First Report of Anthracnose Leaf Spot Caused by Colletotrichum americae-borealis on Potentilla anserina in China.

Potentilla anserina L. has an abundance of bioactive compounds and is widely recognized for its diverse applications in traditional medicine and as a food. In August 2023, typical symptoms of anthracnose were observed in 80% of P. anserina plants in Harbin, China. Symptoms, characterized by reddish-brown spots, tend to occur more frequently on leaves closer to the ground. They initially appeared as oval or irregular circles, measuring 1 to 3 mm in diameter, and later merged into larger patches surrounded by chlorotic areas on the leaves. Twenty leaves exhibiting characteristic symptoms were sampled. Each leaf was sectioned into 5×5 mm pieces at the interface between the diseased and healthy tissues. The sections were disinfected sequentially with 75% ethanol for 30 s, followed by 1% NaClO for 2 min, rinsed three times in sterilized distilled water. Post air-drying, samples were cultured on potato dextrose agar (PDA) plates and incubated at 26°C in the dark for 5 d, yielding nine morphologically similar single-spore isolates (JTC1 to JTC9). The colonies initially displayed gray aerial mycelia, becoming pale brown, accompanied by numerous black microsclerotia. The acervuli appeared black, protruded from the surface of the medium, and were adorned with dark brown setae. Setae (n=50) ranged from 58.4 to 188.2 μm in length, appearing dark brown to black, with smooth walls, rounded tips, swollen bases, and containing 1 to 4 septa. The conidia were hyaline, aseptate, cylindrical to spindle-shaped, with blunt and rounded ends, measuring 13.7 to 18.3 μm in length and 3.4 to 4.3 μm in width (n=50). Morphological analysis indicated a close affinity with Colletotrichum americae-borealis (Damm et al. 2014). For molecular identification, genomic DNA was extracted from three representative isolates (JTC1, JTC2, and JTC3).The ITS, HIS3，GAPDH, and ACT genes were amplified and sequenced using the primers described previously by Damm et al. (2014). The sequences were submitted to GenBank (ITS: PP338190 to PP338192; HIS3: PP355770 to PP355772; GAPDH: PP355773 to PP355775; ACT: PP355776 to PP355778). BLAST analysis showed 99 to 100% identity with C. americae-borealis type strain CBS 136232 (GenBank accessions: KM105224, KM105364, KM105579, and, KM105434, respectively). Multigene phylogenetic analysis positioned the three isolates close to C. americae-borealis. Pathogenicity tests were performed twice on 6-week-old P. anserina seedlings (cv. Qinghai Juema 1) in a greenhouse. A conidial suspension of the JTC1 isolate (1×10⁵ conidia/ml) was sprayed applied to ten pots, each containing two seedlings, and the plants in the control pots were sprayed with sterile distilled water. Two weeks after inoculation under greenhouse conditions (26/22°C day/night temperature, 12-hour photoperiod, 90% relative humidity), the inoculated seedlings exhibited brown spots and necrotic lesions similar to those observed in the field, C. americae-borealis was successfully reisolated from these symptomatic tissues. To the best of our knowledge, this is the first report of C. americae-borealis causing leaf spot on P. anserina in China. Anthracnose caused by C. americae-borealis is associated with leaf spot disease in oats (Wang et al. 2022), alfalfa (Li et al. 2021), and licorice (Lyu et al.2020). However, C. americae-borealis poses a significant threat to P. anserina in China as well, highlighting the urgent need to develop effective disease management strategies.