First report of Pythium ultimum causing crown rot in greenhouse grown Cannabis sativa (L.) in California

In April of 2020 cuttings of Cannabis sativa (L.) in a greenhouse in San Mateo County, CA were observed collapsing, and further observation revealed: water-soaked stems, tan discoloration to the cortex, and discolored roots. The greenhouse irrigation system was supplied by a local stream. We collected one-liter water samples from: intake pond, reservoir tank, irrigation lines, and local potable water tap. Water samples were filtered and plated as described previously (Rollins et al., 2016). Filter papers were removed after 24 hours. Crown sections from four symptomatic plants and one asymptomatic plant were surfaced sterilized in 10% bleach for five minutes, rinsed in sterile deionized water, cut into four-millimeter long sections, and plated onto V8 media, then incubated at room temperature for three days. White mycelial growth was observed from foci within the print of the filter paper from all irrigation water samples but not the potable water supply sample. Similar mycelial growth was observed from plated crown tissue from symptomatic plants only. Observation under light microscope revealed characteristics congruent with P. ultimum, including aseptate hyphae and globose sporangia (Watanabe, 2002). Mycelia was collected for DNA extraction from each of the water and plant sample plates with DNA extractions performed using Quick DNA Fungi/Bacterial Kit (Zymo Research Irvine, CA, USA) and PCR amplified using primers ITS100/ITS4 as described by Riit et al. (2016). All amplicons were Sanger sequenced, aligned using SnapGene software (from GSL Biotech; available at snapgene.com), and compared to barcode referencPe sequences to identify the species using the BarCode of Life Database (BOLDsystems) within the National Center for Biotechnology Information nucleotide database. After trimming and aligning, all amplicons were found to be identical, yielding the 810-nucleotide long consensus ITS amplicon (accession MW114807), which aligned with Pythium ultimum ITS sequences (e.g., accession HQ643886.1) with 100% identity and homology. We then completed Koch’s postulates by using pure cultures from root sections of P. ultimum to stem inoculate C. sativa plants. We used a three-millimeter corer to remove a disc of epidermis and applied a plug of pure culture to the wound. We inoculated 10 plants, with two plants mock-inoculated using clean V8 agar. Inoculation sites were wrapped in parafilm, and plants were grown in the greenhouse for 20 days. Stems of mock and oomycete inoculated plants were examined for callus formation and 30 centimeters of stem were excised from each plant. The mock inoculated plants had fully callused inoculation sites and were discolored only where wounded. P. ultimum inoculated plant inoculation sites were partially callused over and had tan discoloration of the cortex that extended 6.0 mm +/- 2.0 mm above and below the inoculation site. Stem segments above and below inoculation sites were surface sterilized and plated on V8 media as previously described and P. ultimum recovered from inoculated plants, confirmed as identical to the inoculum by ITS amplification and sequencing. Mock inoculated plant stem cultures yielded no oomycete growth. Together, these results indicate that P. ultimum has the ability to cause crown rot in C. sativa in greenhouse cultivation.