LAUSR

Fraxinus rhynchophylla Hance, is a deciduous trees cultivated on a commercial scale focused on medicinal and wood production. In September 2021, leaf spot was observed on F. rhynchophylla in Heilongjiang Province (127.34°E, 45.19°N), China. These symptoms were observed on 100% F. rhynchophylla plants and the incidence of diseased leaves per plant reached 70% in fields measuring 90 ha. Disease symptoms were small yellow flecks initially, and then turned to gray necrotic spot. Ten diseased leaves were collected randomly from 5 plants and surface disinfested. Tissue samples (2 × 2 mm) were cut at the disease-health junction of the leaves, surface sterilized in 75% ethanol for 30 s, submerged in a 7% NaOCl solution for 3 mins, and rinsed three times with sterile water. Leaf segments were placed onto potato dextrose agar (PDA) and incubated at 26℃ for 5 days. After isolation and purification of monospore, the colonies of the all isolates were inky black, with aerial fluffy mycelium, and concentric whorls on PDA. The conidiophore is septate, single-branched, brown, smooth and 35 - 313 × 2 - 5 μm in size (n = 50), while the conidia are brown, bow-shaped, mostly four cells, with three septa and 12 - 385 × 5 - 20 μm in size (n = 150). The morphological characters matched those of Curvularia muehlenbeckiae (Madrid et al. 2014). DNA was extracted from isolates HQLa and HQLb and used for PCR amplification of RNA polymerase II gene (RPB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequences using the primer fRPB2-SF/fRPB2-7Cr (Schoch et al. 2009), gpd1/gpd2 (Berbee et al. 1999), respectively. The RPB2 (OM984674, OM984675) and GAPDH (OM984672, OM984673) were deposited in GenBank. The phylogenetic tree was constructed by combining other published sequences of RPB2 and GAPDH genes using the maximum likelihood method, and the results showed that the obtained isolates clustered into the same clear branch as C. muehlenbeckiae CBS 144.63 (HG779180, HG779108), with 100% bootstrap support. Combining morphological characteristics and phylogenetic analysis of the fungus, the obtained isolates were identified as C. Muehlenbeckiae. To fulfill the Koch's postulates, pathogenicity tests were carried out on newly grown leaves of F. rhynchophylla. Conidia of the selected isolates grown on PDA plates were flooded with sterile distilled water. Spore suspension was adjusted to 105 spores/mL with the hemocytometer. Three leaves of each plant were disinfected with 1% NaOCl for 2 min, washed with sterilized distilled water three times, and dried with sterile paper towels. Three plants were randomly selected for inoculation under field conditions and each leaf was sprayed with 2 mL of the spore suspension for a total of nine leaves, then the plants were bagged and moistened for 48 h. However, control leaves were sprayed with distilled water. Symptoms were observed nine days after inoculation. No symptoms were observed on control leaves. The same fungus was successfully re-isolated from the lesions. The experiment was replicated three times with the same results and C. muehlenbeckiae identification was confirmed by morphological observations and RPB2 and GAPDH sequencing, indicating that the fungus is the causal pathogen of leaf spot disease on F. rhynchophylla. This is the first report of C. muehlenbeckiae determined as fungal pathogens on F. rhynchophylla plant in China. The results of the study laid the foundation for the future occurrence and epidemiological pattern of the disease and scientific control.