Location: Subtropical Insects and Horticulture Research
2023 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.
Field tests are showing that topical applications of plant signaling hormones can be used to attract predaceous and parasitic insects to citrus shoots infested with immature psyllids, resulting in significant reductions in immature psyllid survivorship. The tests also indicate that topical applications of plant signaler hormones can also reduce the psyllid’s oviposition activity.
Over 50 different protein constructs were tested to improve systemic movement of proteins produced in Agrobacterium-induced symbionts. Three of these fusions demonstrated more efficient export and movement within the plant’s vascular tissue.
Optimization of plant “symbiont” cell culture as an in vitro ‘biofactory’ was conducted and used to demonstrate that this culture system could produce desired molecules. This included nanobodies with important implications in human medicine and disease control in plants.
An ARS researcher at Fort Pierce, Florida, was awarded an ARSX prize to demonstrate the ability to use stingrays to produce nanobody-like antibodies that could help control diseases in animals and plants.
Plasmid vectors were developed and optimized for more efficient production of beneficial Agrobacterium-induced symbionts to produce therapeutics 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.
Eight-eight molecules/compounds/formulation were injected into field trees in the largest trial ever conducted to screen for treatment efficacy in improving overall tree health, fruit hold, and recovery of citrus trees with citrus greening disease.
Accomplishments
1. Demonstrating the importance of residential citrus as a source of infective psyllids for commercial citrus groves. Citrus trees growing in residential areas are a source of Asian citrus psyllid that can fly to nearby commercial citrus groves and infect them with citrus greening disease. However, the level of psyllid movement between residential and commercial citrus trees is unknown. A ‘Mark-Release-Recapture’ was conducted in which psyllids were marked with brightly colored fluorescent powders and released either in a neighborhood with many citrus trees or a commercial citrus grove growing on three sides of the neighborhood site. The psyllids were recaptured on yellow sticky card traps hung in both sites. By marking psyllids with differently colored powders, the original release site and date of recaptured psyllids could be determined. A total of 15,300 marked psyllids were released during the study. Most of the marked psyllids recaptured in residential trees were from the neighborhood trees while only 15% were from the commercial grove. In contrast, ca. 50% of psyllids recovered in the grove were released in residential trees. About 40% of the psyllids changed habitats, but the change was skewed toward movement from the residential to commercial grove habitat. These results showed that there is a constant exchange of psyllids between the two habitats, with residential citrus trees functioning as a source habitat of psyllids that emigrate into adjacent commercial groves. The data highlight the necessity of implementing effective psyllid management practices in residential habitats to reduce source populations of the psyllid.
2. Fulbright scholar’s visit accelerates research on plant signaling hormones. The United States Department of State Fulbright Program, the Unites States government’s flagship program of international education and cultural exchange, sponsored a three-month visit by a scientist from the National Council for Scientific and Technical Research of Argentina (CONICET) to work with a scientist at ARS-Fort Pierce, Florida. The joint ARS-CONICET research team conducted field experiments that demonstrated for the first time that spray applications of plant signaling hormones could be used to induce citrus to emit ‘distress signal’ aromas to attract the natural enemies of the psyllid, and which resulted in significant reductions in immature psyllids (Subobjective 1d). A CRADA is being developed to continue collaborative studies on optimizing topical applications of plant signaling hormones for the purpose of reducing psyllid populations.
3. Four genes were identified that could potentially improve the health of citrus trees sickened by huanglongbing. Huanglongbing (HLB) has caused a greater than 70% reduction in commercial citrus production in Florida and threatens the citrus industries in Texas and California. There is currently no cure for this disease, and to save the citrus industry, a solution must be delivered to infected trees in citrus groves. A bacterium named Agrobacterium tumefaciens was used to develop small, localized growths, called ‘galls’, on citrus trees. Galls were genetically engineered using the identified genes to produce molecules that potentially could confer tolerance to the host citrus tree. The engineered galls are called ‘symbionts’ because they produce molecules that provide therapeutic benefits to their tree hosts. The therapeutic molecules produced by the symbiont were demonstrated to move systematically in the tree. This new ‘symbiont’ strategy could mitigate some regulatory concerns about the release of genetically engineered crops because the genetically engineered component is the symbiont, not the tree. This technology would be applicable on any plant that Agrobacterium could infect and form a gall.
Review Publications
Borovsky, D., Rougé, P., Shatters, R.G. 2022. Bactericidal properties of Proline-Rich Aedes aegypti Trypsin Modulating Oostatic Factor (AeaTMOF). Life. 13(1), 19. https://doi.org/10.3390/life13010019.
Santos Silva, M., Patt, J.M., De Jesus Barbosa, C., Fancelli, M., Ribeiro Mesquita, P.R., Demedeiros Rodrigues, F., Selbach Schnadelbach, A. 2023. Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae) responses to plant-associated volatile organic compounds: A mini-review. Crop Protection. https://doi.org/10.1016/j.cropro.2023.106242.
Setamou, M., Patt, J.M., Tarshis Moreno, A.M. 2022. Source or sink? the ole of residential host plants in Asian citrus psyllid infestation of commercial citrus groves. Journal of Economic Entomology. 115:438-445. https://doi.org/10.1093/jee/toab249.
Borovsky, D., Deckers, K., Vanhove, A., Verstraete, M., Shatters, R.G., Powell, C.A. 2021. Crowdsourced identification of potential target genes for CTV induced gene silencing for controlling the citrus greening vector Diaphorina citri. Frontiers in Physiology. https://doi.org/10.3389/fphys.2021.571826.
Kohlrausch Távora, P.T., Santos Diniz, F.A., Rêgo-Machado, C.M., Greitas, N.C., Monteiro Arraes, F.B., Andrade, E.C., Furtado, L.L., Osiro, K.O., Sousa, N.L., Merz Henning, L.M., De Oliveira Molinari, P., Feingold, S.E., Hunter, W.B., Grossi De Sá, M.F., Kobayashi, A.K., Nepomuceno, A.L., Santiago, T.R., Correa Molinari, H.B. 2022. CRISPR/Cas- and RNAi-based technologies for crop improvement: Reviewing the risk assessment and challenges towards a more sustainable agriculture. Frontiers in Bioengineering and Biotechnology. https://doi.org/10.3389/fbioe.2022.913728.
Kennedy, J.P., Wood, K., Pitino, M., Mandadi, K., Igwe, D., Shatters, R.G., Widmer, T.L., Niedz, R.P., Heck, M.L. 2023. A perspective on current therapeutic molecule screening methods against ‘candidatus liberibacter asiaticus’, the presumed causative agent of citrus Huanglongbing. Phytopathology. https://doi.org/10.1094/PHYTO-12-22-0455-PER.
Rhodes, B.H., Stange, R., Zagorski, P., Hentz, M.G., Niedz, R.P., Shatters, R.G., Pitino, M. 2023. Direct infusion device for molecule delivery in plants. The Journal of Visualized Experiments (JoVE). https://dx.doi.org/10.3791/64008.
