First report of Diaporthe pescicola causing leaf spot on tea (Camellia sinensis) in China.

Duyun Maojian Tea mainly cultivated in Duyun City is one of the ten famous teas in China. A brown leaf spot was discovered in March 2023 in Qiannan Normal University for Nationalities' Maojian Tea experimental field (26.29°N, 107.52°E). The incidence of the disease is about 40% surveyed on about 300 m2 of tea plants. Infected leaves showed light brown, round or irregular lesions. With disease progression, they enlarged and merged, creating dark brown necrotic zones along leaf edges. To identify the responsible pathogen, three symptomatic leaves from the tea nursery base were collected. The samples were cut 3 mm x 3 mm tissues with 75% ethanol for 30 s and 5% sodium hypochlorite for 60 s, rinsing three times with sterile water. Tissues were taken to potato dextrose agar (PDA) medium and incubated dark at 28 °C for 3 days. Three isolates (TF1, TF3 and TF5) with similar morphology were obtained from three samples, respectively. The colony showed white, with short, villous hyphae expanding radially. Notably, mycelium near the disc was erect and sparse 2 cm away. After 30 days, conidia were observed. Alpha conidia (5.9 to 8.6 μm × 2.0 to 2.8 μm (n = 50)) were colorless and transparent, no septum, ellipsoid to spindle-shaped. Beta conidia (11.1 to 25.3 μm × 1.0 to 1.8 μm (n = 50)) were transparent, no septum, filiform, hooklike, tapering towards the ends. The isolates were identified as Diaporthe pescicola based on the morphology (Zhang et al. 2022). Genomic DNA was extracted, the internal transcribed spacer region (ITS), β-tubulin (tub2), and translation elongation factor 1-alpha (TEF) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively, and sequenced. The ITS (PQ163865, PQ685064 and PQ685065), tub2 (PQ197032, PV055438 and PV055439), and TEF (PQ197029, PV055440 and PV055441) sequences of isolates (TF1, TF3 and TF5) were deposited in GenBank, respectively. BLAST searches showed >97% nucleotide identity to sequences of D. pescicola MFLUCC 16-0105 (ITS, 98.40% to KU557555.1; tub2, 97.40% to KU557579.1; and TEF, 97.63% to KU557623.1). The phylogenetic tree of ITS, tub2, and TEF gene sequences was constructed using MEGA 6 software with the neighbour-joining method (NJ), and the bootstrap method was employed for 1,000 repeated tests. Isolates TF1, TF3, TF5, and D. pescicola DT 1-1 were grouped together in a node with a bootstrap value of 99. Isolates were identified as D. pescicola. Since large quantities of conidia are difficult to produce in cultures, pathogenicity on the wounded leaves of 5-year-old tea plants of Duyun Maojian Tea local population was conducted by inoculating 6 mm mycelium discs of TF1. The control group and the treatment group were inoculated with 3 tea plants, repeated 2 times. All tea plants were incubated at 25 ± 1 °C, 70% humidity, and natural daylight. The onset of symptoms appeared 7 days later. Lesions were brown, similar to field symptoms. In contrast, the control were healthy. A strain exhibiting the same morphology as TF1 was re-isolated from the diseased portion. ITS, tub2, and TEF gene sequences were consistent with TF1, completing Koch's postulates. D. pescicola as a pathogen has caused leaf spot on Kerria japonica (Zhang et al. 2022), dieback on Prunus persica (Dissanayake et al. 2017) and Vitis vinifera (Manawasinghe et al. 2019), and shoot canker on Pyrus plants (Guo et al. 2020). To our knowledge, this is the first report of tea leaf spot caused by D. pescicola in China and worldwide. It is imperative to develop and implement control measures to manage the disease.