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.

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.

Smooth bromegrass (Bromus inermis Leyss.) is an important forage crop in northern China. In July 2021, leaf spot symptoms were observed on smooth bromegrass in Ewenki Banner, Hulunbuir, Inner Mongolia. In an area of approximately 0.12 hectares, 95% disease incidence was observed. Ten diseased plants were collected for pathogen isolation. Leaf tissues near the lesions were cut into 5 × 5 mm pieces, surface-disinfested in 75% ethanol for 3 min, and rinsed with sterile distilled water. The pieces were placed on water agar in petri plates and incubated at 25℃ for three days. The resulting colonies were flushed with sterile water and a spore suspension was serially diluted and plated on potato dextrose agar (PDA). A single-spore colony was obtained. Ten isolates were obtained and designated HE1 to HE10. The colony morphology was identical for all isolates, grayish white in color on the upper surface and light black on the underside. The mycelia were light gray and velvety. Conidia were light brown to brown in color and oblate, oblong or oval. The conidial dimensions were typically between 15 to 43 μm by 8 to 9 μm in size. The conidia possessed one to six transverse septa, with slight to distinct constrictions at each division, and zero to two longitudinal septa. These morphological characteristics resembled Alternaria alternata (Fr.) Keissl.. DNA was extracted from three isolates, HE3, HE4 and HE5, using the CTAB method. Polymerase chain reaction (PCR) was performed on the extracted DNA with a set of primers ITS1/ITS4, H31a/H31b, gpd1/gpd2, TEF1-728F/TEF1-986R, and RPB2-5F2/fRPB2-7cR. The amplicon sequences from the three isolates were analyzed using the BLAST in GenBank (https://www.ncbi.nlm.nih.gov/). The results showed a high sequence identity, ranging from 99 to 100%, with the A. alternata strain YTMZ-20-2 across all the genetic markers tested. The strong match reinforced the identification of the strains as A. alternata. The sequences were deposited in GenBank (Table S1). The three fungal isolates were identified as A. alternata based on their morphological and genetic data. To conduct Koch's postulates, the representative isolate HE4 was used. Smooth bromegrass seed was soaked in water for four days and sown in potting soil contained in plastic pots (10 cm diameter × 15 cm height, five seeds/pot) in a greenhouse under a 16-h photoperiod at temperatures between 20 to 25°C and 60% relative humidity. When the plants reached a height of approximately 20 cm, the plants in three pots (replicates) were sprayed with a spore suspension (106 conidial/ml) at 10 ml/pot, and three pots were sprayed with sterile water for control. Five days after inoculation, the plants exhibited leaf spot symptoms similar to those previously described, while the control plants remained unaffected. The causative fungus was successfully re-isolated from the diseased plants and confirmed morphologically and molecularly on its identity as described above. This experiment was independently conducted three times. This is the first report of A. alternata causing leaf spot on smooth bromegrass in China. Since there is risk that the disease could seriously reduce the yield of the forage crop smooth bromegrass, further research is needed.