Drought limits forage productivity and causes physiological dysfunction in plants. Melatonin (MT) can enhance stress tolerance, but the optimal dose and the mechanisms by which it mitigates drought-induced physiological and metabolic disturbances in Leymus chinensis remain unclear. A pot experiment under controlled soil moisture was conducted to screen the optimal MT dose for alleviating drought stress in L. chinensis seedlings and to elucidate the key physiological and metabolic mechanisms involved. Adding 100 μM MT significantly improved growth and photosynthetic performance under drought (p < 0.05). Specifically, DM100 increased plant height, root length, stem diameter, aboveground fresh weight (FW), aboveground dry weight (DW), and leaf relative water content (RWC) by 51.91%, 20.95%, 38.40%, 192.57%, 192.41% and 12.52%, respectively. Gas-exchange parameters were likewise enhanced (Gs: 183.38%, Tr: 270.37%, Pn: 114.24%), whereas intercellular CO₂ concentration (Ci) decreased by 113.34% (p < 0.05). Under drought, activities of antioxidant enzymes-superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT)-were significantly elevated, and DM100 further increased these activities; conversely, drought-induced proline (Pro) accumulation was reduced by MT treatment. Untargeted metabolomics showed that drought markedly upregulated biosynthetic pathways for tryptophan, phenylalanine, phenylpropanoids and flavonoids. DM100 selectively attenuated excessive activation of tryptophan and phenylalanine metabolism, modulated phenylpropanoid/flavonoid responses, and coordinately regulated antioxidant and osmotic-adjustment metabolism. In summary, foliar MT at 100 μmol·L-1 appears to rebalance drought-induced metabolic perturbations by selectively modulating stress-responsive pathways rather than broadly activating metabolism, thereby improving photosynthetic performance, antioxidant capacity and growth in L. chinensis.

Soil nutrient transformation capacity is a critical determinant of sustainable productivity in perennial cropping systems; however, the extent to which high-yielding crops actively regulate rhizosphere microbial assembly to maintain nutrient availability remains poorly understood. We investigated whether root exudates from high-yielding alfalfa (Medicago sativa L.) selectively recruit plant growth-promoting rhizobacteria (PGPR) to enhance nutrient transformation. In an 8-year continuous alfalfa system (2018-2025), high-yielding cultivars increased soil organic carbon by 8.64%, total nitrogen by 6.01%, and moderately labile phosphorus fractions by 1.62%. Rhizobox experiments demonstrated that root exudates enhanced growth only with an active microbiome. High-yielding alfalfa enriched PGPR communities, specifically Ensifer, Pseudomonas, and Bacillus. Isolated strains exhibited N fixation, P solubilisation, and IAA production. Metabolomic profiling revealed that exudates were enriched in specific sugars and amino acids. Maltopentaose, maltotetraose, taurine, N-acetyl-L-leucine, and asparagine functioned as chemoattractants, stimulating PGPR proliferation and biofilm formation. These findings demonstrate that root exudate-mediated, targeted recruitment of functional PGPR enhances N fixation and P transformation, thereby supporting sustained high alfalfa productivity. This study demonstrates a key rhizosphere mechanism underlying the long-term sustainability of high-yielding perennial legume systems and provides a mechanistic basis for microbiome-informed sustainable alfalfa production and management.

Salt and heat stresses often occur simultaneously in arid regions, restricting the distribution and productivity of alfalfa (Medicago sativa L.). However, the mechanisms underlying alfalfa responses to combined salt and heat stress remain unclear. Here, we integrated phenotypic, physiological, transcriptomic, and metabolomic analyses to investigate the regulatory mechanisms involved in combined stress responses. Combined stress more strongly inhibited plant height, stem diameter, fresh weight, net photosynthetic rate, and transpiration rate than single stresses. Salt stress had a predominant effect on several agronomic and physiological traits, whereas heat stress mainly affected chloroplast ultrastructure. Multiomics analysis identified flavonoid metabolism, linoleic acid metabolism, and amino acid biosynthesis as key pathways associated with combined stress responses. Moreover, CHS, CHR, P5CS, and LOX genes expression was closely correlated with metabolites such as naringenin, naringenin chalcone, and proline. These findings provide insights into alfalfa adaptation to multiple abiotic stresses.

IntroductionThe rising threat of antimicrobial resistance necessitates sustainable alternatives to synthetic antibiotics, driving interest in plant-derived compounds for livestock feed. Cyperus esculentus L. (tiger nut) exhibits promising antibacterial properties, yet gaps persist in understanding its specific bioactive components, synergistic mechanisms, and metabolic impact on bacterial pathogens.MethodsThis study employed metabolomic profiling combined with bioassays to evaluate the efficacy of tuber, stem/leaf, and composite extracts against Staphylococcus aureus. Methods included Kirby-Bauer assays, scanning electron microscopy (SEM), growth curve analysis, response surface optimization, and wide-targeted metabolomics.ResultsKey findings revealed: (1) Stem/leaf extracts outperformed tuber extracts in antibacterial activity, with their combination yielding synergistic effects; (2) Flavonoids (naringenin, acacetin, diosmetin, silybin A) demonstrated dose-dependent inhibition, disrupting cell wall/membrane integrity via SEM-confirmed morphological damage; (3) Optimal synergistic mix (2.84 μg/mL naringenin, 2.68 μg/mL acacetin, 3.04 μg/mL diosmetin, 3.08 μg/mL silybin A) achieved potent suppression across bacterial growth phases; (4) Metabolomic perturbations implicated sulfur metabolism, oxidative stress responses, and lipid remodeling in antibiosis.DiscussionThis work uniquely establishes C. esculentus stem/leaf extracts as high-value, resource-efficient antibiotic alternatives for animal feed, leveraging synergistic phytochemistry and mechanistic insights to combat antimicrobial resistance.

Paper mulberry (Broussonetia papyrifera) is a high-protein forage, but its high buffering capacity and low water-soluble carbohydrate (WSC) content hinder silage fermentation. This study evaluated the effect of tea tree (Melaleuca alternifolia) essential oil (TTO) as a natural additive for paper mulberry silage. TTO was added at 0 (CK), 500 (CSD), and 1000 mg/kg (CSG) fresh weight, with samples collected on days 7, 15, 30, and 90. After 90 days, CSD had the lowest pH and the highest lactic acid content. NH3-N/TN was significantly lower than in CK (p < 0.05), and the retention of dry matter, crude protein and water-soluble carbohydrate was improved (p < 0.05). Compared with CK, CSD exhibited the highest lactic acid bacteria (LAB) counts, along with the lowest mold and aerobic bacterial counts (p < 0.05). However, CSG did not further improve fermentation quality, and most of the indicators were not significantly different from CSD (p > 0.05). The in vitro gas production test showed that CSD significantly increased the theoretical maximum gas production and the maximum gas production rate (p < 0.05). Additionally, 16S rRNA sequencing revealed a distinct bacterial succession in CSD, which enriched beneficial lactic acid bacteria (LAB) such as Pediococcus while outcompeting less competitive taxa. This microbial reshaping redirected the community toward acidification and nutrient retention. Collectively, these results demonstrate that TTO at 500 mg/kg is a promising natural additive for improving paper mulberry silage quality through selective reshaping of the bacterial community.

Salt stress severely restricts plant growth and productivity. Alfalfa (Medicago sativa L.) is one of the most important forage crops worldwide. However, the physiological and molecular responses of alfalfa to salt stress remain unclear. Therefore, physiological, transcriptomic, and metabolomic analyses were integrated to investigate the responses of Xinmu No. 4 alfalfa to salt treatments. The results showed that key growth parameters and photosynthetic efficiency decreased significantly with increasing NaCl concentration. Low salt treatment (100 mM, S1) did not cause significant changes in the levels of carbohydrates or reactive oxygen species (ROS). In contrast, high salt treatment (300 mM, S3) triggered massive accumulation of organic osmolytes (proline and soluble sugars) and ROS levels, accompanied by a significant increase in antioxidant enzyme activity. Transcriptomic analysis showed significant enrichment of linoleic acid metabolism and flavonoid biosynthesis under the S1 treatment, whereas photosynthesis, amino acid metabolism, and the MAPK signaling pathway were predominantly enriched under the S3 treatment. Integrated multi-omics analysis further indicated that the genes and differentially accumulated metabolites involved in starch and sucrose metabolism and amino acid metabolism pathways exhibited coordinated responses to high salt stress. Specifically, under the S3 treatment, the upregulation of genes such as SUS and TPP was associated with the accumulation of sucrose and trehalose, whereas the upregulation of P5CS and P5CR was associated with increased proline accumulation. These findings improve our understanding of the physiological and molecular mechanisms underlying alfalfa responses to salt stress and provide valuable information for future studies on the breeding of salt-tolerant cultivars.

IntroductionSaline-alkali soil significantly inhibits the growth and development of Leymus chinensis, and root exudates are vital components in plant responses to abiotic stress. At present, whether L. chinensis accumulates specific root exudates to adapt to saline-alkali environments remains poorly understood.MethodsTherefore, in this study, the relatively saline-alkali-tolerant L. chinensis cultivar 'Huise' (HS) and the relatively saline-alkali-sensitive cultivar 'Dongbei' (DB) were selected as experimental materials. Three saline-alkali stress treatments were established: non-saline-alkali soil (Control, CK), moderate saline-alkali soil (MS), and severe saline-alkali soil (ES), to explore cultivar differences in physiological characteristics and root exudate metabolism under saline-alkali stress.ResultsThe results showed that cultivar differences in aboveground biomass were observed between HS and DB L. chinensis under saline-alkali stress. Intracellular Na+ accumulation and K+ decline occurred, which aggravated membrane lipid peroxidation and a 31-38% decrease in root vitality. Compared with HS, DB suffered more severe membrane lipid damage and a larger decline in root vitality. The two cultivars alleviated saline-alkali stress by simultaneously increasing the activities of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) and accumulating proline (Pro), soluble sugars (SS), and soluble protein (SP). In total, 5014 metabolites were identified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Thirteen differentially accumulated metabolite (DAM) markers (including [8]-Shogaol, Pikuroside, L-dihydroanticapsin) were screened. Distinct cultivar differences were also observed in the number of differential root exudate metabolites under saline-alkali stress. KEGG pathway enrichment analysis revealed that the pentose phosphate pathway (PPP) and the tricarboxylic acid cycle (TCA) were significantly enriched in L. chinensis under saline-alkali stress. Compared with the DB cultivar, relevant biosynthetic pathways for leucine and glycine were additionally enriched in the HS cultivar.DiscussionThis study identifies inter-cultivar differences in the content, composition and enriched metabolic pathways of root exudates in L. chinensis. These findings offer fundamental references for further exploring how saline-alkali stress correlates with physiological traits, growth performance and root exudate profiles of regreening stage L. chinensis.

Sediment microbial communities in arid inland rivers are important for biogeochemical cycling, but the assembly mechanisms and network responses across the transition from irrigated oases to desert margins remain unclear. We investigated bacterial and fungal communities in Yarkand River sediments along salinity and nutrient gradients using 16S rRNA and ITS sequencing, sediment chemistry, iCAMP null-model analysis, and co-occurrence networks. Downstream increases in electrical conductivity, total nitrogen, and pH were associated with reduced bacterial α-diversity and turnover from nutrient-responsive Pseudomonadota in the oasis midstream to stress-tolerant Chloroflexota and Actinomycetota in the desert downstream. Fungal diversity showed high inter-site variability without a linear decline. Despite strong physicochemical gradients, ecological drift dominated assembly in both kingdoms, while desert networks showed higher connectivity. These findings support a hierarchical assembly model in which salinity filters community composition, but stochastic processes and network densification shape benthic microbiota in arid river sediments.

Lactic acid bacteria (LAB) and cellulase have been used as additives to improve the fermentation quality of mulberry silage. This study investigated the dynamics of fermentation characteristics and bacterial communities during 60-day ensiling through three established treatment groups: Control (no inoculation), Lactiplantibacillus plantarum (LP) inoculation as well as combination of L. plantarum and cellulase inoculation group (LPC). The results showed that compared with the Control group, the LP and LPC treatments significantly reduced the loss of dry matter, soluble carbohydrates, and crude protein (p < 0.05), effectively promoted the accumulation of lactic acid and acetic acid (p < 0.05), but significantly elevated ammonia nitrogen (NH3-N) production. Inoculation was beneficial to the stability of the bacterial community in mulberry branch and leaf silage because it can maintain a high level of beneficial bacteria (Lactiplantibacillus) and inhibit the growth of harmful bacteria (Escherichia-Shigella). The combination of the inoculation of L. plantarum and cellulase may improve the quality of mulberry branch silage.

The experiment aimed to investigate the species of Clostridium in whole-plant corn silage and the metabolics they produced. Taking Clostridium was isolated from whole-plant corn silage as the research object, through strain enrichment culture, isolation and purification, 16S rDNA sequence determination, and the analysis of its physiology, biochemical and metabolic products. The results showed that one strain of Clostridium beijerinckii SHZ-8 was isolated and identified. The optimal growth pH value was 7.0 and the optimal growth temperature was 30 ℃. Decamethylcyclopentasiloxane was a potential metabolic indicator of it. The study indicates that strain SHZ-8 is a mesophilic and neutral bacterium, and the production of butyric acid and decamethylcyclopentasiloxane is an early warning signal of silage spoilage.

ABSTRACT Nettle showed several benefits for animals as an unconventional feed source, but it is difficult to ensile in order to be better utilized. Recently, nettle has been utilized to alternate alfalfa as ruminant feed, but it is usually poor in ensiling quality. To determine the endogenous component influencing ensiling, we investigated the effect of Pediococcus on the characteristics of nettle silage and the mechanism of action of these substances against Pediococcus. Inoculation with Pediococcus pentosaceus decreased the relative abundance of Clostridium sensu stricto 15 by 85.95% in the middle fermentation stage of nettle silage (30 d), with a 24.74% decrease in the ammonia content (P＜0.05). In the correlation analysis, 4-pentenoic acid showed an extremely significant negative correlation with Pediococcus spp. in nettle silage (P < 0.001). After 4-pentenoic acid treatment, the most downregulated proteins were involved in the ribosome pathway (30 differentially expressed proteins), and then in the glycolysis/gluconeogenesis, pyruvate metabolism, and fatty acid synthesis pathways (particularly for accA, accD, and fabG). The mechanism of action of 4-pentenoic acid against P. pentosaceus mainly involves inhibition of fatty acid synthesis and decreased the expression of acid tolerance proteins. The present study will give new insights into silage fermentation and provide new clues for better ensiling of nettle.IMPORTANCENettle has attracted the attention of scientists due to its several benefits for animals as non-conventional feed sources. However, as for challenge, nettle is difficult to ensile (poor quality), which is an obstacle for nettle use. In the present manuscript, we investigated the effect of Pediococcus on the characteristics of nettle silage and clarified the mechanisms of 4-pentenoic acid against Pediococcus. Our findings suggested that P. pentosaceus could improve nettle silage quality at a significant level through decreased production of ammonia (decline percentage was 21.41%–31.73%) during ensiling, while it could not well improve the quality of nettle silage due to the interference effect of 4-pentenoic acid as an antibacterial substance. The mechanism of 4-pentenoic acid against P. pentosaceus was mainly through inhibition of fatty acid synthesis (fabG) and expression of acid tolerance protein (accA), resulting in destruction of the cell wall in P. pentosaceus. Our finding could give a new clue for better use of nettle silage.

The alfalfa weevil (Hypera postica) is a significant pest in alfalfa cultivation, with both adults and larvae causing significant damage by feeding on stems, leaves and leaf mesophyll, respectively. This study utilized a multi-omics approach to systematically investigate the differential defense mechanisms activated in alfalfa in response to weevil infestation. The findings revealed that larval feeding primarily activates the jasmonic acid (JA) signaling pathway which upregulates the LOX-AOS-AOC gene cluster to facilitate the synthesis of direct defense compounds. In contrast, adult feeding preferentially stimulate the phenylpropanoid pathway leading to the emission of specific volatile. For the first time, we revealed the formation of a synergistic defense barrier involving flavonoid and lignin biosynthesis, with larval-induced γ-aminobutyric acid (GABA) metabolism and adult-specific glutamate signaling contributing to distinct defense responses. Volatile analysis showed an 8.9-fold increase in phenylethanol (adult-induced) and a 2.57-fold increase in green leaf volatiles (larval-induced). These findings provide a comprehensive understanding of the stage-specific defense strategy employed by alfalfa, mediated through precise regulation of JA signaling, secondary metabolism, and volatile synthesis. This study provides a theoretical basis for breeding insect-resistant alfalfa varieties and developing environmentally sustainable pest control strategies.

This study investigated whole-plant maize at three harvest stages: one-third milk line (ML, 1/3 ML), two-thirds ML (2/3 ML), and the mature stage. Two packing densities were applied: 350 kg/m3 (low-density group) and 700 kg/m3 (high-density group). Results showed that starch content increased significantly as the maize matured. The 2/3 ML stage exhibited a 34.0% increase in starch content compared to the 1/3 ML stage (27.96 g/kg dry matter: DM vs. 20.87 g/kg DM, p < 0.01), while the mature stage showed a 13.4% increase compared to the 2/3 ML stage (31.70 g/kg DM vs. 27.96 g/kg DM, p < 0.01). After 60 days of ensiling, DM loss was significantly lower in the high-density group compared to the low-density group (3.37% vs. 9.39%, p < 0.05). From day 7 to day 60 of fermentation, the lactic acid content in the high-density group was consistently higher than in the low-density group by 14.29%, 10.00%, 8.33%, and 9.68%, respectively (p < 0.01). The relative abundance of Clostridium in both groups gradually increased during the first 30 days of fermentation, peaking on day 30 (0.05% in the high-density group vs. 0.12% in the low-density group, p < 0.05), and declined thereafter. On day 30, the abundance of Ruminiclostridium was significantly lower in the high-density group compared to the low-density group (0.12% vs. 0.40%, p < 0.05). Clostridium was negatively correlated with lactic acid bacteria (R2 = −0.58, p < 0.01). It also showed negative correlations with pH, lactic acid, and acetic acid (R2 = −0.25, −0.23, and −0.09, respectively; p > 0.05), but a positive correlation with ammoniacal nitrogen (R2 = 0.28, p > 0.05). In conclusion, the 2/3 ML stage is the optimal harvest time for whole-plant maize. Additionally, a higher packing density can suppress spoilage-associated Clostridium and enhance silage quality.

The utilization of nettle (Urtica cannabina) as feed is restricted by its material properties (antibacterial activity and high buffering capacity). This study hypothesized that the use of lactic acid bacteria (LAB) attached to nettles can improve these problems. Lactococcus garvieae (LG), Pediococcus pentosaceus (PP), and LG + PP (LP) isolated from nettles were inoculated into nettle silage to explore nutrient retention and the microbial community structure. The results showed that inoculation significantly delayed dry matter and crude protein loss, inhibited neutral detergent fiber and acid detergent fiber degradation, and reduced ammonia nitrogen (NH3-N) accumulation. There was a significant increase in Firmicutes abundance after inoculation, and the dominant genus, Aerococcus, was negatively correlated with NH3-N accumulation. In the later stages of the PP treatment, Atopistipes synergistically inhibited Clostridia with acetic acid. However, the high buffering capacity and antibacterial components of raw nettle led to increased pH values during the later fermentation stages, limiting sustained acid production by LAB. These results confirm that nettle-derived LAB can effectively improve the quality of silage by regulating the microbial community and the acidification process; however, they must be combined with pretreatment strategies or optimized composite microbial agents to overcome raw material limitations. This study provides a theoretical basis and technical support for the utilization of nettle as feed.

Introduction:Addressing the challenges of inefficient water-fertilizer utilization and suboptimal seed yield in alfalfa (Medicago sativa L.) seed production systems, we investigated the effects of differential irrigation-fertilization regimes on soil nutrient dynamics, photosynthetic performance, and yield parameters. This study aims to optimize seed production while elucidating the response mechanisms linking soil nutrient availability, foliar photosynthetic efficiency, and seed yield outcomes. This experiment employed drip irrigation to address production constraints in alfalfa seed cultivation. Methods:Using 'WL354HQ' and 'Xinmu No.4' as the experimental materials, a two-factor randomized block design was adopted, with three fertilization levels: F0 (no fertilizer), F1 (90 kg·ha-1 N 75 kg·ha-1 P2O5, 12 kg·ha-1 K2O), and F2 (120 kg·ha-1 N, 100 kg·ha-1 P2O5, 15 kg·ha-1 K2O), and combined with three irrigation levels W1 (1650 m3·ha-1), W2 (2500 m3·ha-1), and W3 (3350 m3·ha-1). Results:Water and fertilizer management is a prerequisite for high yield of alfalfa seeds, and the impact of fertilization on seed yield is greater than that of irrigation. Compared to the non-fertilized control (F0W1), the F2W2 treatment significantly increased soil nutrients in the 0-20 cm layer: soil total nitrogen content (+52.17%), total phosphorus content (+18.72%), and organic carbon content (+16.85%), and available phosphorus content (+37.34%), and alkali-hydrolyzable nitrogen content (+17.45%). Notably, F2W2 enhanced net photosynthetic rate by 35.04% despite reduced stomatal conductance (-2.14%) and intercellular CO2 concentration (-9.50%), thereby promoting assimilate partitioning to reproductive organs. Consequently, seed dimensional parameters (width: +53.02%; thickness: +21.75%) and germination rate (+23.11%) were significantly improved (P < 0.05), increasing the seed yields of WL354HQ and Xinmu No.4 by 42.76% and 49.81% respectively. Correlation analysis revealed significant (P < 0.01) positive associations between seed yield and seed length, seed width, seed thickness, chlorophyll a, carotenoids, total chlorophyll content, and net photosynthetic rate. Principal component analysis showed that the optimal fertilization level was N 120 kg·ha-1; P2O5-100 kg·ha-1; K2O 15 kg·ha-1, with an irrigation level of 2500 m3·ha-1 (F2W2) as the optimal model. Discussion:This optimized model significantly enhanced alfalfa seed yield formation, photosynthetic characteristics, and soil nutrient availability, which provided a theoretical basis for high yield cultivation of alfalfa seed production in arid areas.

Background: Nettle is a potential non-conventional feed resource due to its high level of crude protein content, and ensiling is better for utilization in the animal industry. Previous integrated analysis (microbiome and metabolome) suggested that 4-pentenoic acid and malic acid in ensiled nettle may inhibit harmful microorganisms within the system. The present study investigated the effects of these two acids on nettle silage quality through the addition of 1% fresh weight of these acids, then analyzed the characteristics and bacterial communities during 60 days of nettle ensiling. Results: The addition of 4-pentenoic acid increased the content of crude protein (CP) and acetic acid (AA) compared with both control and malic acid-treated groups during 30 to 60 days of ensiling (p < 0.05). Lactic acid (LA) content was highest in the malic acid-treated group (4.21%, dry matter, DM based) compared to the control and 4-pentenoic acid-treated groups after 7 days of nettle ensiling (p < 0.05), but lower compared with the 4-pentenoic acid-treated group after 30 days of nettle ensiling (p < 0.05), and it was not detected in all groups after 60 days of silage. The contents of butyric acid (BA) and ammonia (AN) were the lowest (2.92–4.39% of DM and 9.94–24.28% of total nitrogen, respectively) in the 4-pentenoic-treated group compared with both control and malic acid-treated groups during 30 to 60 days of ensiling (p < 0.05). Both acids increased the relative abundance of Weissella after 30 days of nettle ensiling, with 4-pentenoic acid showing a higher inhibitory capacity. Both acids showed a trend to inhibit the relative abundance of Clostridium sensu stricto 15 after 30 days of nettle ensiling. Clostridium sensu stricto 15 showed significant positive correlation with BA and AN (p < 0.05). Conclusions: The results of the present study suggested that the addition of 4-pentenoic could improve the quality of silage by reducing levels of protein degradation, probably resulting from its inhibited activity against Clostridium spp. However, malic acid was less effective than 4-pentenoic acid in suppressing Clostridium spp. activity and the associated production of BA and AN, resulting in inferior preservation of CP.

Climate change due to global warming increases the susceptibility of plants to multiple combined stresses. Soil salinization and high temperature stresses that co-occur in arid/semiarid regions severely restrict the growth and development of plants. Although alfalfa (Medicago sativa L.) is an important forage grass, the physiological mechanisms driving its responses to combined salt and heat stress are not yet clear. This study aimed to reveal the physiological and biochemical response mechanisms of six alfalfa cultivars to different stresses by comparing plant morphology, agronomic traits, photosynthetic characteristics, and physiological and biochemical responses under control conditions, salt stress (200 mM NaCl), heat stress (38 °C), and combined salt and heat stress. Compared with single stresses, combined stress significantly inhibited the growth and biomass accumulation of alfalfa. Under combined stress, the cultivars presented decreases in plant height and total fresh biomass of 11.87–26.49% and 28.22–39.97%, respectively, compared with those of the control plants. Heat stress promoted alfalfa photosynthesis by increasing stomatal conductance, net photosynthetic rate, and transpiration rate, while salt stress and combined stress significantly suppressed these effects. Combined stress significantly increased the concentration of Na+ but decreased that of K+ and the relative water content in alfalfa leaves. Compared with the control and single stress treatments, combined stress significantly increased the level of membrane lipid peroxidation and accumulation of reactive oxygen species. The proline contents in the leaves of the different alfalfa cultivars were 2.79–11.26 times greater under combined stress than in the control. Combined stress causes alfalfa to redistribute energy from growth and development to stress defense pathways, ultimately leading to a reduction in biomass. Our study provides theoretical guidance for analyzing the mechanisms of grass resistance to combined salt and heat stress.

Maize silage serves as a crucial feed resource for ruminants, yet its quality is frequently compromised during storage by spoilage-associated microbial activity. Clostridium species, particularly Clostridium beijerinckii, are known to induce spoilage by altering fermentation pathways. This study aimed to elucidate the effects of inoculation with C. beijerinckii SHZ-8 on microbial succession, metabolite profiles, and fermentation quality in whole-crop maize silage throughout the spoilage process. Silage samples were prepared with and without C. beijerinckii SHZ-8 inoculation. Microbial community dynamics were assessed via 16S rDNA sequencing, while metabolite alterations were characterized using untargeted metabolomics. Fermentation parameters including nutrient composition, bacterial counts, and organic acid concentrations and ratios were also determined. Correlation analyses between key metabolites and core microbial taxa were conducted. Inoculation with C. beijerinckii SHZ-8 significantly reduced dry matter content by 5.28% (p < 0.01) and lactic acid bacteria counts by 54.51% (p < 0.01), while increasing Clostridium abundance by 3.40 log₁₀ CFU/g FW (p < 0.01). The dominant fermentation mode shifted from homofermentation to heterofermentation, accompanied by an 81.6% decrease in the lactate-to-acetate ratio (p < 0.01). D-galacturonic acid levels exhibited a strong positive correlation with C. beijerinckii SHZ-8 abundance (R2 = 0.87, p < 0.01), suggesting its potential as a biomarker for Clostridium overgrowth. Notably, octanal and D-galacturonic acid emerged as candidate biomarkers in the inoculated group, providing a basis for the development of silage quality monitoring tools. These findings offer valuable insights for improving silage management strategies, enhancing feed preservation, and advancing the sustainability of livestock production.

Repeated fertilizer applications to different monoculture cropping systems can alter soil nutrients and microbial community structure. Here we investigate the impact of long-term (4 year) distinct nitrogen (N) and phosphorus (P) fertilizer treatments on rhizosphere physicochemical characteristic and soil microbial community composition in an alfalfa (Medicago sativa L.) cropping systems. N and P fertilizer significantly influenced the physicochemical properties and stoichiometry of alfalfa rhizosphere soil. Nevertheless, N and P fertilizers application on the rhizosphere bacterial and fungal community structures were inconsistent. Fertilizer application minimally metamorphose the rhizosphere bacteria and fungi richness (Sobs index) and diversity (Shannon index). Non-metric multidimensional scaling analysis (NMDS) revealed that fertilizer treatments have no significant influence the fungal community, however, they significantly altered the bacterial community. Bacterial dominant phyla, Actinobacteriota, Acidobacteriota, Chloroflexi, and Gemmatimonadota changed significantly, indicating that the composition of the bacterial community was more responsive to fertilizer application when compared to fungal community composition. Spearman correlation analysis demonstrated no significant correlation amidst soil factors and bacterial diversity, conversely, bacterial richness, fungal diversity and richness were significantly modified by soil factors (AP, AN, and C/N). Network analysis indicated that N application reduced the positive associations between bacteria and fungi, whereas P application enhanced the positive associations. In conclusion, fertilization changes soil fertility of alfalfa fields and the bacterial community composition. Additionally, tests on phosphate solubilizing bacteria (PSB) isolated from the rhizosphere soil of alfalfa demonstrated that these bacteria could significantly enhance the biomass of alfalfa.