First report of Alternaria brassicicola causing black leaf spot on cabbage in Pakistan

Black spot diseases are caused by necrotrophic fungal pathogens, including economically important cabbage species. During mid-October of 2022 and 2023, leaf spot symptoms were observed on cabbage plants in the experimental field of the Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan. The disease affected approximately 35% of the plants across a two-acre field. Early symptoms were pin-sized black specks distributed on the leaf surface. Specks eventually enlarged concentrically and became dark brown, sunken lesions/spots with a yellow halo. Necrotic lesions were visible on both sides of the leaves. To identify the causal agent of the disease, 15 symptomatic leaves were collected. Small pieces removed from the margin between healthy and diseased tissues were surface disinfected in 70% ethanol for 2 min, rinsed three times with sterile distilled water, and then plated on potato dextrose agar. The plates were incubated at 25 ± 2°C with a 12-h photoperiod. Morphological observations were made on 7-day-old single-spore cultures. The colonies appeared black and green. The conidia were dark, spindle-like to obclavate, measuring 15 to 48 μm in length, with longitudinal and transverse septa. Chains of conidia were found having 6-14 conidia forming germ tubes. The morphological characters matched those of Alternaria brassicicola (Corlett and MacLatchy 1996). For Molecular identification, DNA was extracted using the DNAzol reagent (Thermo Fisher Scientific MA, USA). For molecular identification, the internal transcribed spacer (ITS) region between ITS1 and ITS2, and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of four representative isolates (Br. ol-01,05,08,11) were amplified with primers ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), respectively. BLASTn analysis of these sequences showed 100% identity with the sequences of A. brassicicola for ITS rDNA, and GAPDH, respectively. Based on morphology and sequencing, isolates were identified as A. brassicicola, and sequences were deposited in GenBank (ITS: PV765402.1-PV765405.1; GAPDH: PX136054.1-PX136057.1). To confirm pathogenicity, three plants at the true leaf stage were inoculated in the greenhouse with conidial suspensions (1.5 to 2.0 ml per plant, until runoff) of each of the four isolates of A. brassicicola (106 conidia/ml, prepared from 1-week-old cultures and amended with 0.1% [vol/vol] Tween 20) using an atomizer, while three control plants were sprayed with sterile distilled water. The plants were then incubated at 25 ± 2°C in the greenhouse, and the experiment was repeated twice. Ten days after inoculation, each isolate induced leaf spots resembling those typically observed in the field, whereas the control plants remained symptomless. The fungus was re-isolated from symptomatic tissues. Re-isolated fungal cultures were again morphologically and molecularly identical to A. brassicicola, thus fulfilling Koch’s postulates. Previously, A. brassicicola has been reported to cause black leaf spot on cabbage (Akram et al. 2019). To our knowledge, this is the first report of A. brassicicola causing leaf spot on cabbage in Pakistan. Given the importance of cabbage's traditional cultivation and consumption in Pakistan, this disease could pose a threat to cabbage cultivation. This finding highlights the need for timely recognition and management of this pathogen to prevent potential impacts on cabbage production.