Assessing risk of False Blossom Disease and vector introduction and establishment to Washington and Oregon Cranberry Producing Regions

In order to assess the risk of false blossom in West Coast cranberries bogs, we propose to sample farms located in the major growing regions of Washington and Oregon for the known vector, the blunt-nose leafhopper (Limotettix vaccinii (Van Duzee), called BNLH going forward) and symptoms of false blossom disease (FBD). In addition to generating relevant data for assessments, this research will boost grower buy-in by involving them in data collection and presenting results to increase engagement and interest on this topic.

Research Objective 1. Characterize and determine relative abundance of leafhoppers, including the blunt-nose leafhopper, in Washington and Oregon cranberry growing regions. For each bog sampled, we will measure leafhoppers from samples to genus noting diversity and abundance. Samples can be kept in 70% ethanol almost indefinitely which will allow us to quantify samples in winter once field work has been completed. Samples will be taken and quantified during 2024 and 2025.

Research Objective 2. Determine if false blossom disease is present in cranberry bogs that have the blunt-nose leafhopper and/or vines with symptoms similar to false blossom with molecular diagnostic assays.  All bogs with BNLH present or with symptoms will be tested for presence/absence of FBD phytoplasma using a nested PCR diagnostic test by the USDA team that has experience with plant pathology testing. Any necessary sampling and testing will be done in field seasons 2024-2026 depending on when BNLH or FBD symptoms are found.

Materials and Methods

Research Objective 1. Characterize and determine relative abundance of leafhoppers, including the blunt-nose leafhopper, in Washington and Oregon cranberry growing regions. In New Jersey where BNLH is most prevalent, adult BNLH populations are most abundant in July and August (De Lange and Rodriguez-Saona 2015). Therefore, we propose sampling insects in July to capture any potential adults in the system.

Locations of farms and grower bogs for sampling: Samples (for both Research Objectives 1 and 2) will be taken from grower bogs. Bogs will be selected from the main growing regions on the West Coast, including bogs owned by the producers collaborating on this project. These regions include Long Beach, WA, Grayland, WA, Ocean Shores, WA, Gearhart, OR, and Bandon, OR. Bogs will be selected for their distance geographically from each other (to avoid sampling neighboring bogs). We will request grower records for treatments made in the previous year and current season from bogs sampled. If possible, we will focus on bogs that may not have received an organophosphate insecticide treatment during the current or previous growing season since that insecticide class is known to effectively manage BNLH. Our goal is to capture any positive ID the first year (not currently known or expected). If we find a positive ID in the first year, we will readjust sampling to include more bogs in the area of the positive ID in order to capture a better idea of the geographic spread of BNLH in the area.

Sweep net sampling: Sampling via sweep net poses a unique challenge in cranberries because the fruits are growing on the plants in July, and walking into cranberry fields in July is avoided to prevent from damaging fruit with each footstep. Therefore, we propose sweep netting along the full perimeter of the field and down a cowlick, if it exists. A cowlick is a dividing line often the center of the bog where vines are trained in two opposing directions to make it easier to move farm equipment along the direction of the vines. The cowlick results in a thin path of vines that are slightly taller and are not accessible to harvesting equipment. Consequently, walking on and sweeping along the cowlick would not cause damage that would cause a significant loss of yield. During sweep net sampling, researchers will take 10 pace samples and stop to aspirate all insects in sweep samples. They will continue taking another 10 pace sample while working around the edge and cowlick to complete sampling. To test that the edge and cowlick sampling style is sufficient, we will test within the research bogs at PCCRF by sweeping in a ‘Z’ pattern across the bog and count the total number of leafhoppers present and compare to samples taken around edges and cowlick. If after the first year, we determine that sweep netting along bog edges and cowlicks does not provide a representative sample of insects on vines within the bogs, then we will sweep in a ‘Z’ pattern for all sampled bogs. To conduct sweeps that cross the harvestable plants in a bog, we will to work with OceanSpray to provide yield loss compensation to participating growers. Nonetheless, we anticipate that edge sweeping for insects in cranberry bogs will be adequate. Principle Investigator Kraft visited New Jersey cranberry bogs with false blossom disease and observed that BNLH were clearly visible on vines that were near the edges of plots.

Insect preservation, identification, and enumeration: Samples from each bog will be placed in 70% ethanol until identification can take place. Kraft or another trained researcher will sort through all samples, noting the total number of insects by order (Lepidoptera, Diptera, etc.) except for any leafhopper samples, which will be noted as either BNLH or other leafhoppers. Those leafhoppers that are not BNLH will be identified to genus in case on-going research at the University of Wisconsin and Rutgers University, universities located in states with endemic outbreaks of FBD, shows that other leafhoppers are capable of vectoring FBD. If necessary, samples from OR may be shipped to WSU Long Beach at the end of the season for identification.

Research Objective 2. Determine if false blossom disease is present in cranberry bogs that have the blunt-nose leafhopper and/or vines with symptoms similar to false blossom with molecular diagnostic assays.  During sweep net sampling, we will evaluate whether bogs have symptoms of FBD, which are clearly visible on a field level due to excessive upright vegetative growth and pink or reddening color of vines. Only fields with suspicious symptoms or with a positive identification of BNLH in the bogs will be sampled for diagnostic PCR assays for the false blossom phytoplasma. So far, we remain relatively confident that FBD is not established in the region as neither growers nor Extension agents have reported any suspicious vines from Washington or Oregon.

For vines or insects that will undergo testing, we will use an established nested PCR method to detect the phytoplasma associated with false blossom disease (Lee et al. 2014, Holland et al. 2021). In this method, Stockwell (co-PI) at USDA in Corvallis, Oregon will pulverize cranberry leaf tissue samples or leafhoppers with a Fast Prep instrument (MP BioMedicals, Santa Ana, CA) and then extract genomic DNA from the tissues with the Qiagen DNeasy Plant Mini kit (Qiagen, Valencia, CA).  A two-step nested PCR assay will be used to detect the false blossom phytoplasma as described by Lee, et al. 2014 and Holland et al. 2021.  In the first PCR reaction, general primer pairs are used to amplify full-length 16S rDNA of phytoplasmas.  This first reaction essentially increases the amount of phytoplasma DNA in the sample of DNA from the cranberry plant.  The second reaction, the nested PCR, will amplify a region of the phytoplasma DNA from the first reaction that is specific to the false blossom phytoplasma. The nested PCR assay is an accurate and sensitive method to detect the false blossom phytoplasma in DNA isolated from cranberry plant tissues. Positive controls will consist of verified genomic DNA isolated from cranberries with false blossom disease provided by Dr. Holland, our colleague at the University of Wisconsin that is actively working on false blossom disease.  Negative controls will consist of genomic DNA from non-infected cranberry plants.