First Report of Alternaria alternata causing Leaf Blight in Carrot ( Daucus carota L.) in Bangladesh

In February 2024, we received blighted carrot (Daucus carota L.) leaves of dark brown to tan color with circular necrotic lesions from approximately 2.5 acres of carrot field in Manikganj, Bangladesh (23.4855°N, 90.0833°E). The disease incidence was estimated at approximately 15%, and the severity of the affected leaves was measured at 25-30%. Fifteen symptomatic leaves from 10 randomly selected plants were surface-disinfected in 1% NaOCl for 1 min, rinsed with sterile distilled water for 30 s three times, blot-dried, plated on potato dextrose agar (PDA), and incubated at 25°C in dark conditions. At 14 days, light-green and olivaceous-colored colonies began to grow. Growing hyphal tips were incubated on fresh PDA. Single spores were grown on clarified V8 juice agar for 8 days to obtain a pure culture (Choi et al. 1999). Colonies were identical and light green to olive green. Microscopic observation at 40× magnification revealed brown, pear- or club-shaped conidia measuring 16.5 to 28.3 µm × 6.9 to 11.2 µm; the conidial body consisted of four to nine transverse and longitudinal septa. Conidiophores were septate and hyaline. Mycelia were branched, septate, and transparent. Based on morphological characteristics, the isolates were identified as Alternaria alternata (Fr.) Keissl. (Simmons 2007). Genomic DNAs from three randomly selected isolates were PCR amplified and sequenced for the internal transcribed spacer region (ITS) (White et al. 1990), large subunit (LSU) (Vilgalys and Hester 1990), the second largest subunit of RNA polymerase II (rpb2) (O'Donnell et al. 2009), plasma membrane ATPase (ATPase) (Lawrence et al. 2013), and glyceraldehyde-3-phosphate dehydrogenase (gpd) (Berbee et al. 1999) loci. The respective sequences of the three isolates were identical, so one isolate (ZAACl-1) was submitted to GenBank with accessions PV211330 (ITS), PV441412 (LSU), PV441483 (rpb2), PV469432 (ATPase), and PV441479 (gpd). The respective loci were 100% identical with the reference sequences from MH824256 (ITS), OP579222 (LSU), PP457497 (rpb2), PQ001739 (ATPase), and MN258117 (gpd) of A. alternata. Multilocus phylogenetic analysis showed that ZAACl-1 is related to A. alternata voucher specimen CBS 916.96. For pathogenicity test, carrot (cv. Deshi Gajor) plants at 6 weeks old (at 8-true leaf stage) were grown in 10 × 10 × 8 cm plastic pots containing autoclaved soil. A 1 × 106 conidia/ml suspension was prepared from 10-day-old cultures in 0.025% Tween® 20 (Chen 2018) and sprayed onto the upper surface of carrot plants until runoff. The experiment was conducted twice. Carrot leaves sprayed with sterile autoclaved water served as non-inoculated control. Inoculated plants were kept in a humidity chamber for 5 days, maintaining a temperature of 25°C, relative humidity (RH) >80%, and a 14-h photoperiod, and then transferred to greenhouse with 70% RH, 25°C, and a 14-h photoperiod (600-W high-pressure sodium lamps). Two weeks later, all inoculated leaves developed grayish to dark brown necrotic lesions followed by blighted symptoms, similar to those observed in the field. Mock-inoculated leaves were symptomless. A. alternata re-isolated on PDA from lesions of inoculated leaves were identical to field isolates. To our knowledge, this is the first report of A. alternata as a pathogen of carrots in Bangladesh. This finding will help growers to design integrated disease management practices against this novel pathogen.