First Report of Colletotrichum fioriniae and C. nymphaeae Causing Anthracnose on Cherry Tomatoes in South Carolina

Tomatoes were commercially produced in South Carolina on 2,600 acres in 2017 according to USDA/NASS (2017) and are one of the favorite fruits grown in homeowner gardens. In July 2018, anthracnose fruit rot was observed on an unknown commercial variety of cherry tomato (Solanum lycopersicum var. cerasiforme) fruit in a home garden in Clemson, SC. Symptoms and signs were typical for tomato anthracnose, with sunken necrotic lesions containing orange mucilaginous spore masses. Two single-spore isolates (Cf_Ebel1 and Cn_Ebel2) were obtained by transferring spores from the lesions to water agar using a cotton swab. After 24 h of incubation in the dark, single spores of each isolate were transferred to potato dextrose agar and incubated in the dark at 22°C (7 days); then they were transferred to oatmeal agar (OMA) to induce sporulation (10 days, 22°C, light). Conidia from both isolates were hyaline and straight. However, conidia of Cf_Ebel1 were fusiform with acute apices and measured 14 to 17 × 4 to 6 μm (mean ± SD: 15.2 ± 1.4 × 5.0 ± 0.6 μm), whereas conidia of Cn_Ebel2 were cylindrical with round to acute apices and measured 10 to 16 × 4 to 5 μm (mean ± SD: 14.3 ± 1.5 × 4.6 ± 0.5 μm). Colonies grown on OMA were salmon colored (Cf_Ebel1) or gray (Cn_Ebel2) after 7 days of incubation. For molecular identification of isolates, the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (TUB2) regions were amplified and sequenced for both isolates (Damm et al. 2012). Basic local alignment search (BLAST) revealed 100% similarity between Cf_Ebel1 and C. fioriniae at GAPDH (MH990621, KX161774.1) and TUB2 (MH990620, MG748139.1) and 99% at ITS (MH990622, MH863851.1) regions, whereas Cn_Ebel2 had 100% similarity to C. nymphaeae at GAPDH (MH990624, KT777658.1) and TUB2 (MK372598, LC438768.1) and 99% at ITS (MH990623, MH863847.1) regions. Based on both morphological and molecular characterization, Cf_Ebel1 was identified as C. fioriniae and Cn_Ebel2 as C. nymphaeae. Koch’s postulates were performed by inoculating cherry tomatoes. They were sterilized in 7% bleach for 1 min and rinsed with distilled water. After fruit were dried, they were wounded with a sterile needle (0.07 × 40.0 mm) and inoculated at the wounds with 20 μl of 10⁶ conidia/ml suspensions of each isolate (one wound per fruit). Control fruit did not receive spores. Inoculations were conducted on three fruit per replicate and three replicates per treatment arranged in a complete block randomized design. Fruit were incubated in a moist chamber at 22°C and 12 h light for 5 days. Spores of both species were then reisolated from emerging lesions that were similar to the ones observed on the original fruit and identified to the species level, confirming pathogenicity. To our knowledge, this is the first report of Colletotrichum acutatum or any of its associated subspecies (i.e., C. fioriniae and C. nymphaeae) (Damn et al. 2012). Knowledge about the existence of C. acutatum species complexes will help with chemical control of anthracnose both in homeowner and commercial settings. Both species, including our isolates (data not shown) are intrinsically resistant to thiophanate-methyl and vary in their sensitivity to some demethylation inhibitor fungicides (Chen et al. 2016).