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Molecular identification of Alternaria species associated with imported industrial hemp seed

Russell Wilhelm: Nevada Department of Agriculture

<div>Industrial hemp (<em>Cannabis sativa</em> L.) seed used for crop cultivation is either imported from foreign countries or produced domestically. In a germination test conducted on imported hemp seeds, a high percentage of fungal growth was observed in the germination tray, suggesting a presence of seed-borne fungal organisms. To isolate and identify those fungal species, fifty hemp seeds (var. Canda) were soaked in 1% sodium hypochlorite for 15 min with shaking, rinsed in sterilized water, plated on potato dextrose agar amended with streptomycin sulfate (PDA+strep), and incubated at 22ºC in the dark. While 86% of seeds germinated, 56% of seeds had fungal growth, the majority of which had fungal colonies grown from newly germinated tap roots. A total of 29 <em>Alternaria</em> isolates were obtained and maintained on PDA+strep for morphological observations. One type contained 14 isolates with white to gray colonies and another had 15 isolates with gray to black colonies. DNA was extracted from all isolates and PCR was performed using primers ITS1/ITS4 to amplify the ITS region of rDNA. The size of PCR amplicons obtained from 29 isolates ranged from 542 to 600 bp. A BLASTn search suggested that these two types of isolates were closely related to <em>Alternaria infectoria</em> and <em>A. tenuissima</em>, respectively. The data suggest that hemp seeds of the Canda variety carried two distinct <em>Alternaria</em> species, but the effects of these two fungal species on the health of seeds or seedlings are not known.</div>