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.

Legacy effects of fumigation on soil bacterial and fungal communities and their response to metam sodium application.

BACKGROUND: Soil microorganisms are integral to maintaining soil health and crop productivity, but fumigation used to suppress soilborne diseases may affect soil microbiota. Currently, little is known about the legacy effects of soil fumigation on soil microbial communities and their response to fumigation at the production scale. Here, 16S rRNA gene and internal transcribed spacer amplicon sequencing was used to characterize the bacterial and fungal communities in soils from intensively managed crop fields with and without previous exposure to metam sodium (MS) fumigation. The effect of fumigation history, soil series, and rotation crop diversity on microbial community variation was estimated and the response of the soil microbiome to MS application in an open microcosm system was documented. RESULTS: We found that previous MS fumigation reduced soil bacterial diversity but did not affect microbial richness and fungal diversity. Fumigation history, soil series, and rotation crop diversity were the main contributors to the variation in microbial ß-diversity. Between fumigated and non-fumigated soils, predominant bacterial and fungal taxa were similar; however, their relative abundance varied with fumigation history. In particular, the abundance of Basidiomycete yeasts was decreased in fumigated soils. MS fumigation also altered soil bacterial and fungal co-occurrence network structure and associations. In microcosms, application of MS reduced soil microbial richness and bacterial diversity. Soil microbial ß-diversity was also affected but microbial communities of the microcosm soils were always similar to that of the field soils used to establish the microcosms. MS application also induced changes in relative abundance of several predominant bacterial and fungal genera based on a soil's previous fumigation exposure. CONCLUSIONS: The legacy effects of MS fumigation are more pronounced on soil bacterial diversity, ß-diversity and networks. Repeated fumigant applications shift soil microbial compositions and may contribute to differential MS sensitivity among soil microorganisms. Following MS application, microbial richness and bacterial diversity decreases, but microbial ß-diversity was similar to that of the field soils used to establish the microcosms in the short-term (< 6 weeks). The responses of soil microbiome to MS fumigation are context dependent and rely on abiotic, biotic, and agricultural management practices.

Floral Resources for Trissolcus japonicus, a Parasitoid of Halyomorpha halys.

The egg parasitoid Trissolcus japonicus is the main candidate for classical biocontrol of the invasive agricultural pest Halyomorpha halys. The efficacy of classical biocontrol depends on the parasitoid's survival and conservation in the agroecosystem. Most parasitoid species rely on floral nectar as a food source, thus identifying nectar sources for T. japonicus is critical. We evaluated the impact of eight flowering plant species on T. japonicus survival in the lab by exposing unfed wasps to flowers inside vials. We also measured the wasps' nutrient levels to confirm feeding and energy storage using anthrone and vanillin assays adapted for T. japonicus. Buckwheat, cilantro, and dill provided the best nectar sources for T. japonicus by improving median survival by 15, 3.5, and 17.5 days compared to water. These three nectar sources increased wasps' sugar levels, and cilantro and dill also increased glycogen levels. Sweet alyssum, marigold, crimson clover, yellow mustard, and phacelia did not improve wasp survival or nutrient reserves. Further research is needed to determine if these flowers maintain their benefits in the field and whether they will increase the parasitism rate of H. halys.

Monitoring Nutrient Status of Brown Marmorated Stink Bug Adults and Nymphs on Summer Holly.

Halyomorpha halys (Stål), or brown marmorated stink bug (BMSB), has become a major pest and nuisance for both agricultural growers and homeowners since its arrival in North America and Europe. The nutritional ecology of BMSB is important for understanding its life history and rearing requirements. However, little is known about the nutritional status of wild populations, especially in the U.S. This research monitored the nutrient status of nymphal and adult BMSB collected from English holly in western Oregon. We measured their weight, nutrient index (weight/(prothorax × width)³), lipid, glycogen and sugar levels and egg load from May⁻September/October. First, glycogen and sugar levels of adults were often lowest sometime in June-August with a general increase by September. Meanwhile, their lipid levels varied without a discernible trend. Second, adult females had few eggs in May, with the highest egg load in June and July, and no eggs by September. Lastly, first and second nymphal instars were found in June, and fourth and fifth instars in September. Because nothing is known about the nutrient levels of nymphs, the reported values from this survey can assist future research on physiological responses of BMSB to treatments or environmental impacts in the field.

Phenology of brown marmorated stink bug described using female reproductive development.

Temperature-based degree-day models describe insect seasonality and to predict key phenological events. We expand on the use of a temperature-based process defining timing of reproduction through the incorporation of female reproductive physiology for the invasive pentatomid species Halyomorpha halys, the brown marmorated stink bug. A five-stage ranking system based on ovary development was able to distinguish between the reproductive statuses of field-collected females. Application of this ranking method described aspects of H. halys' seasonality, overwintering biology, and phenology across geographic locations. Female H. halys were collected in the US from NJ, WV, NC, OR, and two sites in PA in 2006-2008 (Allentown, PA only) and 2012-2014. Results identify that H. halys enters reproductive diapause in temperate locations in the fall and that a delay occurs in developmental maturity after diapause termination in the spring. Modification of the Snyder method to identify biofix determined 12.7-hr photoperiod as the best fit to define initiation of reproduction in the spring. Applying the biofix, we demonstrated significant differences between locations for the rate at which the overwintering generation transition into reproductive status and the factors contributing to this difference require further study. For example, after including abiotic variables influencing development such as temperature and photoperiod (critical diapause cue), reproduction occurred earlier in OR and for an extended period in NJ. This data describe a method to investigate insect seasonality by incorporating physiological development across multiple regions that can clarify phenology for insects with overlapping generations.