First report of anthracnose of Lithocarpus polystachyus caused by Colletotrichum fructicola in China.

ABSTRACT

Lithocarpus polystachyus (Wall. ex A. DC.), an economically valuable plant species belonging to the Fagaceae family, has been used as herbal tea to prevent diabetes because of the high content of flavonoids and dihydrochalcones in the leaves (Shang et al. 2022). In July 2022, the severe leaf lesion on L. polystachyus was first observed in Yongshun County, Xiangxi autonomous prefecture (28°45'34''N, 109°40'11''E), Hunan province, China. Yongshun County is characterized by hills and mountains, situated in a subtropical region with a mild and humid climate. A second outbreak in July 2023 was observed in the same area. The observed incident rates in the past two years were 87.3% and 90.6%, respectively. Once infected, almost all plant leaves will be infected, leading to a substantial reduction in the yield of L. polystachyus. The disease presented symptoms characterized by round or irregularly shaped lesions that initially manifested as brown spots. These lesions frequently merged into larger, dark-brown areas along the leaf margins before eventually wilting. To ascertain the pathogenic species responsible for this disease, fungal isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected with 75% ethanol and 0.1% HgCl then small pieces (1×1 cm), were placed onto potato dextrose agar (PDA) medium (Sigma-Aldrich, 70139) and incubated at 28°C for 6-9 days. Colonies were villiform and initially white, becoming gray after 6 days. Sterilized dissecting needles were used to pick single hyphal tips from the edge of the colonies and placed onto PDA for strain purification. After 15 days, the purified colonies grew fluffy white hyphae with abundant conidia. The conidia were cylindrical, had round ends, and ranged from 5.75 to 14.83 µm long and 1.75 to 2.38 µm wide (n=50). According to morphological and cultural characteristics, these isolates were preliminarily identified as Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Damm et al. 2012). To further affirm the identity of the pathogen, DNA was extracted from mycelia using a DNA extraction kit (Sigma-Aldrich, G2N70). The internal transcribed spacer (ITS) region, the transcription elongation factor (TEF), and the actin (ACT) gene were then amplified from genomic DNA extracted from three isolates (Cof1, Cof2, and Cof3) using specific primers. The primers utilized were ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and ACT-512F/ACT-783R (Carbone and Kohn 1999) for ITS region, transcription elongation factor gene and actin gene amplification, respectively. Sequence identity indicated that these isolates were highly homologous to C. fructicola. The ITS (Genbank No. PP002156, OR880553 and OR880554), TEF (No. PP061421, PP061422 and PP061423), and ACT (No. PP061418, PP061419 and PP061420) sequences of the isolates Cof1, Cof2, and Cof3 shared 99 to 100% identity with their counterparts (No. OR083309, MF627961, and OQ427895) in C. fructicola, respectively. A neighbor-joining phylogenetic tree constructed using MEGA11 (Tamura et al. 2021) also indicated that these isolates were C. fructicola. Both morphological and molecular characteristics confirmed the identification of this pathogen as C. fructicola. Colletotrichum species are known to cause anthracnose disease in a variety of economically important crops (Sharma and Kulshrestha 2015). To further validate the ability of the isolated C. fructicola to induce the same symptoms as observed in the field, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspensions (1×105 conidia per mL) from three isolates were individually inoculated onto artificially wounded leaves of 3-year-old L. polystachyus. Negative controls were established by inoculating leaf wounds with sterile distilled water. The plants were incubated in a greenhouse at 28°C and 90% humidity with a 12-h photoperiod. The experiment was replicated three times. Necrotic lesions were observed on all pathogen-inoculated wounds within 6 days after inoculation, whereas controls showed no observable symptoms. Morphological and molecular characterization of re-isolated pathogens from infected leaves indicated that the pathogens were identical. To our knowledge, this is the first report of anthracnose of L. polystachyus caused by C. fructicola in China. Farmers in the local mountainous areas are economically reliant on L. polystachyus production, while anthracnose has caused over half of the trees to lose their commercial value, resulting in significant economic losses. Our findings hold great promise for advancing strategies in the prevention and treatment of anthracnose in L. polystachyus.