First Report of Calonectria ilicicola causing Leaf spot on Camellia sinensis in Yunnan province, China.

Tea (Camellia sinensis (L.) Kuntze) is among the most significant industrial crops due to its distinctive fragrance and flavor generated (Bag et al. 2022). From October to December in 2021, a leaf spot disease affected the quality and yield of tea (C. sinensis var. assamica cv. Yunkang 10), in Pu'er (100.57°E, 22.45°N), Yunnan province, China. Based on the survey, the incidence was approximately 15% in a plantation of 4500 m2 (2050 tea trees approximately). The symptoms on leaves were regular circular, dark brown lesions with black conidiomata in gray centers. Twenty symptomatic leaves were collected from 10 trees. After rinsing and surface sterilization (75% ethanol for 30 s and 3% NaClO for 90 s, rinsed 3 times with sterile distilled water), diseased tissues (5 × 5 mm) were cut at the junction of infected and healthy site and placed on potato dextrose agar (PDA) (3 pieces per plate) and incubated in the dark at 28℃ for 5 days (Mao et al. 2023). Three single-spore isolates 6a-H-1, 6a-H-2 and 6a-H-3 were obtained, which showed identical in morphology and molecular analysis. Therefore, the targeted isolate 6a-H-2 was used for further study. Fungal colonies were white, then gradually turning into goose yellow (Fig.2. A-C). Chlamydospores were dark brown and oval (Fig.2. G). Asci produced after 30 days approximately, were orange-red, nearly spherical, rough-surface, and measured as 470 µm ± 11.68 µm (n = 50) (Fig.2. H). Ascospores were released from the asci orifice (Fig.2. I) which were hyaline, fusoid with rounded ends, straight to slightly curved, two septate, slightly constricted at the septum, and ranged from 48.77 ± 2.76 µm × 6.22 ± 0.41 µm (n = 50) (Fig.2. D-F). Macroconidia were cylindrical (Fig.2. J), rounded at both ends, straight, with an average length of 63.5 ± 0.31 μm × 2.62 ± 0.03 μm without septa (n=50) (Fig.2. M-O). Stipe extension terminated in sphaero-pedunculate vesicles (Fig.2. K-L). The morphological features were consistent with the descriptions of Calonectria ilicicola (Pei et al. 2015; Polizzi et al. 2012). The pathogen was confirmed to be C. ilicicola by amplification and sequencing of the histone (HIS3), translation elongation factor 1-alpha (TEF1) and calmodulin (CAL) genes using primers H3-3F/H3-3R, EF1-728F/EF1-986R and CAL-228F/CAL-2Rd, respectively (Crous et al. 2004). The sequences of PCR products were deposited in GenBank with accession numbers OR188222 (HIS3), OR188223 (TEF1) and OR188221 (CAL). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides), 98.37% (241/245 nucleotides) and 99.58% (472/474 nucleotides) homology with those of C. ilicicola (CBS 190.50) in GenBank (AY725676, AY725726 and AY725764), respectively. Phylogenetic analysis (MEGA 7.0) using the Maximum Likelihood method placed the isolate 6a-H-2 in a well-supported cluster with C. ilicicola. The pathogenicity of 6a-H-2 was tested through a pot assay. Five healthy plants had their leaves scratched with a sterilized needle, then inoculated by spraying 20 mL of spore suspension (105 spores mL-1) of 6a-H-2. Five additional tea plants sprayed with sterile distilled water served as controls. All plants were placed in a growth chamber at 28℃, with 70% relative humidity. The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphology and molecular analyses (HIS3, TEF1 and CAL) from the infected leaves but not from the non-inoculated leaves. To our knowledge, this is the first report of leaf spot on tea caused by C. ilicicola in China. This study provides valuable information for the identification and control of the leaf spot on tea.