New occurrence of Alternaria cinerariae on Farfugium japonicum in China

Farfugium japonicum is an important ornamental landscape plant cultivated across East Asia (Gu et al. 2021). In July 2025, severe leaf spot was observed on F. japonicum in Chongqing municipality, China (29°22′56.49″N, 106°27′48.48″E, 199 m alt.), with a disease incidence of 85.4% (n=5 plots, 25 m² each). Lesions were circular, dark brown to yellowish-brown, surrounded by distinct yellow halos. Severely infected leaves eventually became wilted. Pathogens were isolated from diseased leaves by surface sterilization on potato dextrose agar (PDA, BD Difco, Montreal, Canada) at 25°C and single-conidium isolation on moist filter paper. Sixteen morphologically identical isolates were obtained. Colonies on PDA were olivaceous buff to olivaceous with white margins. Conidial morphology on mycelia cultured on potato carrot agar (PCA) and V8 juice agar (V8A; 8-h light/16-h dark) showed brown, obclavate to pyriform conidia with 3–12 transverse and 0–4 longitudinal septa. Conidial bodies measured 29.6–97.8 × 5.1–7.4 µm on PCA and 31.2–100.8 × 5.3–8.6 µm on V8A; beaks were 15.8–29.9 × 5.4–6.7 µm on PCA and 15.4–31.2 × 5.5–7.1 µm on V8A, conforming to Alternaria cinerariae (Simmons 2007). Genomic DNA was extracted from four representative isolates (YzU 241565, 241816, 241966, 241967) randomly selected from the 16 morphologically identical strains. Four gene regions were amplified using paired primers: RNA polymerase second largest subunit (RPB2) with RPB2-5F/RPB2-7cR (Liu et al. 1999), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with GPD1/GPD2 (Berbee et al. 1999), translation elongation factor 1 alpha (TEF1) with EF1-728F/EF1-986R (Carbone and Kohn 1999), and internal transcribed spacer (ITS) with ITS4/ITS5 (White et al. 1990). The resulting sequences were deposited in GenBank (sequences of representative strain YzU 241816: PV490325 for RPB2, PV490323 for GAPDH, PV490327 for TEF1, and PV484536 for ITS). Best-fit models for each gene were determined under the Akaike Information Criterion (AIC) in MrModelTest v.2.3. Phylogenetic trees were constructed via Maximum Likelihood (ML, RAxML v.8.2.12; 1000 bootstrap replicates) and Bayesian Inference (BI, MrBayes v.3.2.7). For BI analysis, four Markov chains were run for 1,000,000 generations (sampling every 100 generations; 25% of trees discarded as burn-in). Isolates clustered with A. cinerariae CBS 116495 with strong support (99% maximum likelihood bootstrap support / 1.0 Bayesian inference posterior probability). To assess potential recombination signals within the sampled population, the pairwise homoplasy index (PHI) test in SplitsTree v4.14.6 was performed on the isolates, and no significant recombination was detected (P > 0.05). Pathogenicity assays were performed on whole intact plants maintained in a greenhouse at 25°C with relative humidity >80%. For each replicate, three leaves of comparable size were selected per plant, with three plants used per treatment, and the whole experiment was conducted in triplicate. Inoculation was carried out using 6-mm-diameter mycelial plugs. After 7 days post-inoculation, inoculated leaves developed symptoms identical to those observed in the field, whereas control plants remained asymptomatic. Koch’s postulates were fulfilled through morphological observation and RPB2 sequencing of re-isolated fungal strains. A. cinerariae is a known pathogen causing leaf spot diseases on Asteraceae plants, which has been reported from Jacobaea maritima in Japan (Nishikawa and Nakashima 2015) and Ligularia stenocephala in Korea (Ryu et al. 2024). This is the first report of A. cinerariae causing leaf spot on Farfugium japonicum in China, expanding the known host range of A. cinerariae and providing useful information for disease diagnosis and management.