First report of leaf blight disease caused by Pestalotiopsis microspora on golden camellia ( Camellia sect. Chrysantha Chang) in China

Golden camellia (Camellia sect. Chrysantha Chang) is a subgroup of the Camellia subgenus of Theaceae. It is a world-class rare ornamental plant with high medicinal and research value, mainly distributed in southern and southwestern Guangxi province, China and northern Vietnam (Liang, 2007; Nguyen et al., 2019). Leaf spot is the most important fungal disease of golden camellia, which seriously restricts the high-quality production of tea leaf and scented tea. In December 2023, leaf blight were observed on golden camellia in Nanning, Guangxi, with disease incidence ranging from 3% to 10%. The pathogen damaged from margin and the center of leaf, and the lesion in the center of leaf was nearly elliptical. Initial spots were pinhead-sized with a reddish-brown halo margin, then developed into light brown necrotic spots. The necrotic leaf tissue was grayish white with scattered black granules, which were the fruiting bodies of the pathogen, and there was a reddish-brown halo at the junction of disease and health. Infected tissues usually cause leaf blight without shedding. To isolate pathogens from infected leaves, small sections of leaf tissue (5 to 5 mm2) were excised from lesion margins. Five fungi were isolated from five symptomatic trees, respectively. Representative isolate C1 was further characterized. After 5 days of culture on potato dextrose agar (PDA) medium, the fungi produced lush white villous aerial hyphae. After 10 days, a black acervuli containing slimy spore masses formed over the mycelial mats, the back of the petri dish produces a grayish black pigment. Conidia were slightly curved, fusiform to clavate, 5 to 7-celled (mainly 5) with constricted septa. Dimensions were 17.2 to 21.1 × 4.8 to 7.7 µm (n=50), with hyaline apical and basal cells. Apical cells had 1 to 3 (mainly 3) appendages 9.8 to 21.3 µm long . The median cells were brown to olive green. Aerial hyphae of isolate C1 with vigorous growth were collected for molecular identification, partial sequences of the mitochondrial small subunit (SSU), ribosomal DNA internal transcribed spacer (ITS), and nuclear ribosomal large subunit (LSU) genes were amplified and sequenced using the primers pairs of ITS-Fu-F/ITS-Fu-R, ITS1/ITS4 (White et al. 1990) and SRLSU1//SRLSU2 (Kumar et al., 2016), respectively. BLASTn comparison results showed that the SSU (PX048871), ITS (PX048869) and LSU (PX048872) sequences of isolate C1 shared highest similarities with 99.93% (AF346554), 100% (AF377292) and 100% (PQ299649) sequence identity to Pestalotiopsis microspora, respectively. Phylogenetic analysis revealed that C1 shared a common clade with the other strains of P. microspora in GenBank Data Library. Pathogenicity tests were carried out with the following procedure. Plants were maintained in a greenhouse at 28 ± 2°C. After two weeks of inoculation, symptoms identical to those in the field developed on the plants inoculated. In contrast, no symptoms developed on the control. P. microspora has been isolated from tea plants as a endophytic fungus in China (Wei et al, 2007). However, this is the first report of P. microspora infecting golden camellia in China. This work provides crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.