First Report of Bipolaris gigantea Causing Bipolaris Leaf Spot on Hooked Hair Hops ( Humulus japonicus ) in Kentucky

Hooked hair hops, aka Japanese hops, (Humulus japonicus; Cannabaceae), is an introduced invasive vine in North America. The weedy vine was observed growing on the banks of the Kentucky River (37°31′52.1″N, 83°21′04.7″W) in Breathitt County, Kentucky, USA, in September 2024 adjacent to research plots of hemp (Cannabis sativa) and Japanese stiltgrass (Microstegium vimineum). Hemp and stiltgrass are hosts of Bipolaris gigantea and showed leaf spot symptoms consistent with those previously reported (Szarka et al. 2020). Eyespot symptoms were also sporadically found on hops plants, occurring in discrete foci with low per-leaf incidence (less than 5%) and an estimated 1 to 3% disease severity on affected leaves. Symptoms on hops leaves included abundant, round, light-green to light brown colored lesions with light to dark brown borders, and sometimes surrounded by profuse tissue necrosis. Selected lesions within a pooled sample of 30 symptomatic hop leaves were dissected, surface disinfested for 30s in 10% household bleach (v/v) (7.5% sodium hypochlorite), rinsed three times in tap water, and placed on 1.5% water-agar medium for 2 days at 28°C. Large spores (macroconidia) were observed after 48h. Single spores were isolated using a dissecting needle to half-strength PDA for 5 days, and then transferred to half-strength V8 agar medium where they developed colonies with gray to dark gray mycelium and whitish tufts. Macroconidia from culture were hyaline to pale brown, contained one to four septa, and measured 171.85 to 401.7 µm × 21.5 to 35.2 µm (mean 355.8 µm x 28.3 µm, n = 80). These morphological characteristics were consistent with the description of B. gigantea (Lane et al. 2020; Szarka et al. 2023). All isolates observed had the same morphology. One representative isolate, QTH1, was selected for molecular identification of the species, and pathogenicity testing. QTH1 was grown on PDA overlaid with cellophane for 3 days at 28°C for DNA extraction. The internal transcribed spacer (ITS) region of rDNA, RNA polymerase II subunit 2 (RPB2) gene, and the partial translation elongation factor 1-α (TEF1) gene were amplified and sequenced. GenBank BLASTn analysis of all three loci showed > 98% identity with B. gigantea isolates. Sequences were deposited in GenBank under the accession of PX444436 for ITS, PX549308 for RPB2, and PX549309 for TEF1. A maximum likelihood phylogeny based on a concatenated alignment of ITS, RPB2, and TEF1 sequences confirmed the isolate as B. gigantea. To confirm pathogenicity on hops, Koch’s postulates were carried out on fully developed leaves of three plants on greenhouse-grown hops (3–4 months old) and hemp cultivar NWG2730 (6 weeks old). Plants were sprayed with 4 to 5 ml of a 5 × 10⁴ conidia/ml spore suspension, incubated for 48 h in darkness, then kept in a greenhouse (~30°C, 12-h photoperiod, 66% RH). The same lesions observed in the field developed within 13 days after inoculation (DAI) on hops, and Bipolaris leaf spot symptoms developed within 8 DAI on hemp, with conidia recovered from morphologically identical to the inoculum. Control plants inoculated with sterile water remained symptomless. This is the first report of B. gigantea causing Bipolaris leaf spot disease of hooked hair hops. The proximity of hooked hair hops to hemp cultivation highlights the potential for pathogen spillover among invasive and crop plant species, emphasizing the crucial need for effective control and management strategies to protect this valuable agricultural resource.