First report of alfalfa root rot caused by Trichothecium roseum in China.

Alfalfa (Medicago sativa L.) is perennial leguminous forage, which is cultivated throughout the world due to its high yield, high quality, satisfactory palatability, and wide adaptability. With the increase of planting area in China, root diseases caused by Fusarium spp., Sclerotium rolfsii, Phytophthora spp. (Yang et al. 2022), and new pathogens have been found that reduce the yield and quality of alfalfa and cause economic losses (Li at al. 2019). In 2021, an alfalfa disease occurred under conditions of high temperature and high humidity at the Jiaozhou Experimental Base of Qingdao Agricultural University (Jiaozhou Modern Agricultural Science and Technology Demonstration Park, 36.33°N 120.40°E, Qingdao, Shandong, China), and about 2 ha of alfalfa were infected. The disease affected up to 35% of the plants and caused grass spots. Infected plants developed black-brown lesions with irregular shapes on roots with yellowing of the foliage; the leaves of the whole plant turned yellow. In the late stage of the disease, defoliation occurred and the plants stopped growing, wilted and died. Ten infected plants with typical symptom were collected for isolation and identification of pathogen. The infected roots were cut into 3-5 mm2 sections and then soaked in 75% ethanol for 30 s, followed by a 3-minute immersion in 2% sodium hypochlorite for surface sterilization. Next, the tissues were rinsed in sterile water five times and then placed on potato dextrose agar (PDA) medium. After three subcultures and subsequent single spore isolation, one representative strain named as DC1 was isolated from the infected roots. Based on morphological observation, the colony of DC1 was flat, granular, and powdery in appearance. Four days after inoculation on PDA medium, the size of the colony were 2.1-2.6 cm. After 8 to 20 days, the colonies were initially white and gradually change a light pink to peach color. The conidia are two-celled (Hamid et al. 2014), elliptic to pear-shaped, colorless or translucent, smooth to slightly rough with thick walls. The size of conidia ranged from 11.3 to 23.5 µm long × 6.1 to 12.7 µm wide (n =30). For the identification, the rDNA--ITS gene of the fungus was amplified using the primers ITS1/ITS4 (White et al.1990), and the EF1α gene 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 results of the rDNA-ITS (OM049197.1, 515 bp) and EF1α (OM069381.1, 926 bp) sequences were deposited in GenBank. DNA analysis showed that the two sequences were 100% similar to the rDNA-ITS sequence (MN882763.1) and EF1α sequence (DQ676610.1) of Trichothecium roseum, respectively. A pathogenicity test was done by placing one piece (0.5 cm in diameter) of fungal culture (PDA plug) 1cm below the crown of 40-day-old healthy alfalfa (cv. Zhongmu No.3) plants, 3 replicates and 20 plants in each replicate. PDA plug without the pathogen were used for control. All plants were cultivated in a growth chamber at 25±1°C with a light cycle of 15 h (90% relative humidity). After 18 days, the roots of inoculated plants had dark brown lesions and the leaves of their plants turn yellow, while those control plants had no symptoms. To fulfilling Koch's postulates, the same pathogen was re-isolated from necrotic root tissue of inoculated plants and confirmed by morphology and the rDNA-ITS and EF1-α sequences. Based on disease symptoms, morphological characteristics DNA sequences and pathogenicity, the pathogen of alfalfa disease in Jiaozhou Experimental Base of Qingdao Agricultural University was identified as T. roseum. To our knowledge, this is first report of T. roseum causing alfalfa root rot. The newly emerging disease may pose threat to alfalfa production of central and southern China in future.

Transcriptome Analysis of Fusarium Root-Rot-Resistant and -Susceptible Alfalfa (Medicago sativa L.) Plants during Plant-Pathogen Interactions.

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.

Southern Blight on Medicago sativa Caused by Sclerotium rolfsii in North China.

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).