To predict the role of the MsICE gene family in the response to abiotic stress, in this study, bioinformatics analysis and real-time fluorescence quantitative PCR were performed. Alfalfa (Medicago sativa) is one of the most economically valuable crops globally. Inducer of CBF expression (ICE), which is part of the basic helix–loop–helix (bHLH) transcription factor (TF) family, acts as a key regulator of cold tolerance. Despite this, there is little information available about ICE genes in alfalfa. Therefore, we studied the function of ICE TFs in alfalfa. We identified 11 MsICE genes from the alfalfa genome and classified them into two groups. Analysis of the protein motif and gene structure revealed relatively high conservation among subgroups of the tightly clustered MsICE genes. Through synteny analysis, we detected duplication events in the MsICE gene family, suggesting that the ICE gene family was formed through fragment duplications. All the MsICE proteins were located in the nucleus according to subcellular localization predictions. The promoter cis-regulatory elements of MsICE genes are largely involved in light (Box 4), hormone (ABRE), and stress (MYB) responses. The MsICE01/MsICE07/MsICE09/MsICE10/MsICE11 genes contained MYB- and MYC-binding motifs, indicating an association with abiotic stress. The specific expression patterns of MsICE genes in leaves were revealed by examining their expression patterns in different tissues. These findings suggest that these genes may sense external environmental changes through leaves. Abiotic stress can cause striking upregulation of MsICE07 (PCA score: −4.03) and MsICE10 (PCA score: −4.05) expression. In this study, candidate genes associated with cold stress were identified, and subsequent molecular biological analyses allowed elucidation of the biological functions of these genes in alfalfa. This research provides a theoretical foundation for enhancing alfalfa yield and quality under cold conditions.

A high salt environment seriously affects the physiological metabolism and yield of plants. Hordeum brevisubulatum (Trin.) Link has high biomass and important ecological, feeding and economic values, but its growth conditions have serious saline-alkali effects. The NHX gene family plays a vital role in regulating intracellular Na⁺/K⁺ balance, pH homeostasis, and vesicle and protein transport in plants. In this study, the HbNHX2 gene was cloned from Hordeum brevisubulatum and functionally characterized through phenotypic, physiological, and molecular analyses in transgenic tobacco. Expression profiling revealed that HbNHX2 was most abundant in spikes and least abundant in root tips, and the expression level was significantly induced under salt stress. Overexpression of HbNHX2 led to decreased malondialdehyde (MDA) and superoxide anion (O²⁻) levels, while it enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Additionally, the levels of glutathione (GSH), soluble proteins, proline, and chlorophyll were also increased. Several stress-responsive genes, including CBL1, ERF2, BI-1, Cu/Zn-SOD, Mn-SOD, POD, GR1, KC1, TPK1, TIP, P5CS, BAS1, STN7 and LTP1, were significantly upregulated, while SERK3B was downregulated. These findings suggest that HbNHX2 enhances plant salt tolerance by maintaining osmotic balance, scavenging reactive oxygen species (ROS), and regulating stress-responsive gene expression. This study provides new insights into the molecular mechanism of salt tolerance in Hordeum brevisubulatum and lays a foundation for breeding salt-tolerant forage crops.

Oat, an important cereal and forage crop, is significantly affected by drought stress during production. However, the molecular mechanisms underlying oat’s response to drought stress remain largely unknown. In this study, K-means clustering classified 28 oat varieties into drought-tolerant (Muda, Mengshi No. 1) and drought-sensitive (Heike, Haywire) groups, with grey relational analysis further verifying MS as the most drought-tolerant and HK as the most drought-sensitive variety. Under drought stress, drought-tolerant and drought-sensitive varieties showed notable differences in leaf chlorophyll content, osmoregulation substances, and the activities of antioxidant enzymes. Transcriptomic analysis showed that 1915 differentially expressed genes (DEGs) were shared among all comparisons between treatment groups and the control group. KEGG pathway analysis revealed enrichment in pathways such as plant–pathogen interactions, plant hormone signal transduction, and starch and sucrose metabolism. In the signal transduction of plant hormones, eight PP2C genes associated with ABA signaling were increased, indicating that oats might respond to drought by enhancing metabolic activities via the ABA signaling pathway. WGCNA identified gene modules significantly associated with physiological traits. Notably, Mantel tests revealed that six core genes exhibited a positive correlation with CAT activity in the drought-tolerant variety, while showing an opposite trend in the sensitive variety. This study provides insights into the mechanisms of drought tolerance in oats and aids in the molecular breeding of drought-tolerant varieties.

Vicia amoena is renowned for its high protein content and nutritional value, making it significant in animal production and traditional Chinese medicine production. In July 2023, typical anthracnose symptoms were observed on V. amoena leaves in Suihua City (125°82'E, 46°22'N), Heilongjiang Province, China, affecting approximately 40% of the plants (a total of 200 plants were surveyed). Symptoms initially appeared as black-purple spots, gradually developing into larger, sometimes irregular reddish-brown lesions surrounded by yellow margins, with easily perforated necrotic lesions. Samples were collected from 20 leaves exhibiting distinct symptoms. Each leaf was cut into 5 x 5 mm sections at the junction of diseased and healthy tissues. The sections were surface sterilized: first, in 75% ethanol for 30 s, then in 1% NaClO for 2 mins, followed by three rinses with sterile distilled water. After air-drying, the samples were placed onto potato dextrose agar (PDA) plates and incubated at 26°C in the dark for 5 d. Subsequently, 15 isolates were obtained using the single-spore method, with nine (SYW1 to SYW9) showing similar morphological characteristics. On PDA, colonies appeared cottony and pale yellow from above, with a black-brown center on the reverse side. The conidia were hyaline, aseptate, subcylindrical with broadly rounded ends, measuring 13.8 to 18.6 μm in length and 4.8 to 6.4 μm in width (n = 50). Appressoria were subcylindrical or irregular, with a few lobes, brown to dark brown, and measured 9.2 to 12.3 μm in length and 5.1 to 7.2 μm in width (n = 50). Morphological analysis suggested a close resemblance to Colletotrichum aenigma (Weir et al. 2012). Genomic DNA was extracted from three representative isolates (SYW1 to SYW3) for molecular identification. The ITS, GAPDH, TUB2, CHS-1, and ACT genes were amplified and sequenced using primers previously described by Weir et al. (2012). Sequences were submitted to GenBank (ITS: PP949783 to PP949785; GAPDH: PP952315 to PP952317; TUB2: PP952318 to PP952320; CHS-1: PP952321 to PP952323; and ACT: PP952324 to PP952326). BLAST analysis revealed 100% identity with C. aenigma (GenBank accessions: OM663726, KY820889, MW387032, MN075525, and LC815246). Phylogenetic analysis placed the three isolates and C. aenigma in the same clade. Pathogenicity tests were conducted twice on 4-week-old V. amoena seedlings in a greenhouse. A conidial suspension of the SYW1 isolate (1 × 106 conidia/ml) was sprayed onto five pots, each containing four seedlings, while sterile distilled water was sprayed on the control pots. All plants were grown in a light incubator with a 12-h photoperiod, 100% relative humidity, and 26°C temperature. Two weeks post-inoculation, the inoculated seedlings showed reddish-brown spots and typical anthracnose lesions on inoculated leaves, with C. aenigma successfully reisolated from symptomatic tissues. To the best of our knowledge, this is the first report of C. aenigma causing anthracnose leaf spot on V. amoena in China. Although C. aenigma has previously been associated with anthracnose in crops such as grapes (Kim et al. 2021), mulberry (Zhu et al. 2023), and pecan (Zhao et al. 2024), its identification as a pathogen of V. amoena highlights a significant threat to this plant species in China. This finding underscores the urgent need for targeted disease management strategies to prevent further spread and damage, particularly in regions where V. amoena is economically or ecologically important.

Crop productivity around the world is largely constrained by salt-induced stress, a key abiotic factor. Although oat (Avena sativa L.) can withstand challenging environmental conditions, the physiological and molecular responses underlying salt tolerance during germination and early seedling development remain insufficiently understood. To investigate these responses, 28 oat varieties were evaluated at the germination stage, and two contrasting varieties, the salt-tolerant Mengshi No. 1 (MS) and salt-sensitive Morgan (MG), were selected for detailed analysis under a severe NaCl treatment (300 mM) during early seedling stages. Under severe salt stress, the two oat varieties exhibited distinct growth and physiological responses, including changes in growth traits, chlorophyll content, membrane stability, osmotic adjustment, and antioxidant responses. Transcriptomic analysis revealed 14,109 differentially expressed genes (DEGs) between salt-treated MG and its respective control (CK), 19,405 between salt-treated MS and its CK, and 6161 between salt-treated MG and salt-treated MS, suggesting different transcriptional response patterns between the salt-tolerant and salt-sensitive varieties under severe salt stress. Weighted gene co-expression network analysis (WGCNA) revealed a salt-responsive module associated with MS, from which five hub genes, AVESA.00010b.r2.1CG0087930 (MGL), AVESA.00010b.r2.19DG0180280 (MGL), AVESA.00010b.r2.4CG1272260 (BCH1), AVESA.00010b.r2.5DG0989800 (GPAT7), and AVESA.00010b.r2.6CG1124100 (TPR10), were identified as candidate genes potentially associated with salt tolerance and stress responses.