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.

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.

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.

[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 community during 60 days of nettle ensiling. [Results] Addition with 4-pentenoic acid increased the content of crude protein (CP) and acetic acid (AA) when compared with both control and malic acid treated during 30 to 60 days of ensiling (p＜0.05). Lactic acid (LA) content was highest in malic acid treated (4.21 %, dry matter, DM based) when compared with control and 4-pentenoic acid treated after 7 days of nettle ensiling (p＜0.05), but lower when compared with 4-pentenoic acid treated after 30 days of nettle ensiling (p＜0.05), and was not detected in all groups after 60 days of silage. The content of butyric acid (BA) and ammonia (AN) was lowest (2.92%—4.39% of DM and 9.94 % —24.28% of total nitrogen, respectively) in 4-pentenoic treated when compared with both control and malic acid treated during 30 to 60 days of ensiling (p＜0.05). Both acids treated increased relative abundance of Weissella after 30 days of nettle ensiling, especially for 4-pentenoic acid showed the higher capacity. Both acids treated showed trend to inhibited 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). [Conclusion] Present study results suggested that addition of 4-pentenoic could improve quality of silage due to reduction level of protein degradation probably resulting from its inhibited activity against to 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.

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.