First Report of <i>Paraboeremia litseae</i> Causing Leaf Spot on <i>Ardisia crenata</i> in China

Ardisia crenata Sims (Primulaceae) is widely distributed in southern China and commonly used in traditional Chinese medicine (Hu et al., 2020; Liu et al., 2023). In September 2025, leaf spot was observed on A. crenata in a commercial medicinal plant farm in Xiuwen County, Guizhou Province, China (26°54′7″ N, 106°47′43″ E). Approximately 25% of 80 surveyed plants across 3.3 ha showed symptoms. Lesions were initially brown and circular to nearly circular, later enlarging with dark brown to black centers. Twenty symptomatic leaves were collected for pathogen isolation using the tissue isolation method. Small tissue pieces (ca. 5×5 mm) from lesion margins were disinfected with 2% sodium hypochlorite for 90 s and 75% ethanol for 30 s, rinsed three times with sterile water, placed on PDA, and incubated at 28 °C. After 7 days, fifteen fungal strains exhibiting similar morphology were obtained. Colonies on PDA reached 80–90 mm in diam. After 14 days at 28 ℃ in the dark, with regular margins, olive-colored centers, white margins, and flocculent, pale gray aerial mycelia. Conidiophores are reduced to conidiogenous cells; conidiogenous cells are hyaline, ampulliform to doliiform, and smooth-walled; and conidia are oblong to ellipsoidal, thin-walled, smooth, hyaline, aseptate, and measured 4.7–9.6 × 3.2–6.5 µm (mean = 6.7 × 4.9 µm, L/W ratio = 1.37, n = 30), consistent with Paraboeremia litseae (Jiang et al., 2017). Genomic DNA was extracted from 7-day-old mycelia of GUCC 26-0060 and GUCC 26-0061. ITS, LSU, RPB2, and TUB2 were amplified using ITS5/ITS4, LROR/LR5, RPB2-5F2/RPB2-7cR, and Bt2a/Bt2b, respectively (White et al. 1990; Vilgalys and Hester 1990; Liu et al. 1999; Glass and Donaldson 1995). Sequences were deposited in GenBank (ITS: PZ225409–PZ225410; LSU: PZ225413–PZ225414; RPB2: PZ233839–PZ233840; TUB2: PZ233837–PZ233838). The combined dataset was aligned using MAFFT V.7 and manually refined in BioEdit, the best-fit substitution model was selected using ModelFinder, and maximum-likelihood (ML) analysis was performed with IQ-TREE in Phylosuite v1.2.3, and branch support was calculated with 1000 ultrafast bootstrap replicates. Phylogenetic analysis showed that GUCC 26-0060 and GUCC 26-0061 clustered with P. litseae CGMCC 3.18109T and CGMCC 3.18110 with 98% ML bootstrap support. BLASTn search showed high similarity to P. litseae strains CGMCC 3.18109 and CGMCC 3.18110, with identities of ITS: 100% (483/483 bp), 100% (483/483 bp); LSU: 100% (1313/1313 bp), 100% (1313/1313 bp); RPB2: 99% (594/596 bp), 100% (596/596 bp); and TUB2: 99% (332/333 bp), 100% (333/333 bp), respectively. Pathogenicity tests were conducted twice using nine healthy two-year-old A. crenata plants. GUCC 26-0060 and GUCC 26-0061 were grown on PDA at 28 °C for 14 days to prepare conidial suspensions. Leaves were sprayed with conidial suspensions (1 × 10⁶ conidia/mL), with three plants per isolate; three control plants were sprayed with sterile water. The plants were incubated at 28 °C under greenhouse conditions. Typical leaf spots developed on inoculated leaves after 7 days, whereas controls remained asymptomatic. The same fungus was re-isolated from symptomatic leaves and confirmed as P. litseae based on morphology and molecular analysis, fulfilling Koch’s postulates. Paraboeremia litseae has recently been reported causing leaf spot on Camellia sinensis in China (Wang et al. 2024). To our knowledge, this is the first report of P. litseae causing leaf spot on A. crenata.