First report of Septoria leaf spot caused by Septoria cannabis on industrial hemp ( Cannabis sativa ) in Arkansas

Throughout the 2022 growing season, a leaf spot disease was observed in outdoor plots of industrial hemp (Cannabis sativa) at the Milo J. Shult Agricultural Research and Extension Center in Fayetteville, Arkansas. The disease was initially observed in mid-May on volunteer hemp plants (mixed floral varieties of undetermined parentage) that were 6 to 8 weeks old. Leaf spots first appeared on older leaves in the lower canopy before spreading upwards. Nascent leaf spots began as yellow-green specks and developed into irregular, necrotic lesions with tan or brown centers surrounded by yellow halos. Mature lesions were typically 2 to 6 mm in diameter and often contained dark brown or black pycnidia. To isolate the causal agent, leaves with lesions containing pycnidia were collected from diseased plants. After incubation of the leaves in moist chambers for 24-72 h, cirrhi were observed exuding from pycnidia. Cirrhi were picked from pycnidia with a sterile needle and transferred to V8 agar plates amended with carbenicillin (100 μg/ml). Plates were incubated in the dark at 25°C. Resulting colonies were dark gray and morphologically consistent with Septoria. Colonies were subcultured and grown at 25°C under a 12/12 h light/dark cycle to induce conidiation. After 6 to 8 days, colonies produced scattered black pycnidia which exuded grayish to white cirrhi. Conidia in cirrhi were hyaline, needle-shaped, straight or slightly curved, with a truncate base and pointed tip. Septation was indistinct, with conidia usually containing 3 or 4 septa. Conidium lengths ranged from 28 to 55 μm (n=50). Three isolates were single spored and selected for pathogenicity tests and genetic identification. Koch’s postulates were fulfilled via whole-plant inoculations. Spores from each isolate were collected with sterile water and adjusted to 10 ⁶ spores/ml + 0.01% Tween 20. Four-week-old potted hemp plants (cv. Sour Space Candy) were sprayed with spore suspensions until run-off. Sterile water + 0.01% Tween 20 was used for negative controls. Three plants were used per treatment. All plants were incubated in moist chambers with high humidity for 48 h and maintained in a greenhouse with a 12/12 h light/dark cycle and an average temperature below 26°C. Symptoms consistent with field observations began to appear on all inoculated plants at 7 days post inoculation (dpi). By 14 dpi, leaf abscission was observed in the lower canopy of inoculated plants, while control plants remained healthy. Septoria was reisolated from inoculated plants but not control plants. Pathogenicity tests were repeated once with the same results. For molecular identification, segments of the ribosomal internal transcribed spacer (ITS), large subunit (LSU), RNA polymerase II (RPB2), translation elongation factor 1-alpha (TEF), calmodulin (CAL), β-tubulin (TUB), and actin (ACT) genes were sequenced (GenBank: PZ017032-PZ017034, PZ017039-PZ017041, and PZ024376-PZ024390). Barcoding loci sequences from each isolate were identical to each other and shared 100% (ITS, LSU, RPB2), 98% (TEF), and 99% (TUB, ACT, CAL) identity with those from Septoria cannabis (Rahnama et al. 2021; GenBank: MW556611, MW556614, MW556602, MW556605, MW556608, MW526952, MW526955). A multilocus phylogeny was created with MEGA v12 and IQ-TREE v2.4.0 with ModelFinder (Kumar et al. 2024; Kalyaanamoorthy et al. 2017). All three Arkansas hemp isolates grouped with Septoria cannabis. This is the first report of S. cannabis causing Septoria leaf spot on industrial hemp in Arkansas, and the second report to include multilocus sequence data for S. cannabis isolated in the US.