Soil salinity is a severe abiotic stress that damages plant growth and development. As an antioxidant and free radical scavenger, melatonin is well known for helping plants survive abiotic conditions, including salinity stress. Here, we report that the salt-related gene MsSNAT1, encoding a rate-limiting melatonin biosynthesis enzyme, is located in the chloroplast and contributes to salinity stress tolerance in alfalfa. We found that the MsSNAT1 overexpressing alfalfa lines exhibited higher endogenous melatonin levels and increased tolerance to salt stress by promoting antioxidant systems and improving ion homeostasis. Furthermore, through a combination of transcriptome sequencing, dual-luciferase assays and transgenic analysis, we identified that the basic leucine zipper (bZIP) transcription factor, MsbZIP55, is associated with salt response and MsSNAT1 expression. EMSA analysis and ChIP-qPCR uncovered that MsbZIP55 can recognize and directly bind to the MsSNAT1 promoter in vitro and in vivo. MsbZIP55 acts as a negative regulator of MsSNAT1 expression, thereby reducing melatonin biosynthesis. Morphological analysis revealed that overexpressing MsbZIP55 conferred salt sensitivity to transgenic alfalfa through a higher Na+/K+ ratio and lower antioxidant activities, which could be alleviated by applying exogenous melatonin. Silencing of MsbZIP55 by RNA interference in alfalfa resulted in higher expression of MsSNAT1 and promoted salt tolerance by enhancing the antioxidant system enzyme activities and ion homeostasis. Our findings indicate that the MsbZIP55-MsSNAT1 module plays a crucial role in regulating melatonin biosynthesis in alfalfa while facilitating protection against salinity stress. These results shed light on the regulatory mechanism of melatonin biosynthesis related to the salinity stress response in alfalfa.

5-Aminolevulinic acid (5-ALA) is a plant growth regulator, but its effect on alfalfa (Medicago sativa L.) tolerance to salinity stress is limited. The objective of this study was to investigate the impact of foliar application of 5-ALA on alleviating NaCl-induced salinity stress in alfalfa. Four seedlings' treatments in soil culture, including control (CK), 0.1 mmol L-1 5-ALA, 150 mmol L-1 NaCl, and 150 mmol L-1 NaCl + 0.1 mmol L-1 5-ALA, were conducted for measurement using methods at morphological, physiological and ultrastructural levels. The results showed that salinity stress inhibited leaf size, leaf number, shoot height, and biomass. Similarly, salinity stress decreased photosynthesis by degrading pigments, reducing photosynthetic gas exchange parameters, increasing stomatal closure and damaging leaf ultrastructure. Additionally, salinity-induced disruptions in ion homeostasis, osmotic balance, and oxidative equilibrium exacerbated plant stress. However, foliar application of 5-ALA proved instrumental in mitigating these detrimental effects. Notably, 5-ALA treatment bolstered growth metrics, enhanced pigment biosynthesis, improved photosynthetic performance, facilitated stomatal regulation, and preserved leaf morphology. Moreover, 5-ALA treatment effectively modulated ion transport, osmotic regulation, and redox balance by attenuating Na+ accumulation, reactive oxygen species production, and lipid peroxidation, while bolstering cellular membrane integrity, osmoprotective mechanisms, and antioxidant defenses. Correlation and principal component analyses underscored the interplay and synergistic effects of these pathways. 5-ALA has a multifaceted role in mitigating salinity stress in alfalfa, and this study underscores the efficacy of 5-ALA as a proactive strategy for enhancing salinity tolerance, growth, and productivity in alfalfa cultivation.

Repeated wet swelling and dry shrinkage of soil leads to the gradual occurrence of cracks and the formation of a complex fracture network. In order to study the development characteristics and quantitative analysis of cracks in root-soil complex in different growth periods under dry-wet cycles, the alfalfa root-loess complex was investigated during different growth periods under different dry-wet cycles, and a dry-wet cycle experiment was conducted. The crack rate, relative area, average width, total length, and the cracks fractal dimension in the rootsoil complex were extracted; the crack development characteristics of plain soil were analyzed under the PGDWC (dry-wet cycle caused by plant water management during plant growth period), as well as the crack development characteristics of root-soil complex under PG-DWC and EC-DWC (the dry-wet cycles caused by extreme natural conditions such as continuous rain); the effects of plant roots and dry-wet cycles on soil cracks were discussed. The results showed that the average crack width, crack rate, relative crack area, and total crack length of the alfalfa root-loess complex were higher than those of the plain soil during PG-DWC. The result indicated that compared with plain soil during PG-DWC, the presence of plant roots in alfalfa root-soil complex in the same growth period promoted the cracks development to some extent. The alfalfa root-soil complex crack parameters during different growth periods were relatively stable during PG-DWC (0 dry-wet cycle). During ECDWC (1, 3, and 5 dry-wet cycles), the alfalfa root-loess complex crack parameters increased with the number of dry-wet cycles during different growth periods. Unlike PG-DWC, the EC-DWC accelerated crack development, and the degree of crack development increased with the number of dry-wet cycles. The existence of plant roots promoted crack development and expansion in the root-soil complex to a certain extent, and the dry-wet cycle certainly promoted crack development and expansion in the root-soil complex. This result contradicts the improvement in the root-soil complex's macro-mechanical properties during plant growth, due to differences in the mechanical properties of roots and soil. The research results will provide reference for the root soil complex crack development law and the design of slope protection by vegetation.

Plastic packaging has increased concerns about human health and the ecosystem due to non-biodegradability. Several biopolymers, such as cellulose, starch, and proteins, are being explored, and cellulosic residue from agricultural biomass is suitable to overcome this predicament. Herein, cellulosic residue fibers (ACR) extracted from alfalfa were used to prepare biodegradable films by solubilizing them in ZnCl2 solution and crosslinking the chains with calcium ions (Ca2+) and sorbitol. Box Behnken Design optimized the ACR, CaCl2, and sorbitol amounts against the responses of water vapor permeability (WVP), tensile strength (TS), and elongation at break (EB). The optimized film combination was found to be 0.5 g ACR, 461.3 mM CaCl2, and 1.05% sorbitol, making a 12 x 12 cm2 film, with a TS of 16.9 +/- 0.4 MPa, EB of 10.1 +/- 0.3%, and WVP of 0.47 +/- 0.11 x 10- 10 g.m- 1.s- 1. Pa- 1. It was translucent, blocked UVB light, followed Peleg's water absorption kinetics, displayed anti-oxidant activity, and biodegraded within 35 days at 24 % soil moisture. The ACR film extends the shelf life of strawberries by two more days compared to polystyrene film. The outcome offers a novel path to utilize and conserve natural resources and mitigate plastic perils, promoting a circular bioeconomy and sustainability and a win-win situation between the environment and farmers.

Grassland degradation and reduced yields are often linked to the root soil composite of perennial alfalfa roots. This study introduces a novel modeling approach to accurately characterize root biomechanical properties, assist in the design of soil-loosening and root-cutting tools. Our model conceptualizes the root as a composite structure of cortex and stele, applying transversely isotropic properties to the stele and isotropic properties to the cortex. Material parameters were derived from longitudinal tension, longitudinal compression, transverse compression, and shear tests. The constitutive model of stele was Hashin failure criteria, accounting for differences in tensile and compressive strengths. Results reveal that root tensile strength mainly depends on the stele, with its tensile properties exceeding compressive and transverse strengths by 4-10 times. In non-longitudinal tensile stress scenarios, like shear and transverse compression tests, the new model demonstrated superior accuracy over conventional models. Results of shear tests were further validated using non-parametric statistical analysis. This study provides a finite element method (FEM) modeling approach that, by integrating root anatomical features and biomechanical properties, significantly enhances simulation accuracy. This provides a tool for designing low-energy consumption components in grassland degradation restoration and conservation tillage.

Soil salinization negatively affects plant growth and threatens food security. Halotolerant plant growth-promoting bacteria (PGPB) can alleviate salt stress in plants via diverse mechanisms. In the present study, we isolated salt-tolerant bacteria with phosphate-solubilizing abilities from the rhizosphere of Salix linearistipularis, a halophyte distributed in saline-alkali soils. Strain A103 showed high phosphate solubilization activity and was identified as Enterobacter asburiae based on genome analysis. In addition, it can produce indole-3-acetic acid (IAA), siderophores, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Genome mining has also revealed the presence of several functional genes involved in the promotion of plant growth. Inoculation with A103 markedly improved alfalfa growth in the presence of 100 mM NaHCO3. Under alkali stress, the shoot and root dry weights after bacterial inoculation improved by 42.9 % and 21.9 %, respectively. Meanwhile, there was a 35.9-37.1 % increase in the shoot and root lengths after treatment with A103 compared to the NaHCO3-treated group. Soluble sugar content, peroxidase and catalase activities increased in A103-inoculated alfalfa under alkaline stress. A significant decrease in the malondialdehyde content was observed after treatment with strain A103. Metabolomic analysis indicated that strain A103 positively regulated alkali tolerance in alfalfa through the accumulation of metabolites, such as homocarnosine, panthenol, and sorbitol, which could reduce oxidative damage and act as osmolytes. These results suggest that halophytes are valuable resources for bioprospecting halotolerant beneficial bacteria and that the application of halotolerant growth-promoting bacteria is a natural and efficient strategy for developing sustainable agriculture.

Background: Cold resistance in alfalfa (Medicago sativa L.) is significantly influenced by root system type. The root system plays a crucial role in water absorption and soil stress response. This study investigates the physiological and biochemical responses of creeping-rooted and taprooted alfalfa to cold stress during early winter and mid-winter periods. Methods: Samples were collected on November 3, 2023 and January 7, 2024, from the experimental plot. Roots at a depth of 20 cm were cleaned with distilled water and stored in cryopreservation tubes at ultra-low temperatures. Free proline, soluble sugars, soluble proteins, malondialdehyde (MDA), superoxide dismutase (SOD) activity and catalase (CAT) activity were measured using standard biochemical methods. Result: Results indicated that with decreasing temperatures, the contents of soluble sugars, soluble proteins, free proline, MDA and CAT activity increased, whereas SOD activity decreased. In the early overwintering stage, creeping-rooted alfalfa exhibited higher soluble sugar content and SOD activity compared to taprooted alfalfa. During the overwintering period, creeping-rooted alfalfa maintained higher levels of soluble sugars, soluble proteins, MDA and CAT and SOD activities. Principal component analysis identified CAT, MDA, soluble sugars and soluble proteins as key indicators for evaluating cold resistance in alfalfa.

Atrazine, a commonly employed herbicide for corn production, can leave residues in soil, resulting in photosynthetic toxicity and impeding growth in subsequent alfalfa (Medicago sativa L.) crops within alfalfa-corn rotation systems. The molecular regulatory mechanisms by which atrazine affects alfalfa growth and development, particularly its impact on the microbial communities of the alfalfa rhizosphere, are not well understood. This study carried out field experiments to explore the influence of atrazine stress on the biomass, chlorophyll content, antioxidant system, and rhizosphere microbial communities of the atrazine-sensitive alfalfa variety WL-363 and the atrazine-resistant variety JN5010. The results revealed that atrazine significantly reduced WL-363 growth, decreasing plant height by 8.58 cm and root length by 5.42 cm (p < 0.05). Conversely, JN5010 showed minimal reductions, with decreases of 1.96 cm in height and 1.26 cm in root length. Chlorophyll content in WL-363 decreased by 35% under atrazine stress, while in JN5010, it was reduced by only 10%. Reactive oxygen species (ROS) accumulation increased by 60% in WL-363, compared to a 20% increase in JN5010 (p < 0.05 for both). Antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase (CAT), were significantly elevated in JN5010 (p < 0.05), suggesting a more robust defense mechanism. Although the predominant bacterial and fungal abundances in rhizosphere soils remained generally unchanged under atrazine stress, specific microbial groups exhibited variable responses. Notably, Promicromonospora abundance declined in WL-363 but increased in JN5010. FAPROTAX functional predictions indicated shifts in the abundance of microorganisms associated with pesticide degradation, resistance, and microbial structure reconstruction under atrazine stress, displaying different patterns between the two varieties. This study provides insights into how atrazine residues affect alfalfa rhizosphere microorganisms and identifies differential microbial responses to atrazine stress, offering valuable reference data for screening and identifying atrazine-degrading bacteria.

Alfalfa spring black stem and leaf spot disease (ASBS) is a cosmopolitan soil-borne and seed-borne disease caused by Phoma medicaginis, which adversely affects the yield, and nutritive value and can stimulate production of phyto-oestrogenic compounds at levels that may adversely affect ovulation rates in animals. This review summarizes the host range, damage, and symptoms of this disease, and general features of the infection cycle, epidemic occurrence, and disease management. ASBS has been reported from over 40 countries, and often causes severe yield loss. Under greenhouse conditions, reported yield loss was 31-82% for roots, 32-80% for leaves, 21% for stems and 26-28% for seedlings. In field conditions, the forage yield loss is up to 56%, indicating that a single-cut yield of 5302 kg/ha would be reduced to 2347 kg/ha. P. medicaginis can infect up to 50 species of plants, including the genera Medicago, Trifolium, Melilotus, and Vicia. ASBS is more severe during warm spring conditions before the first harvest than in hot summer and cooler winter conditions, and can infect alfalfa roots, stems, leaves, flowers, pods, and seeds, with leaf spot and/or black stem being the most typical symptoms. The primary infection is caused by the overwintering spores and mycelia in the soil, and on seeds and the cortex of dead and dry stems. The use of resistant cultivars is the most economical and effective strategy for the control of ASBS. Although biological control has been studied in the glasshouse and is promising, chemical control is the main control method in agriculture.

Triaxial compression tests were conducted on the alfalfa root-loess complex at different growthperiods obtained through artificial planting. The research focused on analyzing the time variation law of the shear strength index and deformation index of the alfalfa root-loess complex under dry-wet cycles. Additionally, the time effect of the shear strength index of the alfalfa root-loess complex under dry-wet cycles was analyzed and its prediction model was proposed. The results show that the PG-DWC (dry-wet cycle caused by plant water management during plant growth period) causes the peak strength of plain soil to change in a V shape with the increase of growth period, and the peak strength of alfalfa root-loess complex is higher than that of plain soil at the same growth period. The deterioration of the peak strength of alfalfa root-loess complex in the same growth period is aggravated with the increase of drying and wetting cycles. Compared with the 0 days growth period, the effective cohesion of alfalfa root-loess complex under different dry-wet cycles maximum increase rate is at the 180 days, which are 33.88%, 46.05%, 30.12% and 216.02%, respectively. When the number of dry-wet cycles is constant, the effective cohesion of the alfalfa root-loess complex overall increases with the growth period. However, it gradually decreases comparedwith the previous growth period, and the minimum increase rate are all at the 180 days. For the same growth period, the effective cohesion of the alfalfa root-loess complex decreases with the increase of the number of dry-wet cycles. This indicates that EC-DWC (the dry-wet cycles caused by extreme natural conditions such as continuous rain) have a detrimental effect on the time effect of the shear strength of the alfalfa root-loess complex. Finally, based on the formula of total deterioration, a prediction model for the shear strength of the alfalfa root-loess complex under dry-wet cycles was proposed, which exhibits high prediction accuracy. The research results provide useful guidance for the understanding of mechanical behavior and structural damage evolution of root-soil composite.