First Report of Didymella rhei causing leaf spot on rhubarb in New York.

First Report of Didymella rhei causing leaf spot on rhubarb in New York E. J. Indermaur1, C. T. C. Day1, and C. D. Smart1† 1School of Integrative Plant Science, Section of Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva NY 14456 †Corresponding author: C. D. Smart; Email: cds14@cornell.edu Rhubarb (Rheum spp.) is a perennial grown across the northern United States for petiole production (Foust & Marshall 1991). In August 2021, leaf spots were observed on rhubarb growing in a two-acre field in Erie Co., NY (Fig. S1). Approximately 30% of the plants in the field had leaf spot with disease severity of 5%. Initial symptoms on leaves were light brown, circular lesions with red margins that later coalesced into irregular spots. Lesion centers were dry with concentric rings, often perforating as they enlarged. Lesions on petioles were light brown, fusiform, and sunken with red margins. To identify the causal agent(s), symptomatic leaves and petioles from 50 plants (cultivar unknown) were collected with a W-shape sampling scheme. Lesion margins were surface sterilized with 70% ethanol for 60 s, 10% bleach for 60 s, rinsed in sterile water, plated on acidified potato dextrose agar (PDA), and incubated for two to four days at 20˚C. Hyphal tips from colony edges were transferred to new PDA plates. After 20 days, colonies (n=53) were olivaceous buff to grey olivaceous, producing white to grey, sparse aerial mycelium. Brown to black pycnidia were produced within five days in concentric rings around plate centers. Pycnidia were globose to subglobose, with one to two non-papillate or slightly papillate ostioles, and with mean diameter 75.8 (30.8 to 113.5) µm (n=20). Conidia were hyaline, ellipsoid or allantoid, and aseptate with mean ± SD dimensions of 6.2 ± 0.4 (4.9 to 8.1) x 2.2 ± 0.4 (1.3 to 3.3) µm (n=30) (Fig. S2). Based on these morphological characteristics, the isolates were initially identified as Didymella rhei [Ellis & Everh] (Qian Chen & L. Cai) (Boerema 2004). To confirm the identity, mycelia were scraped from PDA plates and homogenized using a TissueLyser II (Qiagen Inc.). Genomic DNA was extracted with a DNeasy Plant Mini Kit following manufacturer's instructions (Qiagen Inc.). PCR assays with primers ITS 4 and ITS 5 and fRPB2-7cR and RPB2-5F2 (Liu et al. 1999; Sung et al. 2007) were used to amplify the internal transcribed spacer (ITS) and the rpb2 gene regions of one representative isolate (strain RHU21204). Products were sequenced using Sanger chemistry. The sequences were deposited in GenBank with accession numbers OM903952 (ITS) and OM925897 (rpb2). The ITS and rpb2 sequences exhibited 99% (492/494 bp) and 100% (846/846 bp) identity with D. rhei accessions KF531831.1 and KP330428.1, respectively. Based on morphological and molecular characteristics, the pathogen was identified as D. rhei. To fulfill Koch's postulates, healthy leaves and petioles of four rhubarb seedlings (cultivars unknown) were spray-inoculated with a conidial suspension (1 × 107 conidia/ml) containing 0.2% Tween-20 from strain RHU21204. A tween suspension with no conidia was used as a control. Each treatment had three replicates. 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%. Initial symptoms were observed five days post inoculation (dpi), while control plants were asymptomatic. The pathogen was isolated 21 dpi from inoculated leaves and petioles with symptoms as described above (Fig. S1) and identified morphologically and molecularly as D. rhei. A representative isolate was deposited in the Cornell Plant Pathology Herbarium as CUP-070923. To our knowledge, this is the first report of D. rhei causing rhubarb leaf spot in New York and reducing the health and marketability of its host. Funding Source This project was funded by the College of Agriculture and Life Sciences, Cornell University. Literature Cited Boerema, G. H. et al. 2004. CABI Publishing. 288. Foust, C. M. and Marshall, D. E. 1991. HortScience 26:1360. DOI: 10.21273/HORTSCI.26.11.1360 Liu, Y. J. et al. 1999. Mol. Biol. Evol. 16:1799. Sung, G. H. et al. 2007. Mol. Phylogenet. Evol. 44:1204. DOI: 10.1016/j.ympev.2007.03.011.

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.