First Report of Causing Leaf Spot on in China.

Chinese prickly ash (Zanthoxylum bungeanum Maxim.), which is a Rutaceae plant as shrubs or small trees and indigenous to China, is widely grown in this country for its flavor, as well as its high economic and ecological value. So far, in China, the cultivated area and yield of Chinese prickly ash rank first in the world. In June 2023, a leaf spot disease with approximately 30% incidence was observed on Z. bungeanum in Zhenfeng County (25°44'21.38″ N, 105°56'47.15″ E, 1,083 m), Guizhou Province, China. Foliar symptoms appeared as irregularly shaped lesions, yellowish-brown with dark brown margins surrounded by yellow halos, which enlarged, resulting in the lesions dropping from the leaves and leaving holes. To isolate and identify the pathogen, symptomatic leaves were taken and cut into 5 mm × 5 mm pieces, surface sterilized with 2% NaClO for 3 min, 75% ethanol for 30 s, rinsed three times with sterile water, and incubated on PDA at 28°C. Ten isolates with identical morphology were obtained. After one week of incubation at 28℃, the colonies on PDA were brown, reverse dark brown, fluffy, reaching 7.0-7.5 cm in diameter. Conidia were straight or slightly curved, narrowly ellipsoidal or fusiform, 1-3 but mostly 3 septate, light or dark brown, with the middle cells usually darker than the terminal cells, smooth, 20.5-31.0 × 9.0-19.0 μm (x̄ = 26.0 × 14.0 μm, n = 30). The morphological features matched the description of Curvularia trifolii (Kauffman) Boedijn (Ellis 1971; Falloon 1976). Additionally, the internal transcribed spacer (ITS), large subunit (LSU) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes were amplified by PCR with primers ITS5/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys & Hester 1990) and GPD1/GPD2 (Berbee et al. 1999), and the ITS, LSU and gapdh sequences of the isolate GUCC 23-321 (PP837870, PP837881, PP855474) were deposited in GenBank. The BLAST showed 98.5% (ITS, HG779023, 598/709 bp), 99.87% (LSU, HG779077, 779/858 bp), and 97.79% (gapdh, HG779124, 543/498 bp) identities with C. trifolii (CBS 173.55). Furthermore, the phylogenetic tree of ML analysis based on the combined sequence data of ITS, LSU and gapdh revealed that GUCC 23-321 clustered with C. trifolii. Both morphology and phylogenetic analyses supported the identification of GUCC 23-321 as C. trifolii. Pathogenicity tests were carried out twice according to Koch's postulates. Five healthy 2-year-old Chinese prickly ash plants were sprayed with a conidial suspension (1 × 106 conidia/mL) of the isolate GUCC 23-321, while the controls (five other plants) were sprayed with sterile water. All plants were maintained in a greenhouse at 28°C, 80% relative humidity. After 8 days, the inoculated plants developed leaf spots similar to those showed in the field, but control plants were asymptomatic. Re-isolation of pathogenic fungi from the leaf lesions of the inoculated plants and according to molecular analysis and morphology, the fungi were identified as C. trifolii, fulfilling Koch's postulates. C. trifolii is a common fungal phytopathogen that has been reported to infect a variety of plants and cause leaf spot disease, such as Trifolium alexandrinum (Khadka 2016) and Nicotiana tabacum (Chen et al. 2017). This is the first worldwide report of C. trifolii causing Z. bungeanum leaf spot. The report will be beneficial for accurately diagnosing this disease, and proposing specific control measures.