First Report of Stemphylium Leaf Spot of Spinach Caused by <i>Stemphylium vesicarium</i> in New Hampshire

Leaf spots were observed on spinach (Spinacia oleracea) in two commercial farms in New Hampshire. In November 2021, about 10% incidence occurred in a 0.4-ha organic field (cv. Kolibri) in Rockingham County, and in October 2023, 25% incidence was noted in a 0.6-ha conventional field (cv. Sioux) in Strafford County. Affected plants had tan, papery lesions ≤10 mm in diameter with diffuse margins and dark sporulation typical of Stemphylium. Thirty diseased leaves per farm were collected; all lesions were isolated, but only one per leaf from five randomly selected leaves was analyzed. Symptomatic tissue was surface-sterilized in 0.5% NaOCl for 1 min, rinsed in sterile water, dried, and plated on potato dextrose agar (PDA) . All ten isolates developed dense, blackish- to greenish-brown aerial mycelium and produced conidia within 14 days under fluorescent light at room temperature. Conidia were oblong to broadly ellipsoidal, pale to medium brown, verrucose, with up to six transverse and several longitudinal septa, often constricted at the three major septa, measuring 22-36 × 16-24 μm (based on three separate measurements). These features were consistent with Stemphylium vesicarium (Woudenberg et al., 2017). Total DNA was extracted using the DNeasy Plant Mini Kit. The glyceraldehyde-3-phosphate-dehydrogenase (gapdh) and calmodulin (cmdA) genes were amplified with primers from Woudenberg et al. (2017), purified (Qiagen), and sequenced (Eton Bioscience, NC) using ABI PRISM Dye Terminator Cycle Sequencing. Sequences were edited in SeqPup and analyzed by maximum likelihood in PAUP* 4.0b10 (Liu et al. 2020). BLAST searches showed gapdh and cmdA sequences from all ten isolates shared 99% identity with S. vesicarium (KU850702, KU850845) in GenBank. Sequences from isolates NH1 and NH2 were deposited in GenBank (gapdh: PQ045533-PQ045534, 583 bp; cmdA: PQ045535-PQ045536, 693 bp). Phylogenetic analysis (Suppl. Fig. 1) based on combined gapdh and cmdA sequences confirmed that New Hampshire isolates closely clustered with S. vesicarium reference strains (Liu et al., 2020; Woudenberg et al., 2017). For pathogenicity tests, ten spinach seeds (cv. Viroflay) were sown per 10-cm pot with LC1 potting mix, thinned to three plants per pot, with three replicate pots per treatment. The experiment was repeated twice. Three isolates were cultured on PDA, and the conidia within 21 days under fluorescent light at room temperature were suspended in distilled water and adjusted to 1 × 10⁵ spores/ml (Liu et al. 2020). Four-week-old plants were sprayed until runoff, incubated in a mist chamber for 48 h at 24-25°C, and then moved to a greenhouse (20-30°C). Controls were sprayed with sterile water. Disease severity was recorded 5 to 14 days after inoculation. All three isolates caused severe lesions, covering 56-75% of leaf area at 14 days post-inoculation. Disease severity was consistent across both experimental repetitions. No symptoms developed on non-inoculated controls. The greenhouse test produced similar symptoms on leaves as the ones found in the fields, and S. vesicarium was re-isolated from diseased tissue. Based on morphology, molecular and phylogenetic analysis, and pathogenicity, the pathogens causing leaf spot on spinach were S. vesicarium, which is the first report on spinach in New Hampshire, extending the known geographic and host range of this pathogen in New England region.