First Report of Colletotrichum boninense causing anthracnose on Polygonatum odoratum in China

Polygonatum odoratum (Mill.) is a perennial herbaceous medicinal plant and cash crop. Its cultivation area in Hunan and Guizhou Provinces, China, exceeds 130,00 hectares, with an annual output of approximately 0.12 million tons (National Administration of Traditional Chinese Medicine, 2023). In November 2024, anthracnose symptoms were observed on P. odoratum at the Germplasm Nursery (10 m 2 ) of Chinese Medicinal Herbs, Guizhou Academy of Agricultural Sciences (26°30'11"N, 106°39'27"E), with incidence of 40% to 55%. The lesions began as small, round or elliptical leaf spots with light yellow centers surrounded by a brown outer ring, then expanded into irregular shapes not confined by leaf veins (Fig. 1, O-R). To identify the pathogen, symptomatic leaf samples were collected from 10 P. odoratum plants. Tissue subsamples were excised from the lesion margin, immersed in 75% ethanol for 30 s, transferred to 1% NaClO for 60 s, rinsed three times in sterile distilled water, and air-dried. The samples were then cultured on PDA plates at 28℃ for five days. Eight morphologically similar isolates were obtained. The colonies on PDA were covered with white, fluffy aerial mycelium (Fig. 1, A-E). Conidiophores were septate, smooth-walled, and hyaline to pale grey (Fig. 1, F-G). Conidia were cylindrical to ellipsoidal, aseptate, hyaline, smooth-walled (Fig. 1, I), and 16.15 - 19.78 × 6.42 - 7.65 μm (average ± SD: 17.61 ± 1.01 × 7.03 ± 0.35 μm; n=50). Morphological characteristics were consistent with the description of C. boninense (Damm et al. 2012). For further identification, genomic DNA was extracted using the BIOMIGA Fungal Genomic DNA Extraction Kit. Partial sequences of the ITS (White et al. 1990), act (Carbone and Kohn 1999), gapdh (Templeton et al. 1992) and his3 (Crous et al. 2006) genes were amplified using specific primers and sequenced. Sequences of three representative strains (GUCC25-0101 to GUCC25-0103) were submitted to NCBI. BLASTn analysis revealed 100% sequence homology of the ITS (PV953558 to PV953560), act (PV962129 to PV962131), gapdh (PV956154 to PV956156), and his3 (PV956158 to PV956160) sequences from the three representative strains to C. boninense (accession nos. OP723105, ON009367, JQ005240 and JQ005414). A maximum likelihood phylogenetic tree of Colletotrichum species demonstrated that three strains clustered in a clade with C. boninense (Fig. 2). For pathogenicity assays, the isolates were grown on PDA for 7 days and then used to prepare conidial suspensions (1 × 10 5 conidia/mL). Asymptomatic five-leaf-stage P. odoratum plants were used. Six replicate plants were used for each single-spore isolate under study. The plants were subjected to spot inoculation on the adaxial side of the leaf. Three 20-µL drops of spore suspension were deposited 2 cm apart near the central vein of the leaf. The plants were watered twice a week to ensure continuous ambient humidity (80% relative humidity). As controls, six asymptomatic plants per trial received water droplets rather than inoculum on leaves. Fifteen days post-inoculation, all pathogen-inoculated leaves exhibited symptoms similar to those observed on naturally infected plants in the field, while control leaves remained asymptomatic. C. boninense was re-isolated from the lesions, and fungal identity was confirmed based on morphological and molecular characteristics. Consistent results were obtained in three replicate experiments. In addition to C. boninense reported in this study, C. liriopes can also cause anthracnose in p. odoratum (He et al., 2023). This is the first report of C. boninense causing anthracnose on P. odoratum in China. This disease poses a serious threat to P. odoratum cultivation. Our findings will contribute to future monitoring and management of this disease.