First Report of <i>Colletotrichum fructicola</i> Causing Anthracnose on <i>Phoebe zhennan</i> (nanmu) in China

Phoebe zhennan, a vulnerable species (World Conservation Monitoring Centre, 1998) renowned for its “golden-thread nanmu“ timber (~1,500 USD/m³; Xie et al., 2015). In August 2022, a novel twig blight caused a severe outbreak in Xuan'en County, Hubei (29°59′N, 109°28′E), with a 27% incidence rate among 166 surveyed trees. The disease initially appeared as dark brown lesions on twigs, which gradually darkened to black, resulting in blight and ultimately contributing to overall tree decline. Symptomatic tissue samples were collected from twigs, leaves, and buds of six diseased plants. Stem segments were cut to approximately 5 mm in length, while leaf and bud tissues were cut into 5 × 5 mm2 pieces. All segments were surface sterilized with 75% ethanol for 1 min, 2% NaClO for 1 min, rinsed thrice in sterile distilled water, dried with sterile filter paper, and incubated on potato dextrose agar (PDA) at 28°C in the dark. Three morphologically similar isolates (PZ0201 to PZ0203) were obtained. The colonies were initially white with dense aerial mycelium, later turning grayish-white with concentric rings and conidial masses, and eventually becoming gray-black. It exhibited radial growth rate of 5.3 to 8.0 mm/day. Conidia were unicellular, cylindrical to elongated-oval with rounded ends, aseptate, smooth, and colorless, measuring (n = 50) 8.0 to 17.5 μm (12.6 ± 1.8 μm) × 4.0 to 6.0 μm (5.1 ± 0.4 μm). Dark brown appressoria were observed. Morphologically, features resembled Colletotrichum spp. (Weir et al. 2012). The amplified sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), partial actin (ACT), β-tubulin (TUB2), and calmodulin (CAL) were deposited in GenBank under accession numbers PX651344 to PX651346 and PV124327 (ITS), PX696810 to PX696813 (GAPDH), PX696802 to PX696805 (ACT), PX696814 to PX696817 (TUB2), and PX696806 to PX696809 (CAL). BLASTn confirmed identities of 100% (560/560 bp) for ITS, 99.6% (250/251 bp) for GAPDH, 99.3% (280/282 bp) for ACT, 100% (450/450 bp) for TUB2, and 100% (500/500 bp) for CAL with the C. fructicola ex-type strain ICMP 18581. Phylogenetic analyses using concatenated sequences of the five loci revealed that the isolates were C. fructicola. Pathogenicity was tested on healthy 3-month-old and 2-year-old potted P. zhennan. A single twig per plant was inoculated with 1 ml of conidial suspension (1.0 × 10⁶ spores/ml) and incubated at 28°C with 70% relative humidity. Control samples were treated with sterile distilled water. Each treatment included five plants with three replications. On day 8, inoculated twigs exhibited browning, which progressed to black discoloration by day 11, along with noticeable wart-like protrusions of embedded hyphae. Control twigs remained asymptomatic. The pathogen was reisolated from the lesions and confirmed as C. fructicola morphologically and molecularly, fulfilling Koch’s postulates. As necrotrophic pathogens, Colletotrichum species typically cause anthracnose diseases, with symptoms including leaf spot (Liu et al., 2024), leaf blight (Su et al., 2024), and twig blight (Soares et al., 2021). Previous studies have reported C. fructicola as the causal agent of leaf spot on P. sheareri (Huang et al., 2021). This study marks the first report of C. fructicola causing anthracnose on P. zhennan, providing fundamental information for diagnosing, preventing and controlling this disease.