Fusarium root rot, caused by Fusarium spp. in alfalfa (Medicago sativa L.), adversely impacts alfalfa by diminishing plant quality and yield, resulting in substantial losses within the industry. The most effective strategy for controlling alfalfa Fusarium root rot is planting disease-resistant varieties. Therefore, gaining a comprehensive understanding of the mechanisms underlying alfalfa’s resistance to Fusarium root rot is imperative. In this study, we observed the infection process on alfalfa seedling roots infected by Fusarium acuminatum strain HM29-05, which is labeled with green fluorescent protein (GFP). Two alfalfa varieties, namely, the resistant ‘Kangsai’ and the susceptible ‘Zhongmu No. 1’, were examined to assess various physiological and biochemical activities at 0, 2, and 3 days post inoculation (dpi). Transcriptome sequencing of the inoculated resistant and susceptible alfalfa varieties were conducted, and the potential functions and signaling pathways of differentially expressed genes (DEGs) were analyzed through gene ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Meanwhile, a DEG co-expression network was constructed though the weighted gene correlation network analysis (WGCNA) algorithm. Our results revealed significant alterations in soluble sugar, soluble protein, and malondialdehyde (MDA) contents in both the ‘Kangsai’ and ‘Zhongmu No. 1’ varieties following the inoculation of F. acuminatum. WGCNA analysis showed the involvement of various enzyme and transcription factor families related to plant growth and disease resistance, including cytochrome P450, MYB, ERF, NAC, and bZIP. These findings not only provided valuable data for further verification of gene functions but also served as a reference for the deeper explorations between plants and pathogens.

Fusarium spp. can cause root rot in alfalfa, leading to the death of the whole plant, which seriously affects the yield and quality of alfalfa. This study used a Fusarium acuminatum strain labeled with green fluorescent protein (GFP) to observe the infection process of F. acuminatum on alfalfa by confocal fluorescence microscopy. The aim of this study was to reveal the infection mechanism of alfalfa Fusarium root rot at the cellular histological level. The results showed that conidia of F. acuminatum attached to the surface of the root and germinated at one day post-inoculation, the mycelium then entered the vascular bundle tissue of the alfalfa root at 5 days post-inoculation, reached the base of the plant stem at 14 days post-inoculation, and colonized the stem of the first and second compound leaf at 28 and 49 days post-inoculation, respectively. Moreover, the experiment, which sprayed a spore suspension, showed that the conidia of F. acuminatum could spread through the air to infect the pericarp and seed coat tissue of the pod. For the first time, we report the infection process of alfalfa Fusarium root rot caused by F. acuminatum and clarify that F. acuminatum can initially infect the root tissue of alfalfa, colonize the bottom stem of the plant through systematic infection, and eventually cause the plant to wilt and die. The results reveal the infection mechanism of F. acuminatum at the cell level via histology and provide theoretical support for the development of control strategies and key control technologies for alfalfa root rot.

Brassinosteroid (BR) signaling is regulated by BRI1-EMS SUPPRESSOR 1 (BES1) transcription factors, which are crucial for plant growth, development, and stress responses. Despite their importance, BES1 gene studies in Medicago sativa L. are limited, hindering our understanding of the BR signaling in this species. This study identified four BES1 genes in M. sativa; characterized their properties, conserved motifs, cis-regulatory elements, and chromosomal location; and explored their functions in development and stress responses. A phylogenetic analysis grouped these genes into two subfamilies. Transcript profiling showed widespread and tissue-specific expression patterns. A qRT-PCR analysis unveiled that most MsBESI genes were upregulated under salt and drought treatments, except MsG0280009980, which was suppressed. This research lays the groundwork for enhancing M. sativa stress resistance and understanding the BES1 gene family’s function.

Oat (Avena sativa) is a significant cereal crop that is extensively cultivated in temperate regions and is suitable for growth at higher elevations. The damage degree and epidemic area of oat spikelet rot caused by Dactylobotrys graminicola are generally increasing and spreading. Mycelium growth rate method was used to identify biological characteristics and in vitro fungicide screenings of D. graminicola. The results show that sorbitol and casein tryptone were the best carbon and nitrogen source for the D. graminicola, respectively. The optimal temperature for mycelium growth and conidia production of the D. graminicola was 20 °C; meanwhile, an acidic environment was shown to be conducive to mycelia growth, and alkaline facilitated conidia production. Among the ten tested fungicides, 20% Pydiflumetofen showed the best inhibition rate, with an EC₅₀ (concentration for 50% of maximal effect) value of 0.005 mg/L; 30% Difenoconazole Propiconazole and 35% Metalaxyl-M Fludioxonil also showed sufficient inhibitory effects against D. graminicola, with EC₅₀ value of 0.05 mg/L and 0.04 mg/L. Furthermore, we used artificial inoculation to determine the effectiveness of fungicide control in field, Trifloxystrobin 10%-Tebuconazole 20% with more than 90% control effectiveness, followed by 20% Pydiflumetofen. The results of this study not only revealed the biological characteristics of D. graminicola, but also provided effective candidate fungicides for the prevention and control of oat spikelet rot disease.

COI1 (CORONATINE INSENSITIVE) is a central regulator of plant defense against biotic stresses, yet its family members remain uncharacterized in the globally important forage crop alfalfa ( Medicago sativa L.). Here, we identified 32 MsCOI1 genes, which were phylogenetically classified into four subfamilies (I–IV). The four subfamilies each contain 5, 6, 9, and 12 MsCOI1 genes, respectively. Structural analysis revealed conserved three-exon architecture and six critical motifs, with Motif 3 exclusively absent in Subfamily IV. Promoter cis-element profiling showed enrichment in stress-responsive elements: 93.8% contained ABRE (abscisic acid response), 68.8% harbored MeJA-responsive elements, and 50% possessed TC-rich repeats (defense/stress response). Expression dynamics demonstrated tissue specificity (e.g., MsCOI1-24/25/26 in nodules/roots) and hormone responsiveness (40.6% induced by MeJA/ABA). Crucially, six of eight tested MsCOI1s were upregulated during Alfamovirus AMV (commonly known as Alfalfa mosaic virus, AMV) infection. However, MsCOI1–8 was significantly inhibited at 5 dpi, indicating that the virus manipulates the COI1-mediated plant response. This study provides the first comprehensive resource of MsCOI1 genes for enhancing viral resistance in alfalfa breeding.