First occurrence of <i>Alternaria alternata</i> species complex causing leaf spot on common hop ( <i>Humulus lupulus</i> ) in Massachusetts

Common hop (Humulus lupulus) is a perennial crop primarily grown as a flavouring agent for beer (Machado, 2019). It has been cultivated in Massachusetts since 1629 (Rumney, 1998). On July 31st, 2025, browning symptoms were observed in a hopyard in Northfield (42.6368 N -72.4820 W), Franklin County, MA, affecting cones, green bines, and sepals. On leaves, overlapping lesions radiated out from the leaf margins and veins. Lesions were grey or brown with a distinct chlorotic halo. Three rows of ‘Teamaker’ and ‘Centennial’ cultivars were evaluated where disease incidence was 100%. All plants showed mild to moderate disease severity, with some bines severely affected. Two samples were taken from a bine and sepal in ‘Teamaker‘ and from a leaf and petiole in ‘Centennial’. Samples were cut into 1 cm sections, surface disinfested for 30 s with 70% ethanol solution followed by 30 s of 1% NaClO solution, rinsed for 30 s in sterile water before plating on PDA. Colonies ranged from brown to dark grey with aerial mycelium. Conidia from all isolates were morphologically consistent with the Alternaria alternata species complex: brown, elliptical, concatenated, with 1-3 transverse septa and 0-1 longitudinal septa (Woudenberg et al. 2015). Genomic DNA was extracted, and PCR was performed using primers targeting the ITS region (ITS1F/ITS4), EF1 (EF1-728F/ EF1-986R), and β-tubulin (Bt2a/Bt2b) (White et al., 1990; Carbone &amp; Kohn, 1999; Glass &amp; Donaldson, 1995). Amplicons of ITS and TEF were obtained for both cultivars, β-tubulin was amplified for ‘Centennial’. The Nanopore sequencing (Psomagen) platform was used for sequencing. TEF sequences were identical (256/256), and ITS sequences showed 544/545 bp similarity. Sequences from the ‘Centennial’ isolate are deposited in NCBI GenBank (accession nos. PX588111, PX610247, PX610248). All sequences shared 99.7-100% identity with type sequences from the Alternaria alternata species complex (ITS 547/547bp [MH861939.1], TEF 240/240bp [KC584652.1], β-tubulin 325/326bp [JQ672038.1]). Pathogenicity was confirmed on rooted cuttings of cultivar ‘Triple Pearle’ and PI 689630. The first pair of leaves below the apical growing tip were wounded at three sites per leaf with a sterile toothpick dipped in inoculum; control wounds were untreated. For PI 689630 only one leaf served as the control. Lesions, consistent with those observed in the field, developed within 24 hours on ‘Triple Pearle’ and within 72 hours on PI 689630. No symptoms developed on control plants. Eight days post-inoculation, A. alternata was reisolated on PDA from 4/12 inoculated wounds and from 0/9 control wounds, fulfilling Koch’s postulates. Contaminants overgrew six of the isolates and one of the controls. Identification was confirmed based on colony morphology and conidial characteristics. This is the first report of Alternaria alternata species complex causing leaf spot of common hop in Massachusetts. The A. alternata species complex has previously been reported on common hop cones (Pethybridge et al., 2001). Care should be taken to minimize mechanical injury during the growing season, and rapid postharvest cone processing is recommended if symptoms are observed.