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ARS Home » Southeast Area » Fort Pierce, Florida » U.S. Horticultural Research Laboratory » Subtropical Insects and Horticulture Research » Research » Research Project #439528

Research Project: IPM Method for Control of Insect Pests and Transmitted Diseases of Orchard Crops

Location: Subtropical Insects and Horticulture Research

2021 Annual Report

Objective 1: Investigate biological control and ecological interactions of invasive pests of subtropical orchard crops (especially citrus) with their natural enemies, including signaler compounds that influence pest and natural enemy behaviors, and use this information to develop biologically based pest control strategies. Sub-objective 1a: Identify plant species that can function as nectar sources or as banker plants (= ‘conservation plants’) to support the natural enemies of ACP in commercial citrus groves and nearby residential areas. Sub-objective 1b: Determine whether the addition of conservation plants to a target landscape results in increased numbers of natural enemies with a concomitant decrease in ACP and, if so, determine if this effect decreases as a function of distance between conservation plants and citrus trees. Sub-objective 1c: Perform scale-up of conservation plant arrays for use in citrus groves and evaluate their effectiveness in reducing ACP populations. Sub-objective 1d: Determine whether plant signaler compounds can be used to: 1) increase recruitment of D. citri natural enemies to citrus; and, 2) influence ACP settling on citrus shoots. Objective 2: Identify structural, physiological, molecular and chemical aspects of the Asian citrus psyllid and its hosts that can be used in the development of novel interdiction strategies such as feeding disruptors and peptide inhibitors of disease transmission that can be deployed either through biotechnology or exogenous application. Sub-objective 2a: Screen dsRNAs in silico. Sub-objective 2b: Identify interdiction molecules that can be expressed in transgenic or PHACT adapted plants for controlling hemipteran insects and their transmitted diseases. Objective 3: Develop delivery methods to control ACP and HLB using approaches such as biotechnology, optimal chemical formulation, plant infusion, and attract and kill devices. Sub-objective 3a: Develop direct delivery strategies for RNAi inducing and peptide interdiction molecules. Sub-objective 3b: Development of transgenic citrus with increased resistance to hemipteran pest insects and/or their vectored diseases. Sub-objective 3c: Plant-Host Activated-Cell Transplantation (PHACT) as a strategy to induce plant resistance to hemipteran insects and their transmitted diseases. Sub-objective 3d: Develop Attract and Kill (AK) devices that will effectively suppress ACP populations in citrus groves and residential citrus. The devices will be capable of being charged with soft pesticides, entomopathogens or other killing agents. They will attract and manipulate psyllids using a combination of sensory stimulants and attractants.

Orchard crops, a major contributor to the U.S. agriculture industry, are long-lived trees that are threatened by the continuous invasion of exotic pests and the pathogens they transmit. This project’s focus is to increase the sustainability of U.S. orchard crops by reducing economic losses to invasive pests and pathogens. Current pest management practices rely on broad-spectrum pesticides, which are problematic because of their adverse effects on the health of humans, beneficial organisms, and the environment. Reliance on pesticides promotes pesticide resistance in the targeted insects. Thus, there is a need for novel tools and alternative control methods. The biotechnology and biocontrol methods proposed here complement existing IPM strategies and will lead to sustainable solutions for insect vectors of crop pathogens. The project will focus on the citrus/Asian citrus psyllid/Candidatus Liberibacter asiaticus crop/pest/pathosystem. Candidatus Liberibacter asiaticus (CLas) is the presumed causal agent of Huanglongbing (HLB), also known as citrus greening, a fatal disease that threatens citrus production worldwide. CLas is vectored only by the Asian citrus psyllid (ACP) (Diaphorina citri), a phloem feeding hemipteran restricted to Citrus and related genera. The objectives of the project are to develop: 1) Sustainable, biologically-based pest control strategies for area-wide management of HLB-ACP; 2) Interdiction molecules, with a focus on RNAi inducing molecules and bioactive peptides, that block key pathosystem processes; and, 3) Novel delivery methods for improved and effective uptake of interdiction molecules, killing agents, and entomopathogens to control ACP and HLB. The deliverables of this research will be sustainable management strategies that will allow citrus to remain an economically viable commodity in the presence of HLB. These approaches are also broadly applicable to a range of subtropical orchard crops.

Progress Report
ARS scientists at Fort Pierce, Florida, suggests field trials are identifying which plant species can be used to attract and sustain ladybugs and other natural enemies of the Asian citrus psyllid. This ‘conservation biological control’ strategy can be used in commercial citrus groves as well as residential landscapes. The expectation is that, by improving the local habitat of the psyllid’s natural enemies, they will remain and reproduce in the area. This, in turn, will lead to increased predation of the psyllid, by both the predators and their offspring. Experiments to demonstrate the veracity of this concept are ongoing. A method was developed by ARS scientists that permits the use of adult psyllids to produce heritable gene-edits. The method produced a significant reduction in psyllid egg production: treated female psyllids produced an average of only 6 eggs versus wildtype female controls produced an average of 165 eggs. In addition, adult psyllid life span was reduced from an average of 23 days to 8 days. ARS scientists will continue to develop this approach into a practical strategy to suppress psyllid populations and limit spread of bacteria in citrus trees causing Huanglongbing. An ARS scientist at Fort Pierce, Florida, worked with commercial partner to co-develop a commercial kit for improving gene editing in living organisms. The method transforms efforts to produce gene edits across arthropods. Researchers are using the kit in 7 countries including the United States. ARS scientists will continue to develop this approach into a practical strategy to suppress psyllid populations and limit spread of bacteria in citrus trees causing Huanglongbing. Transgenic expression of antimicrobial peptides in citrus was shown to induce greater than 80% mortality in Asian citrus psyllid adults feeding on leaves from these plants. Plants are being propagated for field evaluations and HLB resistance. Plant-Host Activated-Cell Transplantation (PHACT) symbionts were developed that express a Bacillus thuringiensis toxin that is active against Lepidopterans. These symbionts were harvested by ARS scientists at Fort Pierce, Florida and samples were mixed with larval diet. One hundred percent of the Lepidopteran larvae that fed on this died after 3 days; while no mortality was observed. ARS scientists developed devices that can be attached to citrus trees and used to deliver bioactive defense molecules to whole citrus trees for the purpose of controlling HLB. Field studies have led to the development of optimal solution chemistries that support rapid and repeatable uptake from these devices. This finding allows the ability to use the same device over a long period of time, potentially years, for continued periodic delivery of defensive compounds. An ARS researcher intiated the first year as the lead investigator of a 5-year grant to develop field deliverable therapeutic solutions for citrus greening disease. This is a multi-disciplinary systems approach requiring extensive coordination of researchers form USDA, University and private industry located in 5 different states. ARS researchers continued to lead the ARS Citrus Grand Challenge. This Grand Challenge was awarded and has resulted in a collective multidisciplinary ARS team working together to integrate research efforts. Though this collaborative interaction, a grant was developed and submitted for review based on a systems approach to delivery field testable solutions to citrus HLB. Protein modifications were discovered that allow PHACT symbionts to export proteins more efficiently. This discovery will improve the efficiency of PHACT symbionts An improved genome and official gene set of the Asian citrus psyllid were completed. The Open Source Datasets are hosted at and the NCBI database. Key biological pathways were built, and multiple genes annotated for immunity, digestion, salivary gland enzyme, and insecticide resistance. Improved accuracy of gene sequences aided selection of effective biopesticides for psyllid control. Patents were submitted by ARS scientists at Fort Pierce, Florida. Double-stranded Ribonucleic acid was used to produce genetically modified (GMO) citrus to evaluate potential to suppress psyllids, reduce bacteria transmission and improve citrus tree yield. Genomic data were used to produce antisense oligonucleotides that suppress bacteria. The regenerating trees have been rooted in tissue culture and shown to be transgene positive. They have not yet been tested for expression of the inserted construct. A patent has been submitted by ARS scientists at Fort Pierce, Florida. An ‘Attract-and-Kill’ device (AKD) was developed by ARS scientists at Fort Pierce, Florida, to control Asian citrus psyllid in commercial citrus groves and residential settings. Psyllids fly to the AKD, which is hung on the branches of citrus trees, and upon landing, receive a lethal dose of insecticide. The AKD device emits a combination of visual and scent stimuli that are attractive to the psyllid but not to beneficial insects such as bees. The device does not become fouled with dead psyllids and nontarget insects as do traditional yellow sticky card traps. Efficacy tests of prototype AKDs are ongoing.

1. Modifying plant traits without modifying plant genes. For decades, ARS researchers in Fort Pierce, Florida, have been using Agrobacterium, a common soil-dwelling bacteria, to modify plant genes. This method of modifying plant genes results in the generation of a transgenic plant. Transgenic plants have enabled farmers to fortify various crops against harmful pests and pathogens and to change the way their crops grow and respond to their environment. However, transgenic plant adoption in agriculture has been limited, largely due to: 1) concerns over potential environmental impact, and 2) cost and time associated with environmental impact studies needed for deregulation. ARS researchers in Fort Pierce, Florida, and Ithaca, New York, developed a method in collaboration with a small agribusiness in Florida that showed, for the first time, a method to use Agrobacterium to engineer independently growing plant cells, referred to as symbionts, to modify plant traits. When transplanted onto a plant, symbionts impart desirable traits to plants in real-time without the generation of a transgenic plant or modifying plant genes in any way. Benefits include: 1) eliminating concerns over environmental contamination with transgenic plants or the transgenes that can escape in pollen, seed or vegetative propagules; 2) rapid analysis of plant traits of interest; and 3) new ways to produce and farm biomolecules in plants. The symbiont technology may be transformational for plant disease management and rural agriculture. The technology is being used to deliver the solution to citrus growers to combat citrus greening disease.

2. Developing a "Conservation Biological Control" strategy for Asian citrus psyllid. Growers rely on insecticides to control Asian citrus psyllid, but insecticide-resistant psyllid populations are emerging, and control costs are high. As an alternative to insecticide control, ARS scientists at Fort Pierce, Florida, are developing a control strategy called ‘Conservation Biological Control’ in which certain plants are grown to support the insect predators that attack the psyllid. ARS scientists conducted a study which demonstrated that a statistical method called ‘Response Surface Methodology’ (RSM) could be used to optimize mixtures of plants to support the insect predators of the Asian citrus psyllid. RSM analysis showed that predator occurrence was influenced by: 1) Linear mixture effects, which indicated that predator occurrence was driven by the amount of a single plant species in the mixture; or, 2) Nonlinear blending effects, which indicated that the plant mixture itself had emergent properties that contributed to predator occurrence. Predator abundance was highest in the experiments conducted in the spring and both linear mixture effects and nonlinear blending effects were observed. Predator occurrence decreased in subsequent experiments, which were conducted in the warmer summer months. In the summer experiments, only linear mixture effects were observed, indicating that predator occurrence was driven by the amount of a single plant species in the test mixtures. The results showed that not only did the species composition of a plant mixture drive predator occurrence, but that proportionality of species could contribute to the outcome as well. This suggests that, when formulating a plant mixture to aid in biological control of the psyllid, then consideration should be given to the proportion of each plant species included in the mixture. Further experiments by ARS scientists at Fort Pierce, Florida are ongoing.

3. 5-year NIFA grant initiated with Fort Pierce ARS scientist as the lead. A 5-year NIFA grant was awarded to ARS scientists at Fort Pierce, Florida, that will be delivering field deployable therapeutic treatments for citrus greening disease. This grant represents a multidisciplinary systematic approach to that involves synthetic and molecular biologists all the way to agricultural engineers. In this work, a pipeline will be used for rapid screening of potential therapeutic molecules. The most promising molecules that emerge from his pipeline will be evaluated using three different delivery strategies: transgenic citrus expression, direct plant infusion and a new method called PHACT symbiont biofactory delivery. The first year of this project was initiated in November 2020 and has already led to development of two new patents and establishment of field trials at our research farm. This represents a strategy of responding to emerging pest and pathogen threats in a new way that can be adapted to a number of cropping systems.

Review Publications
Murali-Baskaran, R.K., Senthil-Nathan, S., Hunter, W.B. 2020. Anti-herbivore activity of soluble silicon for crop protection in agriculture: a review. Environmental Science and Pollution Research. 28:2626–2637.
Cicero, J.M., Hunter, W.B., Cano, L.M., Saha, S., Mueller, L.A., Brown, S.J. 2020. Reinterpretation of 'sperm pump' or 'sperm syringe' function with notes on other male internal reproductive organs in the Asian citrus psyllid, Diaphorina citri (Hemiptera:Liviidae). Arthropod Structure and Development. 54(2020)100915.
Hunter, W.B., Clarke, S., Sandoval Mojica, A., Paris, T., Miles, G., Metz, J., Holland, C., McCollum, T.G., Qureshi, J., Tomich, J., Boyle, M., Cano, L., Altman, S., Pelz-Stelinski, K. 2020. Advances in RNA suppression of the Asian citrus psyllid vector and bacteria (Huanglongbing pathosystem). Chpt. 17. 352 pages. In Asian Citrus Psyllid. Biology, Ecology and Management of the Huanglongbing Vector. (eds) Phil Stansly and Jawwad Qureshi. Commonwealth Agricultural Bureau International (CABI Press). p. 258-283.
George, J., Kanissery, R., Ammar, E., Cabral, I., Markle, L.T., Patt, J.M., Stelinski, L. 2020. Feeding behavior of Asian citrus psyllid [Diaphorina citri (Hemiptera: Liviidae)] nymphs and adults on common weeds occurring in cultivated citrus described using electrical penetration graph recordings. Insects. 11(1):48.
Murali-Baskaran,, R.K., Sridhar, J., Sharma, K.C., Jain, L., Senthil-Nathan, S., Hunter, W.B., Kumar, J., Kaushal, P. 2020. Kairomones effect on parasitic activity of Trichogramma japonicum against rice yellow stem borer, Scirpophaga incertulas. Journal of Applied Entomology. 00:1-9.
Hosmani, P.S., Flores-Gonazlez, M., Shippy, T., Vosburg, C., Massimino, C., Tank, W., Reynolds, M., Tamayo, B., Miller, S., Norus, J. 2019. Chromosomal length reference assembly for Diaphorina citri using single-molecule sequencing and Hi-C proximity ligation with manually curated genes in developmental, structural and immune pathways. bioRxiv.
Pitino, M., Sturgeon, K., Dorado, C., Cano, L.M., Manthey, J.A., Shatters, R.G., Rossi, L. 2020. Quercus leaf extracts display curative effects against Candidatus Liberibacter asiaticus that restore leaf physiological parameters in HLB-affected citrus trees. Plant Physiology and Biochemistry. 148:70-79.