Location: Subtropical Insects and Horticulture Research
2024 Annual Report
Objectives
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.
Approach
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
Field trials are identifying which plant species can be used to attract and sustain 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 adult predators and their offspring. Experiments to demonstrate the veracity of this concept are ongoing.
APHIS and EPA approval was granted for field evaluation of the Symbiont technology for alleviating citrus greening symptoms in production grove citrus trees. Initial inoculations were conducted, and Symbionts have started to develop. Plant health, yield and fruit quality will be evaluated during the next few years.
A ‘Field First’ concept was developed by ARS researchers at Fort Pierce, Florida to evaluate injection of citrus trees with chemicals that have potential to alleviate citrus greening symptoms. This work led to the identification of several treatments that improved plant health that are now being evaluated in cooperating growers’ citrus groves.
A joint research effort was established with scientists at the USDA, ARS European Biological Control Laboratory in Montpellier, France on using the Symbiont technology to treat diseases induced by Xyllela bacterial species. These bacteria cause serious plant diseases in a number of crops including citrus, olive, grape, coffee, alfalfa and peach. The initial focus is on olive quick decline disease, and we have begun developing Symbionts for these trees and screening antimicrobial peptides.
We have established a collaboration with a private company that has developed a device that rapidly delivers herbicide to invasive woody plant species. As part of this effort, we are also collaborating with the Indian River State College Advanced Manufacturing Hub. We have jointly submitted a grant to USDA National Institute of Food and Agriculture to modify this process and develop a rapid and commercially viable method of delivering Symbiont inoculum directly into citrus trees.
Over 50 different protein constructs were tested to improve systemic movement of proteins produced by Symbionts. Three of these constructs demonstrated more efficient export and movement within the plant’s vascular tissue.
Optimization of the Symbiont as a ‘biofactory’ was conducted and used to demonstrate that this culture system could produce desired molecules. This included nanobodies, which are very tiny molecules with antibiotic properties, which has important implications in human and livestock medicine and disease control in plants.
Vectors of plasmids, which are tiny circular pieces of genetic material found in bacteria, were developed and optimized for more efficient production of Symbionts to produce therapeutic molecules in citrus trees. The optimized plasmid vectors will also be used to develop symbiont cells as biofactories for scalable production of desired biomolecules.
An ARS Researcher at Fort Pierce, Florida demonstrated potential treatments against HLB that combine nanometal with antibiotics to improve bacterial suppression. The combined treatment can improve antibiotic effectiveness against the bacteria that causes HLB. The combined antibiotic plus silver and zinc nanometals treatments showed significant bacterial suppression in plate bioassays. Effective combinations and individual nanometal treatments were injected into field trees for further evaluation on benefits to tree recovery in the field.
Accomplishments
1. Field trials initiated on the use of Symbiont technology to alleviate citrus greening symptoms in production field citrus trees. Citrus greening disease has reduced commercial citrus production in Florida by ~90% and threatens production in Texas and California. The Symbiont technology was developed as a way to allow citrus trees to produce a cure for this disease. It works by using a naturally occurring process in which a common plant-associated bacterium, Agrobacterium tumefaciens, induces plant cells to grow into a tumor-like mass, which is called a ‘gall’. ARS researchers at Fort Pierce, Florida genetically-modified the gall cells so that they produce therapeutic, antibacterial proteins that kill CLas. Because the gall cells are fused with the tree’s vascular system, the therapeutic proteins can be exported from the gall into the tree’s vascular system. Greenhouse studies demonstrated that the proteins produced by the gall are effective in killing CLas in citrus trees. Approval for field evaluation has been received and citrus trees growing in the field have already been inoculated. These trees will be evaluated during the next few years for health and fruit yield and quality. Successful commercial delivery of the Symbiont technology will introduce a revolutionary, highly sustainable method to fight crop pests and diseases in a wide array of cropping systems.
2. Screening multiple antibacterial treatments against HLB. A team of scientists from ARS-Fort Pierce, Florida have used a ‘Field First’ screening pipeline to advance over 80 antibacterial treatments into field trials (June-July 2023). The 80+ treatments have been injected into field trees in the largest trial ever conducted to screen for treatment efficacy in improving overall tree health, fruit hold, and recovery.
Review Publications
Sylvester, T., Adams, R., Hunter, W.B., Li, X., Rivera-Marchand, B., Shen, R., Na Ra, S., Mckenna, D.D. 2023. The genome of the invasive and broadly polyphagous Diaprepes root weevil, Diaprepes abbreviatus (Coleoptera), reveals an arsenal of putative polysaccharide-degrading enzymes. Journal of Heredity. 115:94-102. https://doi.org/10.1093/jhered/esad064.
Durden, S., Cruz, A., Hunter, W.B., Debboun, M., Duguma, D. 2024. Cross-infectivity of Vorticella sp. across genera of mosquitoes for development of biological mosquito control strategies. Journal of Invertebrate Pathology. 203. https://doi.org/10.1016/j.jip.2024.108064.
Nielander, M., Maybank, M., Massimino, C., Fitzgerald, H., Blossum, H., Douthitt, C., Holland, C., Hunter, W.B., Carrol, M., D'Elia, T. 2024. Complete genome sequences of StopSmel and Aussie, two Mu-like bacteriophages of Sinorhizobium meliloti. Microbiology Resource Announcements. 22. Article e0123023. https://doi.org/10.1128/mra.01230-23.