First report of Spiroplasma citri associated with disease symptoms in field-grown hemp (Cannabis sativa L.) in the Pacific Northwest.

Hemp (Cannabis sativa L.) is grown for cannabinoid oil production in Oregon. During the 2021 and 2022 growing seasons, plants with leaf curling, puckering, chlorotic mosaic, fasciation, and vein clearing were observed in disease surveys of Oregon hemp fields (Fig. 1). Symptoms were present on 1-10% of 2-4-month-old plants in fields located in Benton, Clackamas, Deschutes, Jackson, Josephine, Lane, Linn, Marion, Morrow, and Polk counties between July and September. Leaf and stem samples were collected from 38 symptomatic plants. Symptoms resembled those caused by beet leafhopper-vectored (BLH, Circulifer tenellus Baker) curtovirus or phytoplasma infection. Therefore, total nucleic acids were extracted (Dellaporta et al. 1983) from leaf material, and PCR conducted on all 38 samples to detect curtovirus coat proteins (BCTV-1/2 primers; Rondon et al. 2016) and 16S rRNA of phytoplasmas (nested primers P1/P7 followed by FU/RU; Lorenz et al. 1995). No curtoviruses or phytoplasmas were detected in any samples. Spiroplasma citri is also transmitted by BLH, so PCR was conducted with primers targeting the putative P89 adhesin gene and spiralin gene of S. citri (Yokomi et al. 2008). For all samples, PCR using P89F/R primers resulted in a 707 bp amplicon, and a 675 bp amplicon with Spiralin-f/r primers. PCR products were purified with ExoSapIT (Applied Biosystems, Waltham, MA), and two representative samples (ScH1; ScH2) were Sanger sequenced (EuroFins, Lancaster, PA) in the forward and reverse direction. Pairwise aligned P89 sequences were found to be 99 to 100% (ScH1: 633/637 bp; ScH2: 654/654 bp) identical to S. citri accession KT377386. Aligned/span>spiralin sequences were found to be 99 to 100% (ScH1: 661/664 bp; ScH2: 647/647 bp) identical to S. citri accession CP013197 in the NCBI GenBank Database. All sequences were deposited into GenBank (accession no. OQ969983, OQ992766, OQ969984, OQ969986). Frozen leaf material from one sample was used to culture S. citri according to Lee and Davis (1984). Leaf tissue was surface sterilized for 60 s in 1% NaOCl in 70% ethanol, cut into pieces in LD8 broth, and incubated for 25 min at 25˚C. The solution was passed through a 0.45um filter and incubated for 14 days at 30°C with constant shaking (150 rpm). Then, cultures were centrifuged for 2 min at 12,000 rpm to pelletize, resuspended in 100µL sterile distilled water and passed through a 0.2um filter. From this culture, 2 µL of broth was used as template for both the P89F/R and Spiralin-f/r primer sets. Amplicons were purified and sequenced as above (accession nos. OQ969982, OQ969985). Leaf tissue from both representative samples (ScH1 and ScH2) were positive for S. citri using double antibody sandwich ELISA (Agdia, Inc., Elkhart, IN) following manufacturer instructions. These results from sequencing, culturing, and ELISA testing indicate the hemp samples were infected with S. citri. To our knowledge this is the first report of disease symptoms in hemp associated with S. citri in Oregon and the Pacific Northwest. Infection by S. citri can limit yield by reducing photosynthetic capability of the plant and distortion of plant growth. Other pathogens like curtoviruses and phytoplasmas have also been detected in hemp in Oregon and the U.S. (Hu 2021; Rivedal et al. 2022), and the addition of S. citri associated with disease symptoms indicates a need for BLH management research. This discovery has implications for arid regions with other S. citri hosts including cruciferous plants, carrots, and tree fruit crops, all of which were grown near the sampled hemp fields in this study.

Prevalence of Recombinant Strains of Potato Virus Y in Seed Potato Planted in Idaho and Washington States Between 2011 and 2021.

Potato virus Y (PVY) has emerged as the main reason for potato seed lot rejections, seriously affecting seed potato production in the United States throughout the past 20 years. The dynamics of PVY strain abundance and composition in various potato growing areas of the United States has not been well documented or understood up to now. The objective of this study was to find out the prevalence of PVY strains in potato fields in the Pacific Northwest (PNW), including seed potato production systems in the State of Idaho and commercial potato fields in the Columbia Basin of Washington State between 2011 and 2021. Based on the testing of >10,000 foliar samples during Idaho seed certification winter grow-out evaluations of seed potato lots and seed lot trials in Washington State, a dramatic shift in the PVY strain composition was revealed in the PNW between 2011 and 2016. During this time period, the prevalence of the ordinary, PVYO strain in seed potato dropped 8- to 10-fold, concomitantly with the rise of recombinant strains PVYN-Wi and PVYNTNa, which together accounted for 98% of all PVY positives by 2021. In Idaho seed potato, PVYNTNa strain associated with the potato tuber necrotic ringspot disease (PTNRD) was found to increase threefold between 2011 and 2019, accounting for 24% of all PVY positives in 2019. Mild foliar symptoms induced by recombinant PVY strains may be partially responsible for the proliferation of PVYN-Wi and PVYNTNa in potato crops. A spike of another PTNRD-associated recombinant, PVY-NE11, was recorded in the PNW between 2012 and 2016, but after reaching a 7 to 10% level in 2012 to 2013 this recombinant disappeared from the PNW potato by 2019. Whole genome sequence analysis of the PVY-NE11 suggested this recombinant was introduced in the United States at least three times. The data on PVY strain abundance in the PNW potato crops suggest that virus management strategies must consider the current dominance of the two recombinant PVY strains, PVYN-Wi and PVYNTNa.

Potato Virus Y (PVY) Isolates from Solanum betaceum Represent Three Novel Recombinants Within the PVYN Strain Group and Are Unable to Systemically Spread in Potato.

Tamarillo, or tree tomato (Solanum betaceum), is a perennial small tree or shrub species cultivated in subtropical areas for fresh fruit and juice production. In Ecuador, tamarillo orchards are affected by several viruses, with one previously identified as potato virus Y (PVY); however, the specific strain composition of PVY in tamarillo was not determined. In 2015 and 2016, eight tamarillo plants exhibiting symptoms of leaf drop, mosaic, and mottled fruit were sampled near Tumbaco and Quito, Ecuador. These tamarillo PVY isolates were able to systemically infect tobacco, Nicotiana benthamiana, naranjilla, and tamarillo. Seven of the eight PVY isolates from tamarillo exhibited N-serotype, while one of the PVY isolates studied, Tam15, had no identifiable serotype. One isolate, Tam17, had N-serotype but produced asymptomatic systemic infection in tobacco. In tamarillo, four tamarillo isolates induced mosaic and slight growth retardation and were unable to systemically infect pepper or potato. Tamarillo, on the other hand, was unable to support systemic infection of PVY isolates belonging to the PVYO and PVYEu-N strains. The whole genomes of eight PVY isolates were sequenced from a series of overlapping RT-PCR fragments. Phylogenetically, tamarillo PVY isolates were found to belong to the large PVYN lineage, in a new tamarillo clade. Recombination analysis revealed that these tamarillo PVY isolates represent at least three novel recombinant types not reported before. The combination of the biological and molecular properties found in these eight PVY isolates suggested the existence of a new tamarillo strain of PVY that may have coevolved with S. betaceum.

Strain-Specific Resistance to Potato virus Y (PVY) in Potato and Its Effect on the Relative Abundance of PVY Strains in Commercial Potato Fields.

Potato virus Y (PVY) is a serious threat to potato production due to effects on tuber yield and quality, in particular, due to induction of potato tuber necrotic ringspot disease (PTNRD), typically associated with recombinant strains of PVY. These recombinant strains have been spreading in the United States for the past several years, although the reasons for this continuing spread remained unclear. To document and assess this spread between 2011 and 2015, strain composition of PVY isolates circulating in the Columbia Basin potato production area was determined from hundreds of seed lots of various cultivars. The proportion of nonrecombinant PVYO isolates circulating in Columbia Basin potato dropped ninefold during this period, from 63% of all PVY-positive plants in 2011 to less than 7% in 2015. This drop in PVYO was concomitant with the rise of the recombinant PVYN-Wi strain incidence, from less than 27% of all PVY-positive plants in 2011 to 53% in 2015. The proportion of the PVYNTN recombinant strain, associated with PTNRD symptoms in susceptible cultivars, increased from 7% in 2011 to approximately 24% in 2015. To further address the shift in strain abundance, screenhouse experiments were conducted and revealed that three of the four most popular potato cultivars grown in the Columbia Basin exhibited strain-specific resistance against PVYO. Reduced levels of systemic movement of PVYO in such cultivars would favor spread of recombinant strains in the field. The negative selection against the nonrecombinant PVYO strain is likely caused by the presence of the Nytbr gene identified in potato cultivars in laboratory experiments. Presence of strain-specific resistance genes in potato cultivars may represent the driving force changing PVY strain composition to predominantly recombinant strains in potato production areas.