Alfalfa (Medicago sativa L.) is a perennial leguminous forage cultivated globally. Fusarium spp.-induced root rot is a chronic and devastating disease affecting alfalfa that occurs in most production fields. Studying the disease resistance regulatory network and investigating the key genes involved in plant–pathogen resistance can provide vital information for breeding alfalfa that are resistant to Fusarium spp. In this study, a resistant and susceptible clonal line of alfalfa was inoculated with Fusarium proliferatum L1 and sampled at 24 h, 48 h, 72 h, and 7 d post-inoculation for RNA-seq analysis. Among the differentially expressed genes (DEGs) detected between the two clonal lines at the four time points after inoculation, approximately 81.8% were detected at 24 h and 7 d after inoculation. Many DEGs in the two inoculated clonal lines participated in PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) mechanisms. In addition, transcription factor families such as bHLH, SBP, AP2, WRKY, and MYB were detected in response to infection. These results are an important supplement to the few existing studies on the resistance regulatory network of alfalfa against Fusarium root rot and will help to understand the evolution of host–pathogen interactions.

Alfalfa (Medicago sativa L.) is an important perennial forage crop species in many countries around the world, including China. Among the diseases limiting yield, root rots caused by Fusarium can damage the crop since from the second year of the crop cycle that can last up to ten years (Wallenhammar et al. 2008). In China, the main Fusarium pathogens causing root rot are those of the F. oxysporum and F. solani species complex. In May 2012, three-year-old alfalfa plants in a crop grown at Yanggao county, Shanxi province in north China, exhibited typical root rot symptoms. Leaves were chlorotic, stunted and wilted. Necrosis was noticed as red-brown discoloration of vascular tissue within the root zone below the crown (Fig.1b). White mycelium appeared on the root (Fig.1a). In some severely affected plants, the entire root decayed with only the fibrous vascular bundle remaining. Diseased root crown tissues (5×5 mm) were surface-sterilized with 75% ethanol for 30 s and 0.1% HgCl2 for 3 min, rinsed five times w...

In nature, some plant species produce seedpods with spines, which is an adaptive biological trait for protecting the seed and helping seed dispersal. However, the molecular mechanism of spine formation is still unclear. While conducting routine tissue culture and transformation in the model legume Medicago truncatula, we identified a smooth seedpod (ssp1) mutant with a suite of other phenotypic changes. Preliminary analysis showed that the mutation was derived from the tissue culture process. Genetic segregation analysis suggested that ssp1 is a recessive mutant. By combining whole-genome sequencing and bioinformatics analysis, we found that the mutant phenotype was caused by a single nucleotide polymorphism and a 30 bp deletion in the gene locus Medtr4g039430, named SSP1. Complementation of the M. truncatula ssp1 and Arabidopsis twd1 mutants showed complete restoration, indicating that SSP1 is an ortholog of Arabidopsis TWD1 which encodes an immunophilin-like FK506-binding protein 42. The formation of spines on seedpods is associated with auxin transport. The method used in this study offers an effective way for detecting genes responsible for somaclonal variations. The results demonstrate, for the first time, that SSP1 plays a crucial role in the determination of spine formation on seedpods.

Alfalfa (Medicago sativa L.) is one of the most important perennial leguminous forages in many countries, known by its high feed value and yield potential. With the increasing demand for feed, alfalfa has been planted all over China. However, an increasingly serious alfalfa disease was observed and may restrict the development of the alfalfa industry in North China. In August 2019, an emerging alfalfa disease with symptoms resembling southern blight was observed in Jiaozhou experimental base (Jiaozhou Modern Agricultural Science and Technology Demonstration Park) of Qingdao Agricultural University (Qindao, Shandong province, China). The infected plants showed dark brown lesions on the stems and yellowing and wilting of the leaves. The pathogen produced white fluffy mycelia, and later sclerotia on stems and roots; the disease affected up to 25% of the plants and causes bare spots filled with weeds (Figure S1). Typical symptomatic tissues were brought back to the laboratory for pathogen isolation and identification. Fragments (3-5mm2) of root tissues were excised from lesions on the symptomatic roots and their surfaces were disinfested by sequential dipping in 70% ethanol for 30 s and in 2% NaClO for 3 min, then the fragments were rinsed in sterile water five times and cultured on potato dextrose (PDA) medium amended with streptomycin sulfate (0.1mg/mL). Cultures were incubated at 28°C in the dark and purified in PDA medium for three times. A representative strain (coded as CZL1) was isolated from the root rot of the diseased plant. After four days incubation on PDA, CZL1 formed white fluffy aerial mycelium 5.6-6 cm in diameter typical of S. rolfsii. After 15 to 20 days, abundant round sclerotia approximately1-3 mm in diameter were produced on the surface of the culture (Figure S2). The sclerotia were white at first and then gradually turned dark brown. To confirm the identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA region of the fungus was amplified using the primers ITS1/ITS4 (White et al.1990), and the elongation factor-1a gene (EF1a) was amplified using primers EF1-983F/EF1-2218R (Rehner and Buckley 2005). Then the PCR amplicons were cloned into the pCE2 TA/Blunt-Zero vector. The isolate was determined to contain two distinct sequence types for each gene. The results of ITS (MT812692, MT812693) and EF1a (MT846496 and MT846497) sequences were deposited in GenBank. DNA analysis revealed that the two ITS sequences were more than 99% identical to Athelia rolfsii (MN872304) in the NCBI GenBank database, and two EF1a sequences were 99% identical to the A. rolfsii EF1a sequence MN702789 and KP982854. To fulfill Koch's postulates, infected sorghum grain was placed near the roots of 15 40-day-old healthy alfalfa seedlings split into 3 pots with the same number of seedlings receiving a control treatment of sterilized sorghum grain. All plants were incubated in growth chamber at 24±1°C with 14-h-photoperiod (85% relative humidity). After 10-15 days, blight symptoms identical to those in the field were observed on inoculated plants, whereas those control plants were symptomless (Figure S2). S. rolfsii was successfully re-isolated from the inoculated plants and molecularly characterized as described above. Based on disease symptoms, fungal colonies, the ITS and EF1a sequence, and pathogenicity to the host, this fungus was identified as S. rolfsii (teleomorph Athelia rolfsii). To our knowledge, this is the first report of S. rolfsii as the causal agent of southern blight of alfalfa in North China, and it is also the first report of southern blight on alfalfa caused by S. rolfsii in China since 1996 observed in Guizhou province (Mo and Luo 1996).

The legume plant alfalfa (Medicago sativa L.) is a widely cultivated perennial forage due to its high protein content, palatability, and strong adaptability to diverse agro-ecological zones. Alfalfa is a self-incompatible cross-pollinated autotetraploid species with tetrasomic inheritance. Therefore, maintaining excellent traits through seed reproduction is a prime challenge in alfalfa. However, the cutting propagation technology could enable consistent multiplication of quality plants that are genetically identical to the parent plant. The current study aimed to develop a simple, cost-effective, reproducible, and efficient hydroponic cutting method to preserve alfalfa plants and for molecular research. In this study, alfalfa landrace ‘Wudi’ was grown in hydroponics for 30 days and used as source material for cuttings. The top, middle and bottom sections of its stem were used as cuttings. The rooting rate, root length, and stem height of the different stem sections were compared to determine the best segment for alfalfa propagation in four nutrient treatments (H^M, H^M + 1/500H, H^M + 1/1000H and d H^M + 1/2000H). After 21 days of culture, the rooting rates of all the three stem types under four cutting nutrient solutions were above 78%. The rooting rate of the middle and bottom parts in H^M + 1/1000 H and H^M + 1/2000 H nutrient solutions reached more than 93%, with a higher health survey score (> 4.70). In conclusion, this study developed a de novo cutting propagation method that can be used to conserve and propagate germplasm in breeding programs and research. This method is a new report on the cutting propagation of alfalfa by hydroponics, which could supplement the existing cutting propagation methods.

Alfalfa (Medicago sativa L.) is a popular cultivated crop across the world that is grown for forage. After several years of production, root rot often reduces plant vigor and yield. It is estimated that the annual yield loss worldwide caused by root rot is 20%-40% (Pan et al. 2015). In North China, Fusarium oxysporum, Fusarium solani and Rhizoctonia solani are the main pathogens causing root rot. During June and July 2012, 3-year-old alfalfa plants with typical root rot symptoms were collected from Langfang, Hebei province and Linhe, Inner Mongolia Autonomous Region in North China. Leaves of the affected plants were chlorotic, stunted and wilted. Brownish or reddish water-soaked lesions, white mycelium, appeared on the root. In some severe cases, the entire root decayed leaving only the fibrous vascular bundle. To isolate the causal organism, diseased root crown tissues (5×5 mm) were surface-sterilized with 75% ethanol for 30 s and 0.1% HgCl2 for 3 min, rinsed five times with sterile water, dried and incu...