First Report of <i>Fusarium graminearum</i> Causing Head Blight on Hemp ( <i>Cannabis sativa</i> ) in Wisconsin

In September 2024, blight symptoms were observed on hemp grain heads as part of a 0.5-acre grain cultivar trial at the University of Wisconsin-Madison, Arlington Agricultural research station (Latitude 43°18’9.47″N, Longitude 89°20’43.32″W). Grain threshed from infected heads was plated on Nash-Snyder media to isolate Fusarium species (Nash & Snyder 1962). Colonies from Nash-Snyder media were hyphal tip purified onto 1/4-strength potato dextrose agar. After approximately 5-7 days, isolated colonies produced white-pink mycelia, with some having dark red pigmented undersides. Macroconidia generated from Fusarium cultures were slightly hyaline and septate, with between 3 and 5 septa. Pollinated female flowers from the day-neutral cultivar “Pink Kush” were spray-inoculated with a 5 × 105 spores mL-1 suspension in 0.05% (v/v) Tween or mock-inoculated with Tween until runoff, then bagged in clear plastic bags (Yulfo-Soto et al. 2022). After 3 days, plants were uncovered and allowed to grow for an additional 6-9 days under greenhouse conditions (22-25°C with a 16-h photoperiod and 50% relative humidity). Visible mycelial growth was observed on test plants, but not on the mock-inoculated plants. Leaves from test plants were collected and disinfected in 10% bleach for 1 minute and rinsed in sterile DI water before plating on Nash-Snyder media with subsequent purification. Colony and macroconidia morphology were similar to the initial inoculum. Agar plugs taken from the hyphal tip-isolated colony were plated on carrot agar, which induced fertile and mature perithecia (Cavinder et al. 2012). Genomic DNA was extracted from mycelium using CTAB extractions (OPS Diagnostics, Lebanon, NJ, USA). The TEF1 (NCBI Accession Number: PX352985), RPB1 (PX352987), and RPB2 (PX352987) genomic loci were amplified using primers from O’Donnell et al. (2022) and sequenced on a PromethION (Oxford Nanopore Technologies, Oxford, UK). Alignment was performed using minimap2 (2.24-r1122). Variants were called using clair3 (1.0.9) to generate a VCF file that was subsequently used to generate the consensus read. Using polyphasic identification, a comparison of the generated consensus sequence with the Fusarium-ID database revealed significant similarity to Fusarium graminearum (LC13775, 97.89% similarity). Furthermore, the generated FASTA sequences were compared to available sequences in the Fusarium-ID database as follows: FASTA sequences were downloaded from the Fusarium-ID database and then parsed to retain only samples/sequences that possessed all three above loci and belonged to Fusarium. The sequences for each locus were then aligned using MUSCLE (3.8.31) and merged using FASconCAT-G (v1.06.1). Phylogenetic trees were generated using FastTree (2.2.0) and visualized using R packages ape (5.8-1) and ggtree (3.14.0). The consensus sequence grouped most closely with F. graminearum. Therefore, we conclude the isolated fungus likely causing disease is F. graminearum. This report contributes to the existing literature on Fusarium blight previously reported in North Carolina (Thiessen et al. 2020) and Kentucky (Yulfo-Soto et al. 2022). As F. graminearum is known to be a prolific mycotoxin producer, accumulation and concentration of these toxins should be carefully monitored, especially in the context of the recent approval of hemp grain for laying chickens by the Association of American Feed Control Officials.