First Report of <i>Alternaria alternata</i> Causing Leaf Spot on <i>Eucommia ulmoides</i> (Duzhong) in China

Eucommia ulmoides Oliv. (duzhong), a traditional Chinese medicine, whose leaves, bark, and male flowers are medicinal ingredients. In May 2021, leaf spot symptoms were observed on the leaves of E. ulmodies in their major planting region in Lueyang County (33°20'01.6"N, 105°59'10.9"E) in China. Leaf spot disease was observed in a field of 0.27 hectares in all two-year-old plants, with an incidence rate of 60%. The initial symptoms appeared as small and irregular spots that randomly distributed on the surface of the leaf. As the disease progressed, the lesions gradually developed into brown spots with dark margins and surrounding chlorosis, which were white and easily perforated at the center. To isolate the pathogen, tissues of disease-healthy junctions of 20 diseased leaves were surface sterilized with 75% ethanol for 30s and 3% NaClO for 5min, and rinsed three times in sterile distilled water. These tissues were cut into small pieces (5×5mm) and placed on Rose Bengal Agar plates at 28℃ for 5 days. After picking mycelium tips and placing them on potato dextrose agar (PDA) plates, seven purified isolates were obtained. After 15 days, the colonies all appeared grayish-green and had round white margins on PDA. Two representative isolates (DZY7 and DZY5) were selected for subsequent morphological and ITS identification. Conidia were obclavate or ellipsoid, brown with a short conical beak at the tip. Its size was ranged from 8.6 to 40.0 × 5.1 to 13.5 µm (n = 50), with one to five transverse and zero to three longitudinal septa. These morphological characteristics in colony and conidia were consistent with those of Alternaria sp. (Simmons 2007). For molecular identification, genomic DNA of DZY7 and DZY5 isolates were extracted. The internal transcribed spacer (ITS) regions of them were amplified with the primer pairs ITS1/ITS4 (White et al. 1990). The ITS sequences of DZY7 and DZY5 showed 100% (544bp/537bp) similarity, and were deposited in GenBank with accession numbers PP528868 and PV936609. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1-α), and RNA polymerase second largest subunit (RPB2) genes were amplified with the primer pairs gpd1/gpd2, EF1-728F/EF1-986R (Carbone et al. 1999), and RPB2-5F2/RPB2-7CR (Liu et al. 1999) from the extracted genomic DNA of the DZY7 and DZY5 isolate strains. Their sequences were deposited into the GenBank database with accession numbers PQ072215-PQ072217(DZY7) and PV978089-PV978091(DZY5). The phylogenetic tree generated by maximum-likelihood analysis (MEGA 12) based on the concatenated ITS, GAPDH, TEF-α, and RPB2 loci revealed that isolates DZY7 and DZY5 clustered robustly with A. alternata. For pathogenicity testing, two-year-old plants (n=8, one plant per pot) were sprayed with 5 mL of either DZY7 or DZY5 spore suspensions (106 conidia/ml with 0.2% Tween-20), with sterile water (containing 0.2% Tween-20) serving as the control treatment. Each plant was placed in a separate pot to prevent cross-contamination. This experiment has been repeated twice within a greenhouse with an 80% humidity level. A week later, small necrotic spots in dark brown and black appeared on the leaf surface. Two weeks later, the necrotic spots expanded and easily perforated at the center, which were the same symptoms as those previously observed in the field. While the control leaves did not show any symptoms. The fungus strain was re-isolated from the diseased leaf tissue, and showed the same morphological characteristics as the A. alternata strain, with 100% ITS sequence identity. The pathogenicity test was repeated twice. It was fulfilled according to the Koch’s postulates. A. alternata has already been identified on Eucommia ulmoides in Korea (Yu 2001). To our knowledge, this is the first report of A. alternata causing leaf spot disease on E. ulmoides in China. This report is beneficial for the prevention and control strategies against leaf spot disease of E. ulmoides.