First Report of Exserohilum rostratum Causing Foliar Blight of Industrial Hemp (Cannabis sativa)

In the 2017 and 2018 growing seasons (between May and October), industrial hemp plants of several cultivars including those grown for fiber, seed, and flower from numerous counties in North Carolina showed foliar, stem, and floral blight symptoms. Plants were collected from samples submitted to the North Carolina State University Plant Disease and Insect Clinic. Lesions on leaves were round, brown to black, with dark margins. Inside of each lesion, abundant conidia were found. Conidia were rostrate, ellipsoidal to narrowly obclavate, straight or slightly curved, olive-brown, with a protuberant, cylindrical hilum at the base. Conidia were 7 to 12 septate and 75.64 ± 8.31 × 15.61 ± 1.41 µm. Conidiophores were cylindrical, olivaceous-brown with swollen conidiogenous cells containing circular conidial scars. Isolates were obtained by transferring single spores to water agar and then transferring to potato dextrose agar (PDA). On PDA, mycelia were initially white, turning dark brown to black after 2 to 3 days. To fulfill Koch’s postulates, a representative isolate of Exserohilum rostratum (syn. Setospaeria rostrata) was used to inoculate Cannabis sativa L. ‘Carmagnola’. Two-week-old seedlings (n = 6) were inoculated with a conidial suspension (10⁶/ml) using a PreVal hand sprayer, and inoculated seedlings were compared with a water control plant. Plants were incubated at 23°C for 21 days in a growth chamber with a 12-h photoperiod. After 8 days, dark brown lesions were found on inoculated leaves. Lesions were small and varied from 5 mm to 1 cm. After 3 weeks, lesions were surface sterilized in 10% bleach solution for 1 min, rinsed with sterile deionized water, and placed onto PDA. The pathogen of interest was the only microorganism reisolated from lesions, and spores were identical in morphology to those originally isolated. Molecular identification was conducted by first extracting DNA from a representative pure culture using the DNeasy Powersoil kit (Qiagen, Hilden, Germany). The ITS1, 5.8S, and ITS2 regions were amplified via polymerase chain reaction using ITS1f (Gardes and Bruns 1993) and ITS4 (White et al. 1990) primers, and the RPB2 gene was amplified using the bRPB2-6F and bRPB2-7R primers (Matheny 2005). Amplicons were purified using AmpureXP magnetic beads and sequenced using Sanger sequencing at the North Carolina State Genomic Sciences Lab. Forward and reverse raw sequences were trimmed, and a consensus sequence was generated and aligned with ITS and RPB2 sequences of 19 representative isolates of all Setosphaeria species (syn. Exserohilum) in GenBank using MUSCLE (Edgar 2004). A maximum likelihood phylogenetic tree was constructed using PhyML 3.2.2 (Guindon et al. 2010) with 500 bootstrap replicates. In addition, NCBI-BLAST searches (Altschul et al. 1997) of the ITS and RPB2 sequences showed the greatest identity with Setosphaeria longirostrata (99.8% pairwise identity, syn. E. rostratum) and S. rostrata (99.5% pairwise identity, syn. E. rostratum), respectively. Sequence data for ITS1 from the isolate assessed was deposited to GenBank (accession MH779469). Based on the morphological (Seifert and Gams 2011) and molecular identification using three barcoding regions, the fungal isolates were identified as E. rostratum (syn. S. rostrata). As industrial hemp acreage increases in the United States, this disease could limit yield and quality of industrial hemp flower production.

Duke Scholars

Author Tyler Schappe