First Report of Cucurbit Yellow Vine Disease Caused by Serratia marcescens on Cucurbits in New York.

Cucurbits are one of the most significant commodities in New York, with a value of $92.3 million in 2021 (NASS-USDA 2021). In August 2021, several acorn squash (Cucurbita pepo) cultivar Turbinate plants at Cornell AgriTech research farm in Geneva, NY, had chlorotic, wilting leaves, and older leaves appeared scorched. The phloem of stems, bisected at the crown, had a honey-brown discoloration. The incidence of symptomatic plants was 22% in a one-acre planting field. Most of the symptomatic plants rapidly declined and died. The following year, similar symptoms were observed on muskmelon (Cucumis melo), acorn squash, and winter squash (C. pepo) cultivar Bush Delicata at the same location. These symptoms were typical of Cucurbit Yellow Vine Disease (CYVD) caused by the Gram-negative bacterium Serratia marcescens (Bruton et al. 1998, 2003). Moreover, a high incidence of squash bugs (vector of CYVD) was observed. To identify the causal agent, 45 stems from the symptomatic Bush delicata plants were collected. Each stem was cut into small pieces (2 to 3 mm), surface sterilized with 70% ethanol for 60 sec, 10% bleach for 60 sec, and rinsed with sterile water. The tissue was macerated in sterile water, and the resultant suspension was streaked on King's B (KB) medium (King et al. 1954). Plates were incubated at 28°C for 24 h, and 11 developed white, round bacterial colonies that were smooth and creamy in appearance. Single colonies were transferred to new KB plates and incubated for 24 h. The genomic DNA of two isolates (22212 and 22213) was extracted with the Wizard® Genomic DNA Purification Kit Protocol (Promega, Madison, WI). PCR was carried out using YV1 and YV4 primers specific to the 16S rDNA region of S. marcescens and 79F/R primers specific for S. marcescens causing CYVD (Zhang et al. 2005). The DNA sequence of each PCR product was obtained using Sanger sequencing and submitted to GenBank. Accessions OQ584799 and OQ584800 for YV1/YV4 (isolates 22212 and 22213, respectively) exhibited 100% identity to S. marcescens (384/384 bp, nearest accession identity: CP083754). Accession numbers OQ693911 and OQ693912 for 79F/R showed 99% identity to S. marcescens isolates (309/313 bp, nearest accession identity: CP033623). To fulfill Koch's postulates, Bush Delicata squash plants were grown for two weeks in a greenhouse, and three plants per isolate were inoculated using S. marcescens 22212 and 22213, three plants with Escherichia coli DH5a as a non-pathogenic control, distilled water as a mock-inoculated control, and a noninoculated control. Inoculation was performed by taking a single bacterial colony with a small pin and puncturing the plant's lower stem four to five times (Bruton et al. 2003). Twenty-eight days after inoculation, three of the six plants inoculated with the two S. marcescens isolates (two from 22212 and one from 22213) developed CYVD symptoms as observed in the field. Isolations were made from the stems of symptomatic plants and the mock-inoculated controls. PCR was conducted using YV1/YV4 primers and 79F/R primers (Zhang et al. 2005). Only isolations from symptomatic plants amplified with these primers and PCR products were sequenced. These sequences were identical to the original isolates. To our knowledge, this is the first report of CYVD and phytopathogenic S. marcescens in New York. The impact of CYVD can be substantial, with losses up to 100% (Zhang et al. 2005). Therefore, more knowledge on S. marcescens is needed to determine its biology and prevalence in New York.

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.