Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Entomology
Non Technical Summary
X-disease phytoplasma (Candidatus Phytoplasma pruni), the causal organism of X-disease in stone fruits or "buckskin" and "little cherry disease" in cherries, recently re-emerged in the US and is currently devastating cherry production in the Pacific Northwest. Diseased trees never recover, therefore control methods rely on infected tree removal and broad-spectrum chemical control of the leafhopper vectors. Over 1,000 acres of trees have been removed in the last five years and $18 million lost in revenue in 2020 alone due to X-disease.Research on leafhopper-X-disease phytoplasma interactions is nearly a half-century old leaving imprecise epidemiological knowledge, and hindering outbreak mitigation efforts. One interesting finding is the phytoplasma reduces the leafhopper's lifespan; therefore, enhancing the phytoplasma's virulence through coinfection with another insect pathogencould reduce vector capacity. This project utilizes a combination of molecular, physiological, and ecological approaches to describe and understand the disease cycle of X-disease within the leafhopper vectors and develop sustainable vector control techniques to limit transmission and outbreaks.Through laboratory and greenhouse experiments I will describe the X-disease infection cycle within the leafhopper vector from acquisition to transmission in both X-disease phytoplasma only infected leafhoppers and leafhoppers coinfected with a fungus. Specifically, I will use probes to fluorescently mark pathogen DNA within the vector at various time points post acquisition to visualize and determine the pathway of infection, time to the vector becoming infectious, and interactions of two pathogens within a host. The results from these experiments will determine how long it takes for the vector leafhoppers to become infectious after uptake of the pathogen which is critical to informing X-disease management strategies. Understanding the pathway of infection provides insights into phytoplasma disease ecology and can open new avenues for control options within the leafhopper vector. Assessing effects of coinfection will elucidate the effects of microbial control agents on the vector and X-disease phytoplasma. Accomplishing these objectives will build the foundation for sustainable X-disease management programs and provide insight into other arthropod vectored phytoplasmas.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Goals / Objectives
Project, Training, and Career Development Goals and Objectives:Goal 1: Expand into new dsciplines and pest system to develop interdisciplinary skills working with an insect-vectored pathogen to gain experience in insect physiology, disease ecology, and plant pathology. Objective 1: Create histological cross-sections of Colladonus reductus leafhoppers under guidance of Dr. Harper. 2: Dissect and mount leafhopper alimentary canal, and primary and accessory salivary glands under guidance of Dr. Cooper. 3: Describe the X-disease infection cycle within the leafhopper vector through insect DNA probing the dissected tissues and cross-sections. 4: Describe effects of co-infection with fungal entomopathegen and X-disease phytoplasma through histological cross-sections and dissections. 5: Determine incubation period, and daily mortality of infected vs uninfected leafhoppers to determine pathogen transmission probability.Goal 2: Enhance skills in molecular techniques to support ecological research and address grower challenges. Objective 1: Collect leafhopper and cherry tree tissueand conduct PCR test for phytoplasma infection. 2: Create and use DNA or RNA fluorescent probes to hybridize and visually observe pathogens in tissue.Goal 3: Gain experience communicating, teaching, and mentoring. Objective 1: Publish two scientific manuscripts from the research proposed. 2: Lead a discussion in a University Seminar on Career Development. 3: Assist in mentoring Dr. Northfield's graduate students, and lab technicians.MilestonesDevelop methods to use molecular primers to track phytoplasma in vectorsIdentify incubation period of X-disease phytoplasma for key vectors and relay information to growers to inform management timing intervals.Develop manuscript describing incubation period and molecular probe technique.Evaluate B. bassiana and Hirsutella sp. fungi as entomopathogenic fungiIdentify impacts of entomopathogenic fungi on incubation period using molecular probes.Develop manuscript describing effects of co-infection on pathogen incubation period.Contribute to a class on career development.Disseminate research results through two scientific conference presentations, and four grower extension talks.
Project Methods
METHODSObjective 1: Describe the X-disease infection cycle within the leafhopper vector from acquisition to transmission.Acquisition: Colladonus reductus mid-instar nymphs (15-20 days old) will be obtained from the X-disease free colony and gently brush transferred to a cage containing only phytoplasma-infected cherry trees. After a 48-h acquisition access period, nymphs will be transferred to a final cage containing uninfected host plants (sugar snap pea (Pisum sativum), and white clover (Trifolium repens)) to complete development. Presence or absence of phytoplasma will be confirmed using diagnostic PCR of plant material using X-disease specific primers (Forward: CGTTGTGGCAGCAGTTTCAG, Reverse: GAAGATGCGGTTTTCGATAGT) (Wright, Harper unpublished). All colonies and experimental cages will be maintained at 25°C, 60% relative humidity, and a16:8 (L:D) h photoperiod.Histology & Dissection: Twenty leafhoppers will be removed from the final cage at each of the following times post acquisition feeding: 7, 14, 21, 28, 35, 42, 48, and 55 days after transfer. Leafhoppers from each cohort will be anesthetized with CO2 and dissected at 32 × magnification under a dissecting microscope (Olympus SZX7, Olympus America Inc. Central Valley, PA). A Tissue Track Microscope slide (Polyscience Inc., Warrington, PA) will have four wax-barrier circles drawn onto it using an Aqua hold barrier Pap pen (Scientific Devise laboratory, Des Plaines, IL). Hemolymph will be collected and smeared into a wax-barrier circle, followed by the alimentary canal, and the primary and accessory salivary glands respectively. Slides will be air dried, then warmed to 50°C to adhere tissues to the slides. Tissue will be fixed with Carnoy's solution (Electron Microscopy Sciences, Hatfield, PA), dehydrated in ethanol, and washed in hybridization buffer. An extra 10 nymphs at each, 14, 28, and 42 days after transfer will be fixed in formalin, dehydrated in ethanol, and then infiltrated with and embedded in paraffin wax blocks. The wax blocks will be cut into 10 μm cross-sections using a Leica 2155 microtome, and will be fixed to slides by incubating at 60°C for two hours, overnight at 37°C, and then de-waxed with Histoclear II (National Diagnostics, Atlanta GA,USA). The prepared microtome sections and dissected leafhopper organs slides will be used for X-disease detection through fluorescence in situ hybridization.X-disease Phytoplasma Detection: Samples on the slides will be hybridized with high performance liquid chromatography-purified oligonucleotide probe labeled with Alexa Fluor 488 on the 5' end (Invitrogen, Carlsbad, CA) using the X-disease specific primer sequence for the probe sequence. A no probe (hybridized without fluorescent buffer) and a probe on uninfected leafhopper (clean colony) controls will be included using five leafhoppers for each control respectively. After hybridization the presence of X-disease will be examined on the microtome cross-sections and dissected organs at 200× and 400× using a fluorescence microscope with Zeiss filter set 09 (excitation wavelength = 450-490nm). Cross-sections, hemolymph, and dissected organs will be photographed using a DP74 camera mounted to the microscope to measure the change in percent fluorescence by incubation period. While the paraffin embedded cross-section approach is more time consuming than the dissections, it is complementary, as it provides intercellular location of the phytoplasma and excellent visual aids for scientific and extension publications.Data Analysis: Data will be analyzed using a logistic regression to determine the probability of X-disease phytoplasma infection among leafhopper tissue (PROC LOGISTIC, SAS Institute). We will assess the time from acquisition to being infectious by when the first salivary glands fluoresce with Phytoplasma, and determine the probability of being infectious by the number of leafhoppers with fully fluorescing salivary glands at each time point.Objective 2: Assess effects of polymicrobial infection on phytoplasma acquisition and transmission, vector fecundity and longevity.Polymicrobial effects on X-disease: Mid-instar (~3rd instar) C. reductus nymphs (15-20 days old) will be removed from the clean colony and separated into 20 groups of 10 nymphs in separate cages with non-infected developmental host plants. Groups will be randomly assigned into the following treatments: 1) B. bassiana and X-disease co-infection, 2) B. bassiana infected, 3) X-disease infected, and 4) No infection control, such that each treatment receives 5 groups (50 nymphs total). Treatments will consist of spraying the leafhopper nymphs with B. bassiana at the field rate (treatments 1 and 2) or water (treatments 3 or 4), then moving them to a cage of X-disease infected (treatments 1 and 3) or uninfected (treatments 2 and 4) cherry trees and allowing them to feed for 48 hrs before moving to a new X-disease free cage of developmental hosts. We will monitor mortality at each time point to evaluate the interactive effects of X disease phytoplasma and B. bassiana. The leafhoppers will be collected, FISH DNA probed, and examined at 200× and 400× under the fluorescent microscope. For treatment one, containing both B. bassiana and X-disease, Alexa Fluor 660 will be used to mark B. bassiana and Alexa Fluor 488 will be used to mark X-disease to allow visualization of both organisms within the cross section. B. bassiana forward primer (GAACCTACCTATCGTTGCTTC) and reverse primer (ATTCGAGGTCAACG TTCAG) are 100% specific for B. bassiana and will be used for attaching the fluorescent probes. The control treatment will also use both probes as a control for each disease.Data Analysis: From the FISH analyses we will evaluate proportional presence in the salivary glands over time using a generalized mixed linear model with a binomial (or betabinomial if overdispersed) distribution and logit link function. Leafhopper longevity in each treatment will be analyzed with a generalized linear mixed model (glmmTMB package in R) using a Gaussian, Poisson, or negative binomial distribution as appropriate, measured by AIC scores. If there are significant treatment effects, pair-wise Tukey-style comparisons will be performed to determine treatment differences in the multcomp package in R. If the three-way interaction is significant, we will use a Tukey style test to evaluate the interaction between the Beauveria presence and time, when only considering the leafhoppers exposed to X disease phytoplasma. A significant test suggests that B. bassiana alters the incubation period for X-diasease phytoplasma.EFFORTS & EVALUATIONI will present my research to fellow scientists at the Entomological Society of America (ESA) national and branch meetings. To increase transparency with the public and local growers I will present my research through 1) local grower meetings, 2) regional tree fruit extension events, and 3) my professional Instagram and Twitter accounts. I will meet bi-weekly with the full advising committee (Dr. Northfield, Cooper, and Harper) to evaluate my progress on experiment establishment for the first three months and quarterly thereafter. Once an experiment is complete, I will provide a written report to the full committee for discussion. Each objective will be considered accomplished when the research has been presented at one professional and extension conference and submitted to a peer-reviewed journal. Dr. Northfield will assess my progress of knowledge in ecological theory through my written reports and manuscript drafts. He will also gauge my mentoring and teaching abilities through meeting with my mentees, and reviewing student evaluations from the career development class. Feedback from Dr. Cooper and Dr. Harper will gauge my progress in using molecular techniques and understanding of X-disease ecology, respectively.