First report of root and crown rot caused by Armillaria gallica on Cannabis sativa in California, U.S.A.

In October 2018, symptoms of leaf necrosis, wilted shoots, and stunted growth were observed on the upper portion of the 7-month-old cannabis (Cannabis sativa L.) plants in Mendocino County, California, U.S.A. Foliar symptoms were followed by a rapid death of the plants within 24 hours. Out of 200 affected plants, 80% (160/200) were symptomatic. All affected plants were grown in non-woven polypropylene containers (Smart pots, Oklahoma, USA) set directly on the ground approximately 3 m apart outdoors, surrounded by native forest (Quercus spp., Pseudotsuga menzeisii). Closer examination of the C. sativa plants revealed diagnostic signs of Armillaria root disease: white mycelial fans at the base of the woody stem (root collar) and abundant rhizomorphs on the roots and root collar (Supplementary Fig 1A). Also, both woody roots and the root collar exhibited severely rotted wood. Rotted wood, mycelial fans, and rhizomorphs (n=20) were surface sterilized with 0.6% sodium hypochlorite, rinsed with sterile water, and plated on PDA amended with tetracycline (1 mg/L). Sixteen cultures with morphological characters of Armillaria sp. (regular colony margin, no spore structures, no clamp connections) were recovered (Baumgartner et al., 2011). Species identity was confirmed by sequencing the internal transcribed spacer (ITS) region of rDNA and the translation elongation factor subunit 1-alpha (TEF1a) loci (White et al. 1990, Baumgartner et al 2010). Sequences (GenBank nos. MT248417 and MT259788) were compared with those in the NCBI GenBank database using a BLAST search, revealing 876/881bp matching with Armillaria gallica ITS sequence, GenBank no KP960553, and 146/150bp matching with TEF1a sequence from a North America A. gallica isolate, GenBank no. JF895844 (Brazee et al., 2011). Pathogenicity tests were conducted twice using two A. gallica isolates (15389-1 and 15389-2) by inoculating sterile, 1-month old, rooted tissue-cultured cannabis plants of 'Wedding Cake' with 7.5 ml of homogenized A. gallica liquid inoculum (Baumgartner et al., 2010), added aseptically to the surface of the vermiculite, near the plant stem (Ford et al., 2017). Eight plants were inoculated and two (using sterile water instead of inoculum) were used as negative controls. Plants were incubated at 21-26 °C under 40 to 80 μmol·m-2·s-1 from full spectrum light source with an 18/6 photoperiod to support vegetative growth. Plants were watered with 25 ml sterile nutrient solution (Cutting Edge Solutions, Santa Rosa, CA, U.S.A.) at 1 to 2-week intervals, according to the plant's need. At eight weeks post inoculation, all eight inoculated plants showed symptoms of yellowing and wilting. Uptake of the nutrient solution and water had also stopped by this time. The two non-inoculated plants, however, remained healthy throughout the 8-week period (Supplementary Fig 1B). At the end of the experiment, samples were taken aseptically from the crowns and roots of each plant and plated on water agar amended with streptomycin (100 μg/ml) and benomyl (4 μg/ml). Hyphae were subcultured to 0.5X PDA to confirm species identity through ITS and TEF1a. A. gallica was reisolated from affected crowns and stems. This is the first report of A. gallica causing root and crown rot of C. sativa. Considering the expanding cultivation area of Cannabis crops due to legalization of the industry in many U.S. states, A. gallica root and crown rot may become a serious issue affecting the industry, even for plants maintained in non-woven polypropylene containers in direct contact with soil.