First Report of Pythium aphanidermatum Crown and Root Rot of Industrial Hemp in the United States

During June and July 2015, crown and root rot symptoms were observed on industrial hemp (Cannabis sativa cvs. Alyssa and Canda) in research plots in Lafayette, IN. Record setting rainfall in Indiana during June (218.4 mm) and July (162.6 mm) may have factored into this outbreak. Soil type is Crosby-Miami complex alfisol. Symptom development appeared 13 days after sowing with temperatures ranging from 25 to 30°C. Leaves of affected plants were chlorotic, and plants were stunted and often wilted. Brown lesions on roots and loss of feeder roots were observed when symptomatic plants were removed from soil; symptomatic plants often but not always possessed brown, water-soaked stem lesions. A small percentage of affected plants collapsed, but most persisted in stunted growth. Thin, aerial mycelia were visible on the stem surface of some of the infected plants. Tissue fragments were excised from the margins of the affected stem and root lesions, surface sterilized, and plated on 0.25% potato dextrose agar (PDA) and on a medium selective for oomycetes containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP). Plates were incubated at 22°C in the dark. Thirty-eight isolates were tentatively identified as Pythium spp. and were grown on V8 medium (V8 juice 300 g; agar 15 g; CaCO3 1.5 g; distilled water 1 liter) for morphological observation. DNA was extracted with the G-Biosciences OmniPrep for fungi. PCR was performed using the primers ITS1/ITS4 to amplify the internal transcribed spacer (ITS) region of rDNA. Six isolates were identified as Pythium aphanidermatum by BLAST analysis (Altschul et al. 1997) and were morphologically consistent with P. aphanidermatum (Watanabe, 2002). One characteristic isolate with a 738-bp segment showed a 99% homology with the sequence of P. aphanidermatum (GenBank accession JN695786) and has been assigned as GenBank accession KX772239. Pathogenicity tests were performed with this isolate using 20 seeds of ‘Canda’ sown in three 1-liter trays filled with a steam-disinfested soilless medium (Sungro Professional Growing Mix). Plants were fertilized 1 day before inoculation with 0.9 g ammoniacal nitrogen. Plant were inoculated 21 days after sowing (postemergent) with sterilized hemp kernels or 0.25% PDA colonized with the one isolate of P. aphanidermatum. Infested and control inoculum was placed in rows 5 mm from plants. One-liter trays were placed in two-liter trays and watered to flooding to simulate waterlogged conditions. Greenhouse temperature was 25°C; 12 h day night light cycle supplementing and external light. This experiment was replicated three times for both control and challenged treatments. Symptoms developed 18 days after inoculation. After 31 days, all inoculated plants displayed symptoms of chlorosis, crown rot, wilt, dieback, and eventually death. Control plants remained healthy throughout the experiment despite waterlogged conditions. This experiment was repeated 3 days later, with symptoms first developing 14 dpi. P. aphanidermatum was consistently observed based upon morphological observation and/or reisolation from the lesions. To our knowledge, this is the first report of Pythium root and crown rot of C. sativa caused by P. aphanidermatum in the U.S. Although current production of industrial hemp is only 6,900 acres, the importance of the disease could increase with increasing hemp production when it is legalized, particularly in low lying or flood prone areas where hemp is intensively grown (McPartland 1996). © 2017, American Phytopathological Society. All rights reserved.