First Report of Causing Wilt and Fruit Rot of Pepper in Hainan Province, China.

Ornamental pepper (Capsicum annuum L.) is an economically important plant with extensive genetic diversity (Zhang et al. 2020). In September 2022, symptoms of wilt and fruit rot were identified in approximately 0.02 hectares of an ornamental pepper plantation in Haikou, Hainan Province, China (110°32' E, 20°06' N). Disease severity reached 85%, with an incidence rate of 90%. Symptoms started as black foliar spots that expanded into large lesions, spreading to fruits and stems, causing wilting (Fig. S1). Samples from symptomatic leaves, fruits, and stems of 27 plants were surface-sterilized with 75% ethanol for 30 s, rinsed five times with sterile water, air-dried, plated on potato dextrose agar (PDA), and incubated at 28°C for 5 to 6 days. To obtain pure fungal cultures, initial isolates were subcultured onto fresh PDA. Among the 27 fungal cultures, 11 isolates from leaves and fruits consistently formed gray to olivaceous colonies. Isolate LJY224 initially produced grayish-white, fluffy mycelia with radiating aerial hyphae. Over time, the colony became grayish-black and produced black, nearly spherical pycnidia. The conidia were oval, initially transparent, and single-celled. Mature conidia were dark brown, septate, with longitudinal striations, averaging 11.55 ± 0.75 µm in width and 24.93 ± 1.50 µm in length (n=35) (Fig. S2), indicating Lasiodiplodia spp. Genomic DNA was extracted using a fungal DNA extraction kit (OMEGA BIO-TEK, GZ Feiyang Biotech Co., Ltd., Guangzhou, China). Molecular identification involved sequencing the rRNA internal transcribed spacer (ITS) region and genes encoding β-tubulin (TUB) and translation elongation factor 1-α (TEF1) with primers ITS1/ITS4, Bt2a/Bt2b, and EF1-983F/EF1-2218R, respectively (White et al. 1990; Rosado et al. 2016; Rehner and Buckley 2005). BLASTn searches with the obtained ITS, TUB, and TEF1 sequences (GenBank accessions OQ612711, OR039814, OR039813) revealed 98% to 100% identity with Lasiodiplodia theobromae reference sequences from the NCBI database (OR018404, KR260830, MN461169), matching 541/548, 446/446, and 947/955 base pairs, respectively. A phylogenetic tree was constructed using concatenated multi-locus sequence analysis (MLSA) of ITS, TUB, and TEF1 (Fig. S3). To fulfill Koch's postulates, healthy 3-month-old ornamental pepper plants were inoculated by spraying the entire plant with a conidial suspension (50 ml, 107 conidia/L) of LJY224, using sterile water as a negative control. Each treatment included three replicates. Plants were maintained at 25°C, 75% humidity, under a 12-h light/dark cycle, and monitored daily. After 14 days, inoculated plants developed black foliar spots that expanded into large lesions, spreading to fruits and stems, and causing wilting, consistent with initial symptoms, while control plants remained healthy (Fig. S4). The re-isolated pathogen showed identical morphology to the original strain. In contrast, no fungi were isolated or recovered from the plants inoculated with water. Results from disease symptoms, colony and spore morphology, pathogenicity tests, and multi-locus DNA sequence analysis suggest that L. theobromae was the pathogen responsible for the disease symptoms on ornamental peppers. L. theobromae is closely related to Botryosphaeria dothidea, both of which cause fruit rot in peppers (Rui et al. 2023). To our knowledge, this is the first report of L. theobromae causing leaf wilt and fruit rot in ornamental pepper in Hainan Province, China, offering insights to mitigate crop losses.