Research Project: New Technologies and Strategies for Managing Emerging Insect Pests and Insect Transmitted Pathogens of Potatoes

Location: Temperate Tree Fruit and Vegetable Research

2024 Annual Report

Objectives
Objective 1: Develop new tools and approaches for examining landscape-scale movement by Hemipteran vectors and plant pathogens between non-crop plant species and potato fields. • Sub-objective 1A: Identify weedy plant sources of infective potato psyllids and beet leafhoppers entering potato fields of the Columbia Basin growing region. • Sub-objective 1B: Characterize genetic variation in beet leafhopper populations across geographic areas and between host species within the Columbia Basin and use these data to evaluate host-linked dispersal of leafhoppers into potato fields. Objective 2: Describe the biology of Hemipteran vectors of potato pathogens in crop and non-crop habitats, including reproduction and development, feeding ecology, chemical ecology, seasonal phenology, interactions with natural enemies, and transmission/acquisition of pathogens. • Sub-objective 2A: Characterize beet leafhopper feeding behavior and stylet penetration activities to examine acquisition and inoculation of BLTVA across host species. • Sub-objective 2B: Identify predator species important in reducing densities of potato psyllid and beet leafhopper in stands of weedy host plants. Objective 3: Develop new or improved integrated management strategies to control emerging insect pests and insect-transmitted pathogens of potatoes. • Sub-objective 3A: Produce a “risk matrix” that ranks non-crop weedy plants as to importance as sources of infective potato psyllid or beet leafhopper arriving in potato fields and forward those rankings to the potato industry.

Approach
Sub-objective 1A: Identify weed sources of psyllids and leafhoppers. Approach: Molecular gut content analysis will be used to identify plant DNA in insects and define their feeding histories. Both species will be collected as they enter potato fields. Specimens will be tested with PCR for presence of plant pathogens. Presence of a specific plant in insect guts and correlated presence of pathogen DNA will be evidence the plant is a source of infective insects. Contingencies: Plant DNA that cannot be identified to species based on representation in the NCBI database will be identified to genus. Sub-objective 1B: Characterize genetic variation in beet leafhopper populations across regions and host species. Approach: NextRAD sequencing will be used to identify genetically-defined leafhopper subpopulations collected from potato fields and weed hosts. Genetic differentiation among regions and plants will be assessed by analysis of molecular variance. Contingencies: NextRAD sequencing is time intensive which may make it difficult to evaluate all regions and host-sources. We will supplement NextRAD data as needed by analysis of the CO1 gene. Sub-objective 2A: Characterize beet leafhopper feeding behavior to examine acquisition and inoculation of plant pathogens. Approach: Electropentagraphy (EPG) technology to be used to examine how leafhopper feeding behavior affects pathogen acquisition and inoculation in cultivated and weedy hosts. We will record the time required to begin a feeding event and time spent in an event for three behaviors: xylem ingestion, phloem salivation, and phloem ingestion. Probability of pathogen acquisition and inoculation will be evaluated as a function of these time durations. Contingencies: If we encounter difficulties with the EPG assays, we will consult the literature on EPG work with other leafhoppers. Sub-objective 2B: Identify predator species that attack potato psyllid and beet leafhopper in non-crop habitats. Approach: Molecular gut content analysis will be used to identify predators feeding on potato psyllid and beet leafhoppers in non-crop habitats. Insects for molecular assay will be extracted from plant samples in Berlese funnels. The COI gene will be PCR amplified to detect psyllid or leafhopper DNA. We will identify which predatory taxa most readily attack potato psyllid and beet leafhopper by comparing presence vs absence of prey DNA across predator specimens. Contingencies: No difficulties in completing this work is anticipated. Sub-objective 3A: Produce a risk matrix that ranks weedy hosts by importance as sources of infective psyllids and leafhoppers. Approach: Rankings will color-code each plant species according to risk (red, yellow, or green). Host plants color-coded red will be those found to be sources of vectors and pathogens, and to be common in the study region. Rankings will be made available to growers at research meetings and publication in industry newsletter. We will include suggestions of how risk rankings can be used to assist IPM programs through monitoring of at-risk fields or by eradication of high-risk species. Contingencies: No difficulties in completing this work is anticipated.

Progress Report
This report documents FY 2024 progress for project 2092-22000-022-000D, “New Technologies and Strategies for Managing Emerging Insect Pests and Insect Transmitted Pathogens of Potatoes”, which began in October 2020. In support of Sub-objective 1A, ARS researchers in Wapato, Washington, identified a new plant reservoir of the zebra chip pathogen. A relative of potato psyllid, known as bindweed psyllid, has been shown to commonly harbor the zebra chip pathogen. The psyllid develops on field bindweed, but difficulties in locating the pathogen in this host plant complicated efforts to understand the pathogen’s presence in bindweed psyllid. The pathogen is now shown to occur in the below-ground parts of the weed and in stem material at the soil surface, while being undetectable in leaves. These results suggest that population biology of field bindweed and the bindweed psyllid must be considered when searching for the zebra chip pathogen in weedy habitats near potato fields. In a subordinate project related to Sub-objective 1A, specimens of potato psyllid were collected from potato growing regions of Mexico, New Zealand, and Peru, supplemented with specimens from Brazil of Solanaceae-feeding psyllids in the genus Russelliana. DNA has been extracted from psyllids for molecular gut content analysis to identify crop and non-crop hosts of these psyllids and to infer their landscape-level dispersal. In support of Sub-objective 1B, mitochondrial cytochrome oxidase 1 (COI) DNA was sequenced for comparison across beet leafhopper specimens trapped from various non-crop and crop host plants within the Columbia Basin of Washington and southeastern Idaho. The leafhoppers were categorized into maternally-inherited haplotypes by COI sequence analysis. While the genetic relatedness of these specimens was already analyzed using single nucleotide polymorphisms (SNPs) found within nuclear DNA, the inclusion of mitochondrial haplotype data provides a more complete view of beet leafhopper population genetics across host plants and geographical distances. These results suggest beet leafhopper sub-populations readily mix across host plants, but that some differentiation may occur across larger geographical distances requiring long-distance dispersal. In a subordinate project related to Objective 2, ARS researchers in Wapato, Washington, and Prosser, Washington, examined specimens of psyllids in the genus Craspedolepta and Aphalara as reservoirs of ‘Candidatus Liberibacter solanacearum’, the pathogen which causes zebra chip disease in potatoes. Earlier work by this group showed that certain species of Aphalara harbored unique haplotypes of the pathogen; Craspedolepta (a close relative of Aphalara) has been shown to harbor the pathogen in Europe. ARS researchers assayed 120 specimens of Craspedolepta in four species and more than 200 specimens of Aphalara in six species, and detected Liberibacter only in Aphalara. This result fails to document presence of Liberibacter in local specimens of Craspedolepta but does reconfirm local presence in Aphalara. In support of Sub-objective 2A, the feeding and probing behaviors of beet leafhoppers were examined on beet leafhopper-transmitted virescence agent (BLTVA)-infected and uninfected potato plants using an electropenetrograph (EPG) assay to examine whether pathogen infection mediates changes in behavior. Leafhoppers with and without symbiotic Wolbachia infections were included to also assess its effects on feeding and probing behavior, as some sub-populations carry Wolbachia in this region. Additionally, a protocol was developed to generate BLTVA-infectious beet leafhoppers by caging them on infected periwinkle plants. Transmission assays assessing inoculation and acquisition access periods (inoculation access period (IAP) and acquisition access period (AAP), respectively) on radish plants are ongoing. Under Sub-objective 2B, protocols were developed to rear the predator species whirligig mite (Anystis) in continuous culture for use in biological control studies. This mite is an important predator of various pests of potatoes including psyllids, thrips, plant-feeding mites, and aphids. ARS scientists in cooperation with scientists at Washington State University solved rearing problems caused by cannibalism and unsatisfactory egg-laying substrates. The ARS laboratory at Wapato, Washington, is now able to maintain eight to 10 populations of the predator in continuous culture for field trials of biological control. For Sub-objective 3A, stands of matrimony vine again were sampled for presence of potato psyllids. Data were used to update the psyllid prediction model being used by the Potato Pest Alert program of Washington State University for predicting risk of psyllid outbreaks in commercial potato fields. This is the 11th consecutive year that these samples have been taken and added to the psyllid prediction model. In a subordinate project related to Objective 3, the efficacy of different insecticides in preventing aphid-vectoring of viruses to potatoes was evaluated. Inoculation and acquisition rates of potato leafroll virus (PLRV) and potato virus Y (PVY) in aphids were assessed following foliar applications of six insecticides. Aphid feeding/probing behavior following these treatments was also examined using an electropenetrography (EPG) method, as these behaviors are key to virus transmission. This work will evaluate potential chemical management alternatives to neonicotinoids. In a subordinate project related to Objective 3, ARS researchers in Wapato, Washington, and ARS scientists in Ithaca, New York, Gainesville, Florida, and Albany, California, collaborated in using the zebra chip pathogen in potato and tomato as a model system with which to rapidly evaluate smart plant technology (“Symbiont”) for efficacy against the zebra chip pathogen, having aims eventually to test the new technology in citrus against the citrus greening pathogen. The potato and citrus pathogens are closely related, but assays using tomato and potato can be completed in 6-8 weeks whereas assays in citrus require a much longer time. Several “Symbiont” constructs significantly reduced pathogen titers, but results indicate a need to identify sources of variance in “Symbiont” efficacy among individual plants.

Accomplishments
1. New plant reservoir of the zebra chip pathogen is identified. A relative of potato psyllid known as bindweed psyllid has been shown to regularly harbor the zebra chip pathogen. Field bindweed has long been known to be the host plant of the psyllid, but despite extensive testing of foliage bindweed has not been proven to harbor the pathogen. ARS scientists in Wapato, Washington, and scientists at Washington State University, Pasco, Washington, collected foliage, below-ground material (roots and storage organs), and stem samples from psyllid-infested populations of bindweed, and assayed the plant material molecularly for presence of the zebra chip pathogen. The pathogen was detected in the below-ground parts of the weed and in stem material at the soil surface but could not be detected in leaves. Immature psyllids were found feeding on stems right at the soil surface and immediately below the soil surface, which may be how they acquire the pathogen. This research shows that field bindweed and the bindweed psyllid must be considered possible hosts of the zebra chip pathogen in weedy habitats abutting potato fields.

Review Publications
Delgado-Luna, C., Cooper, W.R., Villarreal-Quintanilla, J.A., Hernandez-Juarez, A., Sanchez-Pena, S.R. 2024. Physalis virginiana as a wild field host of Bactericera cockerelli (Hemiptera: Triozidae) and ‘Candidatus Liberibacter solanacearum’. Plant Disease. 108(1):113-117. https://doi.org/10.1094/PDIS-02-23-0350-RE.
Fisher, T.W., Munyaneza, J.E., Brown, J.K. 2024. Sub-optimal temperatures lead to altered expression of stress-related genes and increased ‘Candidatus Liberibacter solanacearum’ accumulation in potato psyllid. Frontiers in Insect Science. 3.Article 1279365. https://doi.org/10.3389/finsc.2023.1279365.
Pitt, W.J., Cooper, W.R., Pouchnik, D., Headrick, H.L., Nachappa, P. 2024. High-throughput molecular gut content analysis of aphids identifies plants relevant for potato virus Y epidemiology. Insect Science. 31;1489-1502. https://doi.org/10.1111/1744-7917.13327.
Serrano, J.M., Cook, R.E., Headrick, H.L., Cooper, W.R. 2023. Dietary history of click beetles and wireworms in the genus Limonius (Coleoptera: Elateridae) revealed by molecular gut content analysis. Environmental Entomology. 53(1):173-179. https://doi.org/10.1093/ee/nvad114.
Chuan, J., Nie, J., Cooper, W.R., Chen, W., Hale, L., Li, X. 2024. The functional decline of tomato plants infected by Candidatus Liberibacter solanacearum: An RNA-Seq transcriptomic analysis. Frontiers in Plant Science. 15. Article 1325254. https://doi.org/10.3389/fpls.2024.1325254.
Foutz, J.J., Cooper, W.R., Swisher Grimm, K.D., Crowder, D. 2024. Seasonal and lifecycle changes in behavior affect the trapping efficiency of an insect vector, Circulifer tenellus (Hemiptera: Cicadellidae). Annals of the Entomological Society of America. 117(3):199-205. https://doi.org/10.1093/aesa/saae011.
Dahan, J., Orellana, G.E., Wald, K., Wenninger, E.J., Cooper, W.R., Karasev, A.V. 2024. Bactericera cockerelli picorna-like virus and three new viruses found circulating in populations of potato/tomato psyllids (Bactericera cockerelli). Viruses. 16(3). Article 415. https://doi.org/10.3390/v16030415.
Felix-Rocha, A.C., Delgado-Luna, C., Cooper, W.R., Villarreal-Quintanilla, J.A., Sanchez-Pena, S.R. 2024. Margaranthus solanaceus, a new weed host plant for Bactericera cockerelli and Candidatus Liberibacter solanacearum on the Gulf Coastal Plain of Northeastern Mexico. Southwestern Entomologist. 49(1):185-191. https://doi.org/10.3958/059.049.0115.