Colletotrichum coccodes causing “black dot” of sugar beet in the Pacific Northwest of the USA

Abstract

Sugar beet (Beta vulgaris) is an important sugar crop in temperate regions. In 2014, sugar beet roots in Washington and Oregon, U.S.A. were found with a shallow, firm, dark rot with minute black “dots” within the tissue (Figs. 1, 2), similar to “black dot” on potato (Davis & Johnson, 2001). Symptoms were observed in freshly harvested roots in the processing factory. Diseased tissue was surface disinfected with 10% bleach, plated on 2% water agar and incubated at room temperature with ambient lighting. All visible growth from ten tissue pieces (five per beet, one each from Washington and Oregon) was fungal, with hyaline mycelia and numerous dark, spherical sclerotia. Pure cultures were obtained by hyphal tip transfer to malt extract agar (MEA), incubated for seven days in the light at room temperature, and examined for morphological characteristics. Yellow spore clusters with dark setae were observed in culture. Conidia (20-25 × 3–4 µm) were hyaline and straight with evenly rounded ends (Fig. 3) matching those of Colletotrichum coccodes (Chesters & Hornby, 1965). For molecular characterisation, DNA was extracted from seven-day old cultures using a CTAB protocol (Anonymous, 2009). PCR amplification used an Invitrogen High Fidelity PCR mix (Thermo Fisher Scientific Inc., Waltham, USA) with primers ITS1 and ITS4 (White et al., 1990) and the resulting amplicons were sequenced by Sanger sequencing. A BLASTn search with the sequence of isolate Co14-1 (GenBank Accession No. KX011522) showed a 99% identity to CBS369.75 (HM171679), the type strain for C. coccodes (Cannon et al., 2012) as well as to additional C. coccodes isolates (JX294043, JX205194, JX205192, and KJ744346). As initial samples were found at harvest, healthy roots of two sugar beet germplasm (C869 and SR102) that had been grown in the field for five months at the Saginaw Valley Research and Education Center (Frankenmuth, Michigan, USA) were used for pathogenicity testing. After removal from the field, roots were kept at 7°C for one-two months then planted in potting mix and grown in a greenhouse at 22°C ±4°C. After growth of new leaves was visible, two plants of each germplasm were treated with a 10 ml drench of a 103 spores/ml suspension that was produced by growing isolate Co14-1 on MEA for seven-ten days, flooding the surface with sterile distilled water, and dislodging the spores with a sterile plastic cell spreader. Plants were grown for four weeks after inoculation and evaluated. Roots of inoculated plants showed shallow sunken lesions with minute black “dots” similar to those on the initial samples (Fig. 4). A fungus morphologically identified as C. coccodes was isolated from lesions on all inoculated beets, completing Koch's postulates. No Colletotrichum sp. was isolated from control beets. The trial was conducted twice with the same results. To our knowledge this is the first report of C. coccodes on sugar beet from the field. Although there was little root loss observed on beet, black dot infection can impact storage in potato (Davis & Johnson 2001). Therefore, testing the potential impact of black dot on storage of sugar beet is warranted. The authors would like to thank Tom Goodwill for his technical assistance.