First Report of Thielaviopsis thielavioides, A Causal Agent of Postharvest Blackening on Daucus carota in Serbia

Carrot (Daucus carota L. subsp. sativus [Hoffm.] Arcang.) is an important vegetable in Serbia, grown on nearly 8,000 ha. In summer 2013, a black fungal mycelium was noticed on the surface of carrot roots stored in polyethylene bags. The symptoms are manifested as irregular black patches in random patterns, increasing in size to cover the entire root surface. Disease development was more severe when carrots were washed prior to packing and stored at room temperature. These features are characteristic of Thielaviopsis thielavioides (Peyr.) Paulin, Harr. & McNew (syn. Chalaropsis thielavioides [Peyronel]), as well as of closely related T. basicola spp., causal agents of postharvest blackening of carrots (Paulin-Mahady et al. 2002). Aleurioconidia were single, thick, dark-walled, and arisen from the tip of specialized hyphae. Dimensions were 8 to 18 × 9 to 14 µm. Phialoconidia were produced from terminal cells on large phialides produced on short, septate, aerial hyphae. Phialoconidia measured 10 to 21 × 4 to 10 µm. Based on the morphological features, we identified the pathogen as T. thielavioides (Paulin-Mahady et al. 2002). Single-spore isolates were produced from aerial phialoconidia placed on water agar and incubated at 25°C in the dark, and after 24 h, single germinated conidia were transferred to individual Petri plates containing PDA. Koch’s postulates were performed on carrot roots to confirm the pathogenicity of three isolates (TT2, TT3, TT4). For each isolate, 10 roots of the commonly used ‘Nantes SP-80’ were inoculated with 10 noninoculated carrots as a control. The roots were sprayed until run-off with a spore suspension of 1.0 × 10⁴ conidia/ml in sterile water. Similarly, the control roots were sprayed with sterile water. All roots were incubated in a dew chamber for one week (t = 25°C; RH = 95% ± 3), when the same symptoms observed in the polyethylene bags developed on all inoculated roots. Control roots remained asymptomatic. The pathogen was reisolated from all inoculated carrot roots. Identity of the reisolated fungus was confirmed based on the morphological characteristics described above. The pathogenicity test was repeated with no important differences in symptoms, or dimensions of conidia among isolates. To further verify pathogen identity the internal transcribed spacer (ITS) region of rDNA was amplified using ITS1/ITS4 primers (White 1990) and sequenced with an automated equipment (Macrogen Inc., Korea). Sequences for the three isolates were deposited in GenBank (Accession Nos. KP231218 to KP231220). BLAST analysis of ITS sequences indicated a 100% identity to the reference ITS sequence of T. thielavioides (HM031127) from Australia retrieved from the NCBI database. This represents the first report of T. thielavioides developing on carrots in Serbia.