First report of Alternaria leaf spot caused by Alternaria brassicae on broccoli in North Carolina, United States.

In September 2023, broccoli (Brassica oleracea var. italica) 'Sweet Bunch' plants on an organic farm in Buncombe County, North Carolina (NC), displayed symptoms of Alternaria leaf spot. Disease affected 10 to 20% of leaf area on all (approximately 30) plants. Lesions were dark brown with chlorotic halos, irregular in shape, and ≤3 cm in diameter. Leaf samples were incubated in a humidity chamber for 24 h, and the pathogen was putatively identified as Alternaria brassicae based on conidial morphology (Rimmer et al. 2007; Supplemental Fig. 1). Leaf pieces (2 x 2 mm) spanning lesion edges were surface-disinfested (1% sodium hypochlorite solution) for 1 min, rinsed in sterile distilled water, embedded in potato dextrose agar, and incubated at 22°C with a 12-h photoperiod. A pure culture was obtained by transferring a hyphal tip onto 20% V8 juice agar and incubated as above. After 10 d, colonies consisted of concentric zones of white to brown mycelia with white, aerial mycelia. Within 7 d, conidia measuring 80 to 150 µm long with 0 to 3 longitudinal septa, 6 to 15 transverse septa, and a long beak were observed, and the isolate was putatively identified as A. brassicae (Rimmer et al. 2007). To confirm identification, DNA was extracted from mycelia and subjected to PCR using primers to amplify a portion of the internal transcribed spacer of rDNA (ITS: ITS1/ITS4) (White et al. 1990), the transcription elongation factor-1 (tef1: EF1-728F/EF1-986R) (Carbone and Kohn 1999), and the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH: gpd1/gpd2) (Berbee et al. 1999). Amplicons were sequenced at Eton Bioscience (Durham, NC) and subjected to BLAST analysis in NCBI GenBank. Sequences matched (99.5 to 100% identity) A. brassicae CBS 116528 accessions KC584185 (ITS), KC584641 (tef1), and KC584102 (GAPDH) (Woudenberg et al. 2013). All sequences were deposited in NCBI GenBank (accessions PP584506, PP584507, PP584508). To confirm pathogenicity on broccoli and kale (B. oleracea var. sabellica), 5-week-old plants of broccoli 'Sweet Bunch', 'Eastern Crown', and 'Green Magic' and kale 'Winterbor', 'Darkibor', and 'Oldenbor' grown in 10-cm-diameter pots were separated by crop type and arranged in four randomized complete blocks by cultivar. Each block contained single-plant replicates of each cultivar that were either inoculated or non-inoculated. Conidia of A. brassicae obtained from 10-d-old cultures (3.5 x 103 conidia/ml) were sprayed onto each inoculated plant until run-off. Non-inoculated plants were sprayed with distilled water. Plants were incubated in a humidity chamber alternated with ambient greenhouse conditions every 48 h for 10 d, with average greenhouse temperatures ranging from 15 to 28°C. The experiment was conducted twice. Dark brown to gray lesions measuring 1 to 3 mm in diameter were observed on leaves of all inoculated plants within 5 d of inoculation (Supplemental Fig. 2), and non-inoculated plants remained healthy. On broccoli plants, lesions expanded in concentric rings, and sporulation of the pathogen was observed on leaf tissue and petioles 10 d after inoculation. Lesions remained small (< 3 mm) on kale plants, and sporulation was not observed. Alternaria brassicae was re-isolated from leaf tissue of all inoculated cultivars using methods described above. Pathogen identity was confirmed via colony and conidial morphology. The larger impact of A. brassicae on brassicas is unknown, but finding this less common Alternaria species in the eastern United States could have implications for host resistance development and fungicide efficacy (Nieto-Lopez et al. 2023).