First Report of Serratia marcescens Causing a Leaf Spot Disease on Industrial Hemp (Cannabis sativa)

Abstract

In the 2017 and 2018 growing seasons (between May and October), several cultivars of industrial hemp plants grown for flowers in greenhouse production from four North Carolina counties showed symptoms of an angular leaf spot on leaves, stems, and flower parts. Lesions were initially small, dark brown, 1 to 3 mm in size, and vein-limited. As the disease progressed, lesions coalesced to form larger regions of necrosis that engulfed large portions of leaves, and whole plants were lost to disease. Red bacterial ooze was observed streaming from the plant tissues. Bacterial colonies growing on potato dextrose agar (PDA) were raised and dark-pink colored. The Gram stain indicated gram-negative bacilli. A representative isolate (called strain T2) was used to inoculate Cannabis sativa L. ‘Carmagnola’. Two-week-old seedlings (n = 6) were inoculated with a bacterial suspension (OD₆₀₀ = 0.1, approximately 10⁸ CFU/ml) using a PreVal hand sprayer; additional seedlings (n = 6) were inoculated with sterile water to serve as the negative control. Plants were incubated at 23°C for 21 days in a growth chamber with a 12-h photoperiod. After 8 days, dark brown-black lesions similar to those described above were observed on inoculated leaves. Control plants remained symptomless. The pathogen of interest was the only microorganism reisolated from lesions, and after transferring onto PDA, colonies were identical in morphology to those isolated from the original diseased plants. Molecular identification was conducted by first extracting DNA from the representative isolate T2 using the DNeasy Powersoil kit (Qiagen, Hilden, Germany). The 16S ribosomal RNA region (Klindworth et al. 2013) and the RNA polymerase β-subunit (rpoB) gene (Mollet et al. 1997) were PCR amplified. Amplicons were sequenced at the North Carolina State Genomic Sciences Laboratory, and sequences for each gene from the isolate assessed (T2) were deposited to GenBank (accessions MK598699 and MN400982 for 16S rRNA and rpoB, respectively). NCBI-BLAST searches (Altschul et al. 1997) showed the highest similarity (99.6% identity) with 16S rRNA of Serratia marcescens subsp. sakuensis and 99.6% pairwise identity to rpoB of S. marcescens strain B3R3 (GenBank accession KU894791). Based on pathogenicity, morphology, and molecular identification, the unknown bacterial isolate was identified as S. marcescens. Because industrial hemp is increasingly grown across the United States for cannabidiol production, this disease could pose significant challenges and cause yield reduction in affected plants. Additionally, this pathogen could be of human health concern should it survive hemp processing practices, because there are strains that affect humans.