First report of Colletotrichum truncatum causing leaf anthracnose of Ficus carica in China

Ficus carica (fig tree) is an economically and ecologically important species in China. In September 2023, leaf spot was observed affecting over 23% of 1,600 fig trees (aged 3–7 years, including cultivars 'Bourjassotte Grise', 'Orphan', 'Green Ischia', and 'Banana') in a fig orchard on Songmu Island, Dalian, China (39°24'N, 121°46'E). Initial symptoms appeared as small, irregular, water-soaked brown spots on the leaves. The spots subsequently enlarged into irregular, gray to dark-brown necrotic lesions, often surrounded by a distinct chlorotic halo. In severe infections, extensive lesion development led to leaf curling, premature defoliation, and a decline in overall tree vigor. To identify the causal agent, tissue blocks (5 × 5 mm) were excised from the margins of lesions. The pieces were surface-sterilized by sequential immersion in 75% ethanol for 30 s and 2% sodium hypochlorite for 1 min, followed by three rinses in sterile distilled water. The sterilized tissues were placed onto full-strength potato dextrose agar (PDA) without antibiotics and incubated at 25°C in darkness for 5 days. Nine isolates with similar colony appearance were obtained. The identified colonies were round with clear, white edges and sparse mycelium. As the culture aged, the center initially appeared light gray but gradually darkened to black. The conidial discs featured black, heap-like protrusions. The conidia were transparent, sickle-shaped structures with rounded tips at both ends, measuring between 19.28 to 25.89 μm in length and 2.01 to 4.77 μm in width (n = 30). Meanwhile, the appressoria were oval to irregular, black, and measured between 6.30 to 13.66 μm in length and 4.81 to 9.59 μm in width (n = 30), with distinctive bristle-like appendages, preliminarily identifying the pathogen as Colletotrichum spp. For molecular identification, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS), actin (ACT), and β-tubulin (TUB2) genes were amplified and sequenced from three isolates (DLFC2301, DLFC2302, and DLFC2303) using ITS1/ITS4 (White et al. 1990), GDF1/GDR1 (Cannon et al. 2012), CHS-79F/CHS-345R, ACT512F/ACT783R (Carbone and Kohn 1999), and BT2A/BT2B primers (Glass and Donaldson 1995), respectively. The amplicon sequences of the representative isolate DLFC2303 were deposited in GenBank under accession numbers: ITS (PQ460263), GAPDH (PQ471494), CHS (PQ471493), TUB2 (PQ471481), and ACT (PQ471491). A maximum-likelihood phylogenetic analysis of five concatenated genes in MEGA 11 identified DLFC2303 as Colletotrichum truncatum. Conidial suspension (1×10⁶ conidia/mL) of isolate DLFC2303 was inoculated onto nine healthy leaves on three separate 3-year-old potted fig trees (three leaves per tree) via needle-prick wounding. To maintain humidity, the inoculation sites were covered with sterile moist cotton and enclosed in plastic bags. For the control, nine leaves on three additional trees were treated similarly with sterile distilled water. After 10 days, lesions resembling those found on field specimens appeared, and C. truncatum was successfully reisolated from diseased leaves, fulfilling Koch's postulates. These findings confirmed the pathogen as C. truncatum. Although C. truncatum has been reported to cause leaf anthracnose of Ficus carica in Malaysia (Nur-Shakirah et al. 2023), this is the first report of fig leaf anthracnose caused by C. truncatum in China.