First Report of Downy Mildew caused by Pseudoperonospora cannabina on Cannabis sativa in New York.

Hemp (Cannabis sativa <0.3% tetrahydrocannabinol) is an emerging crop used for grain, fiber, and cannabinoid production (Fike et al. 2020). In New York, hemp is grown both in controlled environment facilities, including greenhouses, and as a field crop. In August 2020, downy mildew-like symptoms were observed on leaves and inflorescence of hemp plants in a field research trial in Ithaca, NY. Several cultivars, including 'Auto CBD', were affected. Disease was severe with some plants reaching 75% disease severity at the individual plant level. In the most severely affected plots, there was no marketable yield. The disease was characterized by chlorotic and necrotic lesions producing sporangiophores under high humidity. Pigmented sporangia were produced on branched sporangiophores. On artificially inoculated leaves incubated at 18°C, 80% humidity, 12h light for 5d, sporangiophores produced 8-19 pigmented, lemon-shaped sporangia with mean ± SD dimensions of 25.2 ± 3.0 (18.9 to 30.4) x 18.2 ± 2.1 (14.6 to 23.2) µm (n=50). Each sporangium produced 2-5 zoospores after less than 45 min in water at room temperature (22°C). Sporangia were collected from sporulating lesions and DNA was extracted as outlined in Crowell et al. (2020). Fragments of the ribosomal internal transcribed spacer (ITS) region (White et al. 1990), the beta-tubulin ras-associated ypt1 gene (Moorman et al. 2002), and the mitochondrial cytochrome B oxidase subunit 2 (cox2) gene (Hudspeth et al. 2000) were amplified by PCR and sequenced bidirectionally. Sequences were deposited in GenBank under accession numbers OK086084, OM867581, and OM867580, respectively. BLAST searches using the amplified ITS and cox2 sequences resulted in 100% identity to Pseudoperonospora cannabina (HM636051.1, HM636003.1) with ypt1 aligning at 97.95% identity (382/390 bp) with P. cannabina (KJ651402.1). The molecular characterization identified the causal agent as P. cannabina. A representative isolate was deposited in the Cornell Plant Pathology Herbarium as CUP-070922. Sporangia were rinsed from detached leaves and used to confirm pathogenicity on whole plants. Ten 4-week-old 'Anka' plants were spray-inoculated until run off with a suspension of 1x104 sporangia mL-1. Ten control plants were sprayed with water. After inoculation, plants were placed in a 19˚C growth chamber with a 12-h photoperiod and misted for 30 min twice daily to maintain humidity above 80%. Sporangia and previously described symptoms were observed 7 days post-inoculation, while control plants were asymptomatic. The pathogen was reisolated onto detached leaves of 'Anka' from inoculated leaves where both sporangia and oospores were observed. The reisolated pathogen was confirmed morphologically and molecularly, through PCR amplification and bidirectional sequencing of the ITS, cox2, and ypt1 genes, as P. cannabina. To our knowledge, this is the first report of P. cannabina causing hemp downy mildew in New York. Depending on the severity and timing of infections, this disease could pose a significant threat to hemp production in the state. Other members of the genus, P. cubensis and P. humuli cause downy mildew on cucurbits and hops, respectively. As these can cause devastating diseases on their hosts, P. cannabina must be monitored with vigilance as an emerging pathogen (Purayannur et al. 2021; Savory et al. 2011). Literature Cited: Crowell, C. R., et al.2020. Plant Dis. 104:2949. DOI 10.1094/PDIS-04-20-0718-RE Fike, J. H., et al. 2020. Page 89 In: Sustainable Agriculture Reviews, vol 42. Springer, Cham, Switzerland. DOI 10.1007/978-3-030-41384-2_3 Hudspeth, D. S. S., et al. 2000. Mycologia 92:674. DOI 10.2307/3761425 Moorman, G. W., et al. 2002. Plant Dis. 86:1227. DOI 10.1094/PDIS.2002.86.11.1227 Purayannur, S., et al. 2021. Mol. Plant Pathol. 22:755. DOI 10.1111/mpp.13063 Savory, E. A., et al. 2011. Mol. Plant Pathol. 12:217. DOI 10.1111/j.1364-3703.2010.00670.x White, T. J., et al. 1990. Page 315 In: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA. DOI 10.1016/B978-0-12-372180-8.50042-1.

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.