseae, Bacillus megaterium, double inoculated with Funneliformis mosseae and Bacillus megaterium, and given no inoculation bacteria. P₂O₅ was applied under the condition of bacterial inoculation, and the contents were 0, 50, 100, and 150 mg kg⁻¹, respectively, to explore the effect of bacterial inoculation on alkaline phosphatase, organic acid, pH, organic matter, and the relationship between the indicators in alfalfa soil, under different phosphorus application rates. The effect of mixed inoculation was significantly higher than that of the non-inoculated control group (p < 0.05). The organic matter content of rhizosphere soil was higher than that of non-rhizosphere soil. When the phosphorus application rate was 100 mg kg⁻¹, the content of alkaline phosphatase in the soil inoculated with Funneliformis mosseae and Bacillus megaterium was better than that in the single inoculation, and no inoculation. Principal component analysis showed that the top three treatments were: double inoculation bacteria and treatment group with phosphorus application rate of 100 mg kg⁻¹ >; double inoculation bacteria and treatment group with phosphorus application rate of 50 mg kg⁻¹ >; double inoculation bacteria and treatment group with phosphorus application rate of 150 mg kg⁻¹. In addition, when P₂O₅ was 100 mg kg⁻¹, the addition of Funneliformis mosseae and Bacillus megaterium to alfalfa soil could increase the content of organic matter in the soil, promote the metabolism of alfalfa root exudates, and increase the organic acid of the rhizosphere soil, compared with the control without inoculation, and without phosphorus application. At the same time, the phosphatase activity in the soil had a significant positive correlation with malic acid, oxalic acid, acetic acid, total organic acid, and soil pH, thereby improving soil fertility and promoting phosphorus absorption by plants. These findings provide new insights into alfalfa root soils and the effects of Funneliformis mosseae and Bacillus megaterium additions on soil nutrients.

The present study investigated the dynamic profiles of fermentation quality, aerobic stability, and the bacterial community of paper mulberry silage inoculants without (Control) or with Lactiplantibacillus plantarum (LP), Lactiplantibacillus brevis (LB), or their combination (LPLB), which was screened from naturally fermented paper mulberry. The results showed that the inoculated treatments had significantly reduced neutral detergent fiber, water-soluble carbohydrates, and ammoniacal nitrogen contents compared with the control after 60 days of ensiling (the decreased proportion of LP, LB, and LPLB treatments ranged from 7.33%–11.23%, 9.60%–21.44%, and 21.53%–29.23%, respectively, p &lt; 0.05). The pH value of the LP and LB treatments was significantly lower than that of the control after 60 days of ensiling (4.42 and 4.56 vs. 4.71, p &lt; 0.05). The LP treatment promoted lactic acid accumulation and LAB number compared with the control (66.59% vs. 54.12% and 8.71 log₁₀ CFU/g vs. 8.52 log₁₀ CFU/g, respectively, p &lt; 0.05), and the LB and LPLB treatments inhibited the growth of yeast and mold after 14 days of fermentation. After 5 days of aerobic exposure, both the LB and LPLB treatments increased the aerobic stability time and acetic acid content (from 29 to 75 h and 16.14%–48.72%, respectively, p &lt; 0.05), inhibited the growth of yeast and mold, and did not detect butyric acid. Additionally, the bacteria community of each treatment was dominated by Aerococcus on day 3 of ensilage (accounting for 54.36%–69.31%), while the inoculated treatments reduced the abundance of Aerococcus on day 60 (from 59.73% to 85.16%, p &lt; 0.05), and Lactobacillus became the dominant genus (accounting for 54.57%–70.89%). Inoculation of L. plantarum effectively maintained the acidic environment at the end of the fermentation system by maintaining the abundance of Lactobacillus, maximizing the preservation of dry matter and protein, and reducing protein corruption. Inoculation of L. brevis alone or in combination with L. plantarum significantly inhibited the growth of mold and improved the aerobic stability of paper mulberry silage.

Sainfoin (Onobrychis viciifolia) is rich in condensed tannins (CT). CT function includes inhibiting bacterial and fungi activity during the ensiling process. We used polyethylene glycol (PEG) to deactivate tannin activity to find out the effects of CT. The results show that the addition of PEG increased dry-matter loss (8.32% vs. 14.15%, on a dry-matter basis) after 60 d of ensiling, and also increased lactic acid (10.90% vs. 15.90%, on a dry-matter basis) and acetic-acid content (7.32% vs. 13.85%, on a dry-matter basis) after 30 d of ensiling. The PEG-treated group increased its Pediococcus relative abundance (0.37–3.38% vs. 7.82–23.5%,) during the ensiling process, increased its Gibellulopsis relative abundance after 3 d of ensiling (5.96% vs. 19.52%), increased its Vishniacozyma relative abundance after 3 d and 7 d of ensiling (2.36% vs. 17.02%, 3.65% vs. 17.17%), and increased its Aspergillus relative abundance after 7 d, 14 d and 60 d of ensiling (0.28% vs. 1.32%, 0.49% vs. 2.84% and 1.74% vs. 7.56%). However, the PEG-treated group decreased its Alternaria relative abundance during entire ensiling process (14.00–25.21% vs. 3.33–7.49%). These results suggest that condensed tannins inhibit lactic-acid bacteria fermentation though reducing Pediococcus activity, and inhibiting fungi activity depending on different strains.

Alfalfa (Medicago sativa L.) is an important forage legume in arid areas, but limited water resources and low fertilizer utilization have restricted its agricultural development. Meanwhile, studies on the effects of integrated water and phosphorus on production performance and water‐use efficiency and phosphorus‐use efficiency of alfalfa, especially on hay yield, phosphorus accumulation, and total phosphorus uptake are rarely reported under drip irrigation. The treatments were a factorial combination of three irrigation rates (5,250, 6,000, and 6,750 m³/ha per year) and four P rates (0, 50, 100, and 150 kg/ha per year) and consisted of 12 treatments for water and P management, arranged in a randomized complete block design with three replicates. Total hay yield and water‐use efficiency and phosphorus‐use efficiency of alfalfa in P₂ treatment were significantly greater than those in the P₁ and P₃ treatments (p < .05), and the total hay yield of alfalfa with phosphorus application increased by 7.43%–29.87% compared with that in the nonphosphorus (P₀) treatment under the same irrigation amount. The total phosphorus and available phosphorus concentrations in the 0–20 cm soil layer were greater than those in the 20–40 cm and 40–60 cm soil layers compared with those in the P₀ treatment. Correlation analyses showed that total hay yield was significantly positively correlated with total phosphorus uptake and water‐use efficiency (p < .01). The accumulated phosphorus concentration was significantly positively correlated with total phosphorus and available phosphorus concentration (p < .01) and was positively correlated with the phosphorus‐use efficiency (p < .05). The membership function method was used to evaluate all the indicators, and the three treatments that had the greatest influence on the production performance of alfalfa were, in order, W₂P₂ > W₃P₂ > W₁P₂. Therefore, an irrigation rate of 6,000 m³/ha and a phosphorus application rate of 100 kg/ha per year should be considered as the best management for both high yield and water‐use efficiency and phosphorus‐use efficiency of alfalfa.

Condensed tannins (CT) from sainfoin have a high capacity to inhibit proteolysis. A previous study reported that CT from sainfoin can inhibit lactic acid bacteria activity and decrease ammonium-nitrogen (N) content during sainfoin ensiling; however, no study has focused on the metabolome of ensiled sainfoin. The objective of the present study was to investigate the effects of CT [following supplementation of deactivated CT with polyethylene glycol (PEG)] on protease activity, keystone bacteria, and metabolome during sainfoin ensiling. According to the results, PEG amendment increased non-protein N, amino acid, and soluble protein contents significantly (in the 49.08–59.41, 116.01–64.22, and 23.5–41.94% ranges, respectively, p < 0.05) during ensiling, whereas neutral detergent-insoluble protein and acid detergent-insoluble protein were decreased significantly (in the 55.98–64.71 and 36.58–57.55% ranges, respectively, p < 0.05). PEG supplementation increased aminopeptidase and acid protease activity after 3 days of ensiling (p < 0.05) and increased carboxypeptidase activity during the entire ensiling process (p < 0.05). The keystone bacteria changed following PEG addition (Stenotrophomonas, Pantoea, and Cellulosimicrobium in the control vs. Microbacterium, Enterococcus, and Brevundimonas in the PEG-treated group). In total, 510 metabolites were identified after 60 days of sainfoin ensiling, with 33 metabolites annotated in the Kyoto Encyclopedia of Genes and Genomes database. Among the metabolites, phospholipids were the most abundant (72.7% of 33 metabolites). In addition, 10 upregulated and 23 downregulated metabolites were identified in the PEG-treated group when compared with the control group, after 60 days of ensiling (p < 0.05). Pediococcus (correlated with 20 metabolites, R² > 0.88, p < 0.05) and Lactobacillus (correlated with 16 metabolites, R² > 0.88, p < 0.05) were the bacteria most correlated with metabolites. The results suggested antagonistic effects between Lactobacillus and Pediococcus during ensiling. The decreased proteolysis during sainfoin ensiling was mainly attributed to the inhibition of protease activity by CT, particularly carboxypeptidase activity. In addition, proteolysis decreased partly due to CT inhibiting Pediococcus activity during ensiling, with Pediococcus being significantly and positively correlated with dopamine after 60 days of ensiling (R² = 0.8857, p < 0.05).

The productivity of alfalfa is associated with a large amount of nitrogen (N) and phosphorus (P); the addition of exogenous N and P fertilizers can fully exploit the growth potential of alfalfa. However, there is uncertainty about the relationship between changes in alfalfa productivity and photosynthetic physiology and anatomy. We conducted field fertilization experiments on alfalfa in the second and third years under drip irrigation, as well as measurement of the photosynthetic physiology, anatomical structure and agronomic traits of alfalfa at different levels of N (0, 120 kg·ha⁻¹) and different levels of P₂O₅ (0, 50, 100 and 150 kg·ha⁻¹). The results showed that the dry matter yield (DMY), crude protein (CP), net photosynthetic rate (P_n) and specific leaf weight (SLW) were increased by 2.10~11.82%, 4.95~11.93%, 4.71~7.59% and 2.02~7.12% in the N application treatment compared with the non-N application treatment, while the DMY, CP, P_n and SLW were increased by 3.19~17.46%, 1.99~8.42%, 6.15~24.95% and 2.16~11.90% in the P application treatment compared with the non-P application treatment. N and P increase the thickness of the spongy tissue (ST) of alfalfa, which will facilitate the entry and exit of gas and water, and will further affect the photosynthetic indexes, such as stomatal conductance (G_s) and transpiration rate (T_r), of alfalfa leaves. Increased palisade tissue (PT) thickness will also enhance the adaptability of plant leaves to strong sunlight, thereby increasing the maximum net photosynthetic rate (P_max) and light saturation point (LSP). Fertilization treatment showed the highest utilization efficiency for low light and better adaptation to strong light, but the R_d decreased. The comprehensive scores of principal component analysis for anatomical structure, photosynthetic performance and agronomic traits were N₁P₂ > N₀P₂ > N₁P₃ > N₁P₁ > N₀P₃ > N₀P₁ > N₁P₀ > N₀P₀. Therefore, the application of N and P fertilizers contributed to the adaptive changes in alfalfa leaf anatomy and the improvement of photosynthetic capacity, which were beneficial to the improvement of alfalfa dry matter yield, growth traits and nutritional quality, with the most obvious improvement effect obtained with the application of 120 kg·ha⁻¹ of N and 100 kg·ha⁻¹ of P₂O₅.