Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
The Asian citrus psyllid (ACP) has devastated citrus production in Florida due to the ability to vector a bacteria that causes Huanglongbing (HLB) disease and is single handedly responsible for billions of dollars lost stemming from loss of orange production.The bacateria is a phloem restricted pathogen and ACP are unable to acquire or transmit CLas if feeding is interrupted prior to phloem feeding.Thus, we aim to develop technologies that will kill ACP and inhibit phloem feeding by ACP will prevent HLB disease.We have developed inhibitors of inward rectifier potassium (Kir) channels that are highly toxic to ACP and prevent plant feeding and thus, our goal is to develop and optimize commercially viable Kir inhibitors that kill ACP and prevent HLB in field settings.In Obj. 1, we will define toxicity and antifeedant activity of structurally distinct Kir inhibitors through laboratory, greenhouse, and field studies.In Obj. 2, we will quantify inhibition of CLas acquisition/transmission by Kir inhibitors through lab and semi-field assays and lead candidates will be field tested in year 3.In Obj 3, we will utilize unique technology to generate systemic movement of Kir inhibitors throughout the plant.Stakeholder involvement is critical for the development of new technologies and thus, we will work with citrus stakeholders in FL, CA, and TX and agrochemical companies to ensure outputs remain commercially viable.We anticipate data from this study will lead to development of technologies for restoration of citrus production in regions with established disease (Florida) or protect groves in "at-risk" regions for HLB disease (Texas/California).
Animal Health Component
50%
Research Effort Categories
Basic
40%
Applied
50%
Developmental
10%
Goals / Objectives
Thelong-termgoalof this investigation is to increase the sustainability of the US citrus industry through development of novel mechanism insecticides that can be integrated into ACP management programs.This proposal will validate synthetic and/or natural chemicals that increase citrus tree health by inducing ACP toxicityandinhibiting ACP feeding to prevent HLB disease.This Standard Project (SP) proposal combines studies at the laboratory, semi-field, and field levels and we anticipate the data generated will provide the framework for a Coordinated Agricultural Project (CAP) with large scale field testing across FL, TX, and CA in future work.To achieve our goal, we will:Identify commercially viable Kir inhibitors that kill ACP and prevent plant feedingDefine anti-acquisition and anti-transmission activity of Kir channel inhibitors forCLasUse new technology to generate systemic movement of Kir inhibitors through the plantDisseminate our findings to the citrus industries in FL, TX, and CA
Project Methods
General Materials for All ObjectivesACP colonies and infection withCLas:We will maintain ACPcolonies onCLas-free orCLas-infectedC. sinensisL. Osbeck cv Valencia trees in two separate greenhouses with environmental conditions described previously. TheCLas infection rate in each population will be determined monthly by testing 40 adult insects per colony (infected and uninfected) using a TaqMan qPCR assay following the protocol described by Li et al..Citrus Tree stock:All experiments will be performed using 2 year oldCitrus sinensisL. Osbeck cv Valencia grafted onto US-812 rootstocks. Plants will be obtained from Southern Citrus Nurseries and prior to initiation of experiments following previously described methods.Objective 1:Test structurally diverse Kir inhibitors for acute toxicity, inhibition of salivary secretions, and plant feeding by ACPDetermine the acute toxicity and selectivity of Kir channel inhibitors and natural VU041 analogs to ACP in laboratory, semi-field, and field settings.We will quantify the toxicity of ca. 100 structurally diverse Kir channel inhibitors and 75 natural VU041 analogsLaboratory assessments:Acute toxicity of Kir inhibitors to nymphal and adult ACP will be quantified via leaf dip bioassays.Leaf disks (35 mm) will be cut from uninfectedC. sinensisand a discriminatory concentration of 200 ng/cm2 will be pipetted onto the leaf disk. Three replicates on separate cohorts per compound will be performed. Concentration-response curves for determination of LC50values will be generated for compounds that elicit >90% mortality at the discriminatory concentration.semi-field:Compounds identified to be toxic at 30 ng/cm2(3-fold less than imidacloprid) through leaf dip bioassays will be tested at the semi-field levelusing young citrus trees. Three commercial citrus groves in Florida will be designated as sites A, B, and C.Site Ais planted withCitrus sinensiscv. 'Vernia' on 802 rootstalk,site Bis planted withCitrus sinensiscv. 'Valencia' on 942 rootstalk, andsite Cis planted withCitrus sinensiscv. 'Valencia' on a C-54 rootstalk.Kir inhibitors will be applied with a hand-gun sprayer using standard methods and treatment dosage will bebased on efficacious concentrations determined in laboratory assays. Agri Flex (abamectin 3% + thiamethoxam 13.9%) will be applied at established label rates as the industry standard positive controls.Each treatment will be tested in 5-tree plots and replicated four times per site in a randomized complete block design. Two fully expanded, but not hardened, young leaves will be randomly collected from each plot 0, 7, 14, 21, 28, 35 and 42 days after treatment.Leaf disks will be cut from field-collected leaf samples from chemical or control treatments and loaded with ACP.Average toxicity will be determined from a total of 3 replicates where each replicate will consist of 30 (3 sets of 10 individuals) and mortality assessments will be performed at 24h, 48h, and 72h.Field Toxicity assessments:Our data has shown that the Kir inhibitor VU041 is highly toxic to ACP at concentrations near or exceeding commercialized insecticides, prevents phloem feeding, and reduces acquisition ofCLas at sub-lethal concentrations in laboratory assays.When combined with the high selectivity profile of VU041, these data justify testing of VU041 in real-world scenarios via field experiments in year 1.In addition to VU041, we will perform field testing of synthetic or natural Kir inhibitors identified to have >90% toxicity (Obj 1), inhibit plant feeding (Obj 1), and/or preventCLas acquisition/transmission (Obj 2) at concentrations within 2-fold of neonicotinoids.Methods for field trials will follow our published studies. The experiment will follow a randomized complete block design with five replicates and with blocks separated by 50 m. Four trees will be sampled per treatment plot per sampling date.Quantify the influence Kir inhibitors and natural VU041 analogs have on salivary sheath formation.An artificial membrane assay will be used to measure the salivary sheath from individual insects fed sucrose solutions containing Kir inhibitors as described above.Adult or nymphal insects will be allowed to feed on an artificial feeding system with or without a discriminatory concentration of Kir modulators.After feeding, the membrane will be removed, slide mounted, and sheaths counted and measured using Leica LAS × softwareDetermine antifeedant efficacy of structurally diverse Kir inhibitors to ACP with EPG analyses. Electrical penetration graph (EPG) recordings:To compare ACP feeding behavior, EPG experiments will be conducted with adult and nymphal ACP.EPG recordings will be conducted on leaf surfaces and plants that have been age matched.Kir inhibitors will be selected based on potency to saliva secretion determined in Obj. 1.2-1.3 and the 20 most active inhibitors will be evaluated. EPG will be performed on nymphs and adults and feeding waveforms will be distinguished using characteristics listed in our previous publications. Three test plants (1CLas-free control, 1CLas-free + Kir inhibitor, 1CLas free + inactive analog) will be tested simultaneouslyObjective 2:Quantify reduction ofCLas acquisition and transmission by ACP after exposure to Kir channel inhibitors. General Methods for Objective 2CLas source:Uninfected orCLas-infected ACP will be maintained onC. sinensiscv Valencia in two separate greenhouses following standard environmental conditions. TheCLas infection rate is determined in each population monthly by testing a sub-sample of 40 adult insects and 10 plants per colony using methods described inLi, et al..Test plants:At least three citrus genotypes will be tested to ensure generalizability of our resultsCLas detection:We will follow our established protocols for quantification ofCLas in ACP to assess acquisitionChemical selection:The 20 most active Kir inhibitors that inhibited salivary gland function, secretion of proteins relevant to plant immune activity, or prevented phloem feeding will be tested.C. sinensisleaves will be treated with Kir inhibitors at 100 µM or water+solvent (neg. control).Compounds that inhibitCLasacquisition/transmission by >80% at 100 µM will be tested at 1 µM and 10 µM for potency comparisons. Objective 3:Optimization of formulation for increased bioavailability and enhanced systemic activity of Kir inhibitors.Test if formulation of Kir inhibitors with WSC will increase chemical dispersion in aqueous media.Chemical stability and particle size of developed Kir-WSC will be assessed using HPLC and dynamic light scattering (DLS) in the Swale Lab as we have done previously21.We will generate Kir-WSC with the five most active compounds that induce toxicity, prevent phloem feeding, and preventCLas acquisition/inoculation. We will test VU041-WSC for anti-CLas activity in the semi-field inYear 1because VU041-WSC prevents phloem feeding (Fig. 7)andwe will incorporate additional compounds into Obj. 3 as they are validated in Objective 1-2.Assess the systemic activity of solubilized Kir inhibitors in citrus plants.Methods will follow those described in our previous studies with cotton plants.We will treat a single lower leaf with Kir-WSC, incubate the plant for 72 hours, and perform chemical and biological analyses on newly emerged leaves at the top of the plant.