First report of anthracnose on sugar beet (Beta vulgaris) caused by Colletotrichum incanum in Michigan, USA

Abstract

In 2016, sugar beet leaf samples were received at the Michigan State University plant diagnostic laboratory with distinctive oblong lesions on the petiole (Fig. 1). The lesions contained acervuli with dark setae (Fig. 2). Lesions on the blade had light and dark rings that showed similar acervuli in a 90% humidity chamber. Pure cultures from single spore isolation were grown on malt extract agar (MEA). The resulting cultures produced acervuli-like structures with dark setae distributed throughout the conidiomata. Spores were curved and had a distinct gutule (Fig. 3). Based on these characteristics, isolates were identified as a Colletotrichum sp. A representative isolate, Co16-4, was grown in liquid V8 medium with shaking for seven days. Fungal tissue was lyophilised and DNA extracted using a DNeasy Plant Mini Kit (Qiagen, Germany). Partial sequences of the internal transcribed spacer (ITS), actin (ACT), and β-tubulin (TUB2) genes were amplified according to Damm et al. (2009) and submitted to the Research Technology Support Facility (East Lansing, USA) for Sanger sequencing. Sequences were compared to data in GenBank where ITS gave the closest match to C. spaethianum (>98%), while ACT and TUB2 gave the closest match to C. incanum (99 and 96%, respectively). Sequences were submitted to GenBank as Accession Nos. OP628186 (ITS), OP627081 (ACT) and OP627082 (TUB2). Due to the BLAST search ambiguity, sequences of 19 Colletotrichum species were retrieved from GenBank as identified in Yang et al. (2014). Sequences were concatenated and manually adjusted using Geneious Prime 2021.1.1 (Biomatters Ltd., Auckland, New Zealand). Alignment and a maximum-likelihood tree were generated in Mega version 11.0.13 (Tamura et al., 2021) using the Tamura-Nei substitution model with 1000 bootstrap replicates (Fig. 4). Based on this data, isolate Co16-4 was identified as Colletotrichum incanum. Koch's postulates were fulfilled by spraying sugar beet foliage (C869) at the 10- to 12-leaf growth stage with 10ml of a spore suspension (1×104/ml with 0.1% Tween 20). After seven days, an oblong lesion was observed on the petiole and irregular lesions developed on inoculated leaves. No lesions were observed on control plants. A morphologically identical Colletotrichum sp. was isolated from lesions. Colletotrichum incanum has been reported on soybean (Yang et al., 2014), but to our knowledge, no Colletotrichum spp. have been reported causing anthracnose on beet in the United States. The symptoms and morphology of the lesions on sugar beets are consistent with beet anthracnose reported in Japan and Canada which was attributed to C. dematium (Chiko et al., 1984; Gourley, 1966). In Japan, a radish isolate, originally identified as C. dematium, was recently identified as C. incanum (Gan et al., 2016). Leaves with similar lesions were found in commercial sugarbeet fields in 2021 and 2022, indicative of continuing disease presence, but yield effects are unknown.