Search Results (477)

Abiotic stress of cold is one of the limitation factors that hinder the production of alfalfa (Medicago sativa). Although there are a large number of studies suggesting that non-coding RNAs (ncRNAs) play an important role in plant response to abiotic stress, the mechanism by which ncRNAs and competing endogenous RNAs (ceRNAs) influence the low-temperature tolerance of alfalfa remains understudied. In this study, we integrated whole-transcriptome RNA-seq and genome-wide association studies (GWASs) to identify cold stress-related metabolic pathways and candidate genes, differentially expressed (DE) mRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Degradome sequencing was used to verify the ceRNA network under cold stress. A total of 46,936 DEmRNAs were identified. Ribosome (ko03010), amino sugar and nucleotide sugar metabolism (ko00520), ribosome biogenesis in eukaryotes (ko03008), circadian rhythm–plant (ko00270), and starch and sucrose metabolism (ko00500) were the top five KEGG terms with the highest p-value, enriching the most number of DEmRNAs. MS.gene53818 (MsUAM1) was considered to be the critical candidate gene for alfalfa response to cold stress by conjoint analysis of GWASs and DEmRNAs. A total of 223 DEmiRNAs, 1852 DElncRNAs, and 13 DEcircRNAs were identified under cold stress. Functional analysis indicates that they play important roles in GO terms such as leaf development (GO:0048366), DNA-binding transcription factor activity (GO:0003700), central vacuole (GO:0042807), response to auxin (GO:0009733), and water channel activity (GO:0015250), as well as in KEGG pathways such as plant hormone signal transduction, starch and sucrose metabolism, and flavone and flavonol biosynthesis (ko00944). A ceRNA network comprising 28 DElncRNAs, 8 DEcircRNAs, 11 DEmiRNAs, and 23 DEmRNA triplets was constructed. In this study, mRNAs and ncRNAs were identified that may be involved in alfalfa’s response to cold stress, and a ceRNA regulatory network related to cold stress was established, providing valuable genic resources for further research on the molecular mechanisms underlying alfalfa cold stress. Full article

Soil salinization and alkalinization critically constrain alfalfa (Medicago sativa L.) productivity, yet the regulatory mechanisms underlying its responses to salt–alkali stress are not fully understood. In this study, the alfalfa variety “Zhongmu No. 1” was used as experimental material. The seeds were subjected to salt stress (75 mM NaCl), alkali stress (15 mM NaHCO₃), and combined salt–alkali stress (50 mM NaCl + 5 mM NaHCO₃) in dishes, with ddH₂O serving as the control (CK). After 7 days of germination, the seedlings were transferred to a hydroponic system containing Hoagland nutrient solution supplemented with the corresponding treatments. Following 32 days of stress exposure, leaf and root tissue samples were collected for morphological and physiological measurements, as well as mRNA and miRNA sequencing analyses. Physiological assays revealed significant growth inhibition and increased electrolyte leakage under stress conditions. Transcriptome profiling identified over 5000 common differentially expressed genes (DEGs) in both leaves and roots under stress conditions, mainly enriched in pathways related to “iron ion binding”, “flavonoid biosynthesis”, “MAPK signaling”, and “alpha-Linolenic acid metabolism”. MiRNA sequencing detected 453 miRNAs, including 188 novel candidates, with several differentially expressed miRNAs (DEMs) exhibiting tissue- and stress-specific patterns. Integrated analysis revealed 147, 81, and 140 negatively correlated miRNA–mRNA pairs across three treatment groups, highlighting key regulatory modules in hormone signaling and metabolic pathways. Notably, in the ethylene and abscisic acid signaling pathways, ERF (XLOC_006645) and PP2C (MsG0180000476.01) were found to be regulated by miR5255 and miR172c, respectively, suggesting a post-transcriptional layer of hormonal control. DEM target genes enrichment pathway analyses also identified stress-specific regulation of “Fatty acid degradation”, “Galactose metabolism”, and “Fructose and mannose metabolism”. qRT-PCR validation confirmed the expression trends of selected DEGs and DEMs. Collectively, these findings reveal the complexity of miRNA–mRNA regulatory networks in alfalfa’s response to salt–alkali stress and provide candidate regulators for breeding stress-resilient cultivars. Full article

Plant growth-promoting (PGP) bacteria are beneficial microbes that can help plants mitigate various biotic and abiotic stresses through different PGP functions. Flavins (FLs) are involved in flavoprotein-mediated reactions essential for plant metabolism and could act as PGP molecules. The aim of this study was to isolate and characterize potential FLs secreting bacteria from apple (Malus domestica [Suckow] Borkh) roots based on their fluorescence and to evaluate their PGP properties, including FLs secretion. A total of 26 bacteria with increased fluorescence in liquid culture were isolated from the apple roots. Based on 16S rRNA sequencing analysis, 11 genetically different strains mostly from Burkholderia and Rhizobia spp. were identified. All isolates secreted considerable amounts of riboflavin. In vitro plant assays showed that under nitrogen (N) limitation, inoculated alfalfa (Medicago sativa) plants yielded at least 25% more dry mass than non-inoculated plants, and inoculation with AK7 and FL112 enriched plant tissue N content compared to non-inoculated plants. This improved N acquisition was not linked to symbiotic N fixation. Additionally, the isolates exhibited some other PGP properties. However, no specific PGP functions were linked to improved plant N acquisition but could potentially be linked to the FLs secretion. For future investigation, the mechanisms underlying improved plant N uptake should be assessed to gain a more in-depth understanding. Full article

Metal pollution from metallurgical emissions poses serious environmental and public health risks in Kazakhstan. A replicated pot-culture experiment (n = 4) in a completely randomized design under controlled phytotron conditions evaluated biomass production and metal accumulation in six crop and forage species, alfalfa (Medicago sativa), amaranth (Amaranthus spp.), corn (Zea mays), mustard (Brassica juncea), rapeseed (Brassica napus), and sunflower (Helianthus annuus); three ornamental species, purple coneflower (Echinacea purpurea), marigold (Tagetes spp., ‘Tiger Eyes’), and sweet alyssum (Lobularia maritima); and three native wild plants, greater burdock (Arctium lappa), horse sorrel (Rumex confertus), and mug wort (Artemisia vulgaris). Plants were grown in soils collected from the Qarmet industrial zone in Temirtau, central Kazakhstan. Initial soil analysis revealed substantial mixed-metal contamination, ranked as Mn > Ba > Zn > Sr > Cr > Pb > Cu > Ni > B > Co. Mn reached 1059 mg·kg⁻¹, ~50-fold higher than B (22.7 mg·kg⁻¹). Ba (620 mg·kg⁻¹) exceeded FAO/WHO limits sixfold, Zn (204 mg·kg⁻¹) surpassed the lower threshold, and Pb (41.6 mg·kg⁻¹) approached permissible levels, while Cr, Cu, Ni, Co, and Sr were lower. Biomass production varied markedly among species: corn and sunflower produced the highest shoot biomass (126.8 and 60.9 g·plant⁻¹), whereas horse sorrel had the greatest root biomass (54.4 g·plant⁻¹). Root-to-shoot ratios indicated shoot-oriented growth (>1–8) in most species, except horse sorrel and burdock (<1). Metal accumulation was strongly species-specific. Corn and marigold accumulated Co, Pb, Cr, Mn, Ni, Cu, B, and Ba but showed limited translocation (transfer function, TF < 0.5), whereas sunflower, amaranth, and mug wort exhibited moderate to high translocation (TF > 0.8 to <1) for selected metals. Corn is recommended for high-biomass metal removal, marigold for stabilization, sunflower, horse sorrel, and mug wort for multi-metal extraction, and amaranth and coneflower for targeted Co, Ni, and Cu translocation, supporting sustainable remediation of industrially contaminated soils. Full article

Alfalfa (Medicago sativa L.) is an important forage crop with significant economic value. Alternating partial root-zone irrigation (APRI) is a promising water-saving technique that has been shown to improve water use efficiency in various crops. In this study, the effects of APRI on root water uptake, photosynthetic indices, and physiological responses in alfalfa were investigated. Polyethylene glycol (PEG 6000) was used to simulate water stress, and four irrigation treatments were established: conventional irrigation (CI), deficit irrigation (DI), fixed partial root-zone irrigation (FPRI), and APRI. Principal component analysis (PCA) revealed that APRI reduced stomatal conductance (Gs) by 19.82% and transpiration rate (E) by 19.16%, which was associated with increased abscisic acid (ABA) content, thereby enhancing instantaneous water use efficiency (iWUE) by 47.93%. Meanwhile, APRI promoted root growth, leading to a 14.09% increase in root–shoot ratio, which in turn enhanced the photosynthetic rate by 22.06%. APRI enhanced methyl jasmonate (MeJA) content in alfalfa leaves by 45.23%, which was associated with a 24.13% improvement in water absorption capacity. In conclusion, APRI induced positive physiological responses in alfalfa, with the effectiveness ranked as follows: APRI > CI > FPRI > DI. These findings provide a theoretical basis for the rational application of APRI in alfalfa forage production. Full article

Water scarcity and poor soil fertility are major limiting factors constraining agricultural production in the arid and semi-arid regions of Northwest China. Water–nitrogen synergistic regulation is an important approach to improving crop growth and enhancing agricultural productivity. In this study, four irrigation levels—severe water deficit (W1: 45–65% θ_f), moderate water deficit (W2: 55–70% θ_f), mild water deficit (W3: 65–80% θ_f), and full irrigation (W4: 75–90% θ_f)—and four nitrogen application rates—no nitrogen (N0, 0 kg·ha⁻¹), low nitrogen (N1, 80 kg·ha⁻¹), medium nitrogen (N2, 160 kg·ha⁻¹), and high nitrogen (N3, 240 kg·ha⁻¹)—were established to systematically analyze the effects of water–nitrogen coupling on osmotic adjustment substances, yield, and forage quality of alfalfa (Medicago sativa L.) leaves. The results showed that: (1) Proline (Pro) content increased significantly with intensified water deficit, with W1 being 82.29% higher than W4 on average. Soluble protein (SP) and soluble sugar (SS) contents increased with increasing water availability, with their average values under W4 being 26.50% and 36.92% higher than those under W1, respectively. Increasing nitrogen application significantly improved the accumulation of osmotic adjustment substances, with Pro reaching the lowest value at N2, SP peaking at N2, and SS peaking at N3. (2) Yield increased significantly with higher irrigation, and increased first and then decreased with nitrogen application. Yield under W4 was 94.20% higher than under W1, and N2 increased yield by 12.45–50.65% compared with other nitrogen levels. (3) Under the W4N2 treatment, crude protein (CP) content and relative feed value (RFV) increased by 34.54% and 51.10%, respectively, compared with W1N0, while acid detergent fiber (ADF) and neutral detergent fiber (NDF) decreased by 28.74% and 24.44%, respectively. (4) Correlation analysis indicated that Pro content was significantly positively correlated with ADF and NDF but negatively correlated with yield, CP, and RFV. In contrast, SP and SS contents were significantly negatively correlated with ADF and NDF and positively correlated with yield, CP, and RFV. (5) Principal component analysis identified that the combination of full irrigation (W4: 75–90% θ_f) and medium nitrogen application (N2, 160 kg·ha⁻¹) optimizes both yield and forage quality by balancing osmotic adjustment substances. Full article

Oats (Avena sativa L.) are a staple grain and forage crop with substantial market demand. In China, they are the second most-imported forage grass, only after alfalfa (Medicago sativa L.). Enhancing the salt tolerance of oats to facilitate their cultivation in saline areas can thereby increase forage yield and promote the utilization of saline land, which constitutes an important reserve land resource in China. This study aimed to identify the bacterial strain Bacillus sp. LrM2 (hereafter referred to as strain LrM2) to determine its precise species-level classification and evaluate its effects on oat photosynthesis and growth under salt stress through indoor pot experiments. The results indicated that strain LrM2, capable of urease production and citrate utilization, was identified as Bacillus mojavensis. The strain LrM2 had a positive effect on shoot and root growth of oats under 100 mM NaCl stress conditions. Strain LrM2 inoculation modulated osmotic stress in oats under 100 mM NaCl stress by significantly increasing soluble sugar and decreasing proline content in leaves. It inhibited Na⁺ uptake and promoted K⁺ absorption in the roots, thereby reducing Na⁺ translocation to the leaves and mitigating ionic toxicity. Inoculation with strain LrM2 significantly increased photosynthetic pigment content (chlorophyll a, carotenoids), improved gas exchange parameters (stomatal conductance, transpiration rate, net rate of photosynthesis), enhanced PSII photochemical efficiency (maximum quantum yield, coefficient of photochemical quenching, actual photosynthetic efficiency of PSII, electron transfer rate), and reduced the quantum yield of non-regulated energy dissipation. These improvements, coupled with increased relative water content and instantaneous water use efficiency, thereby collectively enhanced the overall photosynthetic performance. In conclusion, strain LrM2 represents a promising bio-resource for mitigating salt stress and promoting growth in oats, with direct applications for developing novel biofertilizers and sustainable agricultural strategies. Full article

The goal of this study was to elucidate the genetic and molecular regulatory mechanisms underlying agronomic traits in elite alfalfa (Medicago sativa L.). Through the analysis of 44 varieties and lines, we measured 19 yield-related traits and performed transcriptome sequencing to investigate the factors driving yield variation. The results indicated extensive variation in agronomic traits among the tested accessions, with the coefficients of variation (CVs) ranging from 7.85% to 42.66%, suggesting substantial potential for genetic improvement. Correlation analysis revealed that seed yield was significantly and positively correlated with the number of reproductive branches and inflorescences at maturity, whereas early vegetative growth was negatively correlated with 100-seed weight. The 44 accessions were categorized into three clusters: Cluster II (the largest group) exhibited balanced traits; Cluster I showed vigorous early growth but low pod yield; and Cluster III was characterized by the highest pod and branch numbers. Principal Component Analysis (PCA) explained 65.88% of the total variation (first six components), identifying GNS31 and GNS43 as the superior and inferior genotypes, respectively. Furthermore, transcriptome profiling detected the highest number of differentially expressed genes (10,089 DEGs) in pod tissues, with 66% being upregulated. Functional enrichment analyses (GO and KEGG) highlighted that varietal differences were primarily enriched in secondary metabolism, lipid metabolism, and plant hormone signal transduction pathways. Notably, within the auxin pathway, the SAUR and GH3 families displayed significant tissue-specific expression in pods. Full article

Phosphorus (P) is a key nutrient limiting crop growth and productivity, particularly in saline-alkali soils with low P availability. Arbuscular mycorrhizal fungi (AMF) have the potential to enhance P uptake in alfalfa (Medicago sativa L.); however, the synergistic effects and underlying biological mechanisms by which AMF improve P acquisition and utilization efficiency under varying P application levels remain unclear. To explore P acquisition strategies associated with AMF status, root morphology traits, rhizosphere carboxylate exudation, soil properties and microbial biomass, we conducted a pot experiment growing alfalfa in saline-alkali soil under four P application levels (0, 5, 10, and 20 mg kg⁻¹), with or without AMF inoculation. Our results showed that AMF colonization and P application synergistically increased alfalfa biomass and shoot/root P concentrations. Notably, at a low P application level of 5 mg kg⁻¹, the mycorrhizal contribution to P absorption and P-utilization efficiency reached their highest levels, while both declined under high P conditions (20 mg kg⁻¹), suggesting an interaction between P availability and AMF efficacy. Structural equation modeling (SEM) and regression analysis revealed that rhizosphere carboxylate concentrations were positively associated with P-utilization efficiency, whereas soil available P, microbial biomass P (MBP) and carbon (MBC) negatively affected it. Among these factors, AMF-induced enhancement of rhizosphere carboxylate exudation played a critical role in promoting P-utilization efficiency in alfalfa under low-P conditions. In contrast, higher P availability reduced rhizosphere carboxylate concentrations, resulting in lower P-utilization efficiency. In conclusion, the combination of AMF colonization and low P application synergistically improves P acquisition and utilization efficiency in alfalfa, providing valuable insights for sustainable nutrient management in saline-alkali soils with limited P availability. Full article

Applying non-thermal plasma (NTP) to seeds prior to sowing is recognized for its ability to enhance germination and promote plant growth. This study investigated the effects of NTP seed treatment on alfalfa seed surface characterization, germination, growth, and biochemical traits under varying water conditions. NTP modified seed surface properties by decreasing water contact angle, roughening the coat, and reducing O–H/N–H and C–H band intensities, while major functional groups remained intact. Short plasma exposures (<2 min) enhanced germination, whereas prolonged treatment (10 min) reduced viability, indicating embryo sensitivity. In pot experiments, both 1 and 5 min treatments improved fresh and dry weight, stem and root elongation, pigment accumulation, and protein content, particularly under normal and moderate water stress, while extended exposure (10 min) offered limited benefits and could be detrimental under severe drought. Root growth was most responsive, suggesting enhanced water and nutrient uptake. Plasma had modest effects on polyphenols and flavonoids but influenced early physiological responses and antioxidant activity. These findings highlight NTP as a promising seed priming tool to improve alfalfa performance, though further studies are needed to clarify the mechanisms and specific contributions of plasma components. Full article

Alfalfa (Medicago sativa L.), a perennial leguminous herb, can tolerate cold and saline–alkaline conditions. In this study, alfalfa cultivars LJ and 218TR were exposed to saline–alkaline, cold, and saline–alkaline–cold conditions and compared in terms of phenotypes, physiological indices, key metabolite contents, and stress-responsive gene expression. Malondialdehyde, soluble sugar, proline contents and phenylalanine ammonia-lyase (PAL), superoxide dismutase, catalase, and peroxidase activities initially increased under individual stress conditions, but decreased when stresses were combined. Photosystem II maximum photochemical efficiency and chlorophyll contents decreased under individual and combined stress conditions. Nitroblue tetrazolium-stained leaves revealed that the combined stress treatment significantly increased cell mortality rates and superoxide anion levels. LJ was more tolerant to saline–alkaline, cold, and combined stress treatments than 218TR. Metabolite analyses indicated that for LJ and 218TR, salicylic acid (SA) was the most responsive metabolite to combined stress conditions. Additionally, the expression of isochorismate synthase (ICS) and PAL genes critical for SA biosynthesis was upregulated under single or combined stress conditions, leading to SA accumulation and improved tolerance to saline–alkaline–cold conditions. This study revealed the physiological indices and molecular changes underlying alfalfa responses to saline–alkaline stress combined with cold stress, providing a theoretical basis for breeding stress-tolerant cultivars. Full article

The Aras Basin, located at the intersection of three major gene centers, represents one of the most important transition zones for the evolution of forage legumes. This study evaluates the genetic diversity and population structure of 74 Medicago sativa genotypes, including wild populations and commercial cultivars, using ISSR markers. The analysis revealed a broad level of genetic variability, reflecting the adaptive potential of alfalfa in this ecologically heterogeneous region. Population structure analyses consistently separated the germplasm into three genetic clusters, demonstrating clear differentiation between wild accessions and registered varieties. Geographical patterns were also evident, with genotypes from western, central, and eastern subregions forming distinct groups. These results highlight the unique genomic composition of alfalfa in the Aras Basin and demonstrate the value of ISSR markers for characterizing multilayered genetic variation in ecological transition zones. The findings provide a complementary genomic perspective that expands existing knowledge of M. sativa diversity and offers useful guidance for breeding programs and genetic resource conservation. Full article

Alfalfa (Medicago sativa) is a globally important forage crop; however, improvements in its biomass yield have stagnated due to its complex genetic architecture and the costly, labor-intensive phenotyping. This study evaluated the potential of the normalized difference vegetation index (NDVI) to predict biomass yield and enhance selection efficiency in alfalfa breeding programs. Specifically, nineteen alfalfa experimental populations (AEXP 1–19) and one control cultivar (OS 66) were evaluated over two growing seasons in Croatia. NDVI was measured at four development stages using a GreenSeeker sensor and compared with forage yield, dry matter yield, and plant height. NDVI values varied significantly among genotypes, years, and growth stages, ranging from 0.23 to 0.87, and increased consistently from early to late vegetative phases. Strong positive correlations were observed between NDVI and forage yield (r = 0.543–0.843) and plant height (r = 0.537–0.738) at early vegetative, late vegetative, and early bud stages. Conversely, NDVI at the mid-vegetative stage correlated negatively with yield and height (r = –0.622 to –0.794). High-performing populations (AEXP 2, AEXP 15, AEXP 18) also exhibited the highest NDVI values. NDVI is a reliable, non-destructive indicator for early selection of high-yielding alfalfa genotypes, although multi-location validation is advised to confirm its broader applicability. Full article

Background: The Songnen Plain in China contains soda saline–alkaline soil, wherein salinity and alkalinity severely constrain crop productivity. Alfalfa (Medicago sativa L.) is a forage legume that has adapted to moderate saline–alkaline conditions, but how its rhizosphere microbial community facilitated this adaptation remains unclear. Methods: Using 16S rRNA gene sequencing, we compared alfalfa rhizosphere bacteria in saline–alkaline soil (AS) and control soil. Bacteria isolated from AS were screened for plant growth-promoting traits, with the most effective strains validated in pot experiments involving 50 mM NaHCO₃. Results: Compared with the control soil bacterial community, the AS bacterial community was significantly enriched with Methylomirabilota and unclassified bacteria (phylum level), with the genus RB41 identified as the most discriminative biomarker. Gene functions predicted using PICRUSt2 reflected the responsiveness of this community to environmental stressors. Inoculations with Pseudomonas laurentiana strain M73 and Stenotrophomonas maltophilia strain M81, which were isolated from AS, significantly improved alfalfa growth and health under NaHCO₃ stress. Conclusions: Saline–alkaline conditions in the Songnen Plain reshape the alfalfa rhizosphere bacterial community, enriching for specific taxa and potentially enhancing microbial functions associated with stress resistance. Strains M73 and M81 can effectively promote stress tolerance, making them useful microbial resources for improving soil conditions. Full article

Alfalfa (Medicago sativa L.) is an underutilized source of phytonutrients and easily digestible protein, containing all essential amino acids, highlighting its potential for food applications. This study aimed to produce alfalfa protein concentrates (APC) from frozen aerial parts and evaluate how conventional extraction and ultrasound-assisted extraction (UAE) affect the extraction yield, physicochemical properties, functional attributes, color parameters, phytochemical composition and antioxidant activity. The influence of extraction pH and the type of acid used for isoelectric precipitation was also evaluated. Paired t-tests (p ≤ 0.05) showed that UAE (37 kHz, 25 °C, 15 min) increased the extraction yield by 20.5–39.7%, the protein content in APC by 2.5–12.1% and the in vitro protein digestibility by 5.6–11.03%, depending on the extraction conditions. Ultrasound treatment decreased the levels of chlorophyll and carotenoids, modified the color parameters and increased the total polyphenols and flavonoids content. Improvements in the textural, foaming and emulsifying properties of APC were also observed. UAE also reduced the scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. However, the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•⁺) scavenging activity significantly increased in aqueous APC extracts, reaching 3118.8 mg TE/100 g DW. Overall, UAE proved effective in improving the yield and functionality of APC, supporting its application in the development of alfalfa-based protein ingredients. Full article