First report of Cercospora leaf spot caused by Cercospora cf. flagellaris on industrial hemp (Cannabis sativa) in Arkansas and Oklahoma.

Abstract

In fall of 2021, a leaf spot disease was observed in outdoor plots of industrial hemp (Cannabis sativa) in Fayetteville, Arkansas. Indistinguishable symptoms were also observed in outdoor plots of hemp in Adair County, Oklahoma. Leaf spots first appeared on older leaves in the lower canopy before spreading to the upper canopy. Nascent leaf spots began as yellow-green specks and developed into round, necrotic lesions 2 to 4 mm in diameter with white, tan, or grey centers and brown boarders. To isolate the pathogen, green leaves with mature lesions were collected and placed in moist chambers. After incubation for 24 to 72 hrs, melanized, erumpent conidiophores bearing hyaline conidia emerged in lesions. Conidia were picked with a sterile needle and transferred to V8 agar + carbenicillin (100 μg/ml). Cultures were incubated in the dark at 25°C. Resulting colonies were light or dark grey with white aerial hyphae. Some colonies produced a reddish-purple pigment consistent with cercosporin. Two isolates from AR (21CD76 and 21CD169) and three isolates from OK (21CD47, 21CD52, and 21CD56) were single spored and selected for pathogenicity tests and genetic identification. Conidiation was induced in vitro by incubation on V8 agar at 25°C in the dark. Conidia were hyaline, needle shaped, straight or slightly curved, truncate at the base, terminal at the tip, with indistinct septa ranging from 5 to 15 per conidium. Conidium lengths ranged from 15 to 101 μm, consistent with descriptions of Cercospora cf. flagellaris (Chupp 1953). Koch's postulates were fulfilled via whole plant inoculations. Spores from each isolate were collected with sterile water and diluted to 10⁵ or 10⁶ spores/ml + 0.01% Tween 20. Three-week-old hemp plants of cv. Sour Space Candy and Red Kross were inoculated by spraying until run-off. Sterile water + 0.01% Tween 20 was used for negative controls. Four plants of each cultivar were used per treatment. Plants were kept in moist chambers for 72 hrs and maintained in a growth chamber with a 16/8-hr light/dark cycle. Symptoms consistent with those seen in the field began to appear after 7 days. After 14 days, all inoculated plants developed lesions, while control plants remained healthy. C. cf. flagellaris was reisolated from inoculated plants but not from control plants. The pathogenicity test was repeated once with the same results. For genetic identification, segments of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-α (TEF), histone H3 (HIS), calmodulin (CAL), and actin (ACT) genes were extracted from whole-genome sequences or sequenced individually (GenBank: PV178994 to PV178998 and PV335254 to PV335273). BLAST queries revealed 99-100% identity with NCBI C. cf. flagellaris strains for all but one sequence (PV335272), which shared 97.76% identity (ITS: KX443947; TEF: MG975845, KX443988; HIS: MG975843, KX443893; CAL: KX443808, KX443803, KX443809; ACT: MG975837). A multilocus phylogeny was created in MEGA12 (Kumar et al. 2024) with sequences from the AR/OK hemp isolates, twenty-four C. cf. flagellaris strains, sixteen other Cercospora spp., and Septoria provencialis as the outgroup. All five AR/OK hemp isolates grouped with C. cf. flagellaris, which has also caused Cercospora leaf spot on hemp in Kentucky and Florida (Doyle et al. 2019, Marin et al. 2020). This is the first report of C. cf. flagellaris causing Cercospora leaf spot on hemp in AR and OK, representing the westernmost known occurrence of the disease at this time.