First Report of Leaf Blight on <i>Lithocarpus litseifolius</i> Caused by <i>Diaporthe amygdali</i> in China

Lithocarpus litseifolius is a third-generation new tea source that integrates "tea, sugar, and medicine" (Wang et al., 2021). In July 2023, a leaf blight on L. litseifolius was observed in the L. litseifolius plantation (0.33 ha) at Mount Dajue, Fuzhou City, Jiangxi Province, China (27.696119°N, 117.172554°E). The disease incidence was estimated to be above 40%. The symptoms on leaves showed brown necrotic lesions at the tips and margin of leaves, and localized areas on leaf blades. As the disease developed, the lesions enlarged and the leaves incurved with time and, in severe cases, the whole leaves became light brown, necrotic, desiccated, papery, dead, and eventually defoliated. We collected 10 leaves from the diseased area for pathogen isolation. The leaf tissue near the lesion was cut into 5 × 5 mm slices, disinfected with 75 % ethanol for 30 s, soaked in 2.5 % sodium hypochlorite solution for 3 min, and washed with sterile distilled water 4 times. The sterilized leaves were placed on potato dextrose agar (PDA) plates and incubated in darkness at 28°C for 3 days to isolate symptom-related pathogens. After 3 days of incubation, hyphal tips from the edge of the growing colony were transferred to fresh PDA plates for further purification. Finally, 10 colonies with similar morphology were isolated from 10 symptomatic tissues. Fungal colonies initially appear white, turning pale gray from the center with dense and felted mycelium with concentric zonation. The alpha conidia were on average 2.56× 5.84 μm ( 2.00 to 3.80 × 3.96 to 7.89 μm) in size and were aseptate, hyaline, smooth, and ellipsoidal (n = 30). Based on these morphological characteristics, the fungi were determined to be Diaporthe species (Gomes et al. 2013). The internal transcribed spacer region (ITS), a partial sequence of β-tubulin gene (Tub2) and translation elongation factor 1-α gene (TEF1) of one representative isolate 2BDJS1 were amplified and sequenced using primers ITS1/ITS4 (White et al., 1990), Bt2a/Bt2b (Glass and Donaldson, 1995), and EF1-728F/EF1-986R (Carbone and Kohn, 1999), respectively. The nucleotide sequences obtained from isolate 2BDJS1 were deposited in GenBank under accession numbers PQ185599 (ITS), PQ358310 (TEF1), and PQ358311 (Tub2). BLASTn analysis revealed that the ITS, TEF1, and Tub2 sequences of isolate 2BDJS1 exhibited 97.98%, 95.16%, and 99.79% similarity, respectively, to those of the D. amygdali isolate DJY-HW-1 (GenBank accession numbers MK511798 for ITS, MK570512 for TEF1, and MK570513 for Tub2). A Maximum-Likelihood (ML) phylogenetic tree was constructed using the concatenated ITS-TEF1-Tub2 multigene sequences in MEGA7. The results indicated that isolate 2BDJS1 clustered independently with two reference strains of D. amygdali, with a bootstrap support value of 99%. Combining these molecular data with morphological characteristics, including colony morphology and alpha conidia morphology, we confidently identified isolate 2BDJS1 as D. amygdali. The pathogenicity test was carried out by inoculating the mycelial disc into the leaves of L. litseifolius by piercing method (Wang et al., 2024). The control was inoculated with sterile PDA plug. Each treatment was repeated 6 times. The inoculated plants and the control plants were placed in a greenhouse at 25°C, relative humidity of 80%, and photoperiod of 12 h. All inoculated leaves exhibited blight symptoms 14 days after inoculation, whereas the control plants remained asymptomatic. Based on morphology and DNA sequence verification, only D. amygdali was re-isolated from symptomatic leaves, which confirmed Koch's hypothesis. Previous studies have shown that D. amygdali can cause canker disease in Juglans regia, Carica papaya and Prunus persica (Meng et al., 2018; Alam et al., 2023; Beluzán et al., 2021). As far as we know, this is the first report of leaf blight disease of L. litseifolius caused by D. amygdali in China. L. litseifolius is an important medicinal and edible tree species, and its planting area is increasing year by year. Therefore, it is essential to further investigate the presence of this pathogen in other planting areas of L. litseifolius within the province, and explore mitigation strategies to reduce losses.