First Report of Diaporthe phaseolorum Causing Stem Canker of Hemp ( Cannabis sativa )

Hemp is an annual herbaceous plant that is used for its fiber and oil in a variety of commercial and industrial products. In Florida, it is currently being explored as a new specialty crop. During a field trial from October to January 2019 in Wimauma, FL, a stem canker was observed on up to 60% of three-month-old plants of 'Eletta Campana', 'Carmagnola Selezionata', and 'Tygra'. Symptoms started on the main stems with light-to-dark brown lesions of different sizes and shapes. Over time, the lesions coalesced into large necrotic areas and bore pycnidia. Isolations were made from diseased stem tissues on General Isolation medium (Amiri et al. 2018) after surface disinfestation (Marin et al. 2020). The plates were placed in a growth chamber at 25°C under a 12/12 photoperiod. A fungus with white, floccose, aerial mycelium and pycnidia producing alpha and beta conidia was consistently isolated. Three single spore isolates were chosen for identification and pathogenicity tests. Pycnidia on PDA were globose to irregular and ranged from 170 to 250 μm long (210 ± 2.5, n = 50) and 140 to 220 μm wide (180 ± 2.7, n = 50). The alpha conidia were unicellular, hyaline, ellipsoidal to fusiform and ranged from 5.3 to 7.7 μm long (6.5 ± 1.6, n = 50) and 1.5 to 4.6 μm wide (2.8 ± 1.8, n = 50). The beta conidia were hyaline, elongated, filiform, straight or curved and ranged from 10.2 to 17.7 μm long (16.1 ± 2.2, n = 50) and 0.5 to 1.8 μm wide (0.8 ± 0.2, n = 50). Perithecia were not observed. Based on morphological features, the fungus was similar to anamorphs of Diaporthe spp. (Santos et al. 2011; Udayanga et al. 2015). DNA from the same three isolates was extracted using the FastDNA kit, and the ribosomal internal transcribed spacer (ITS), β-tubulin (TUB), and calmodulin (CAL) regions were amplified following Udayanga et al. (2014), and Sanger sequenced by Genewiz. Sequences were deposited in GenBank (accession no. MT497039 to MT497047 for ITS, TUB, and CAL). BLASTn searches revealed isolates 20-58, 20-59, and 20-60 were 96.34% identical to the epitype isolate AR4203 for ITS (KJ590738.1, 527 bp out of 547 bp), 100% for TUB (KJ610893.1, 459 bp out of 459 bp), and 100% for CAL (KJ612135.1, 522 bp out of 522 bp) (Udayanga et al. 2015). Their identity was confirmed by phylogenetic analyses using maximum likelihood and Bayesian inference methods. To complete Koch's postulates, pycnidia of the same three isolates were harvested and crushed in 2 mL Eppendorf tubes containing 0.01