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

Location: Subtropical Insects and Horticulture Research

2022 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 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. This approach should also benefit the efficacy of exotic parasitoids that have been introduced to control the psyllid. Experiments to demonstrate the veracity of this concept are ongoing. A CRISPR/Cas9 method was developed that permits the use of adult psyllids to produce heritable gene-edits. The CRISPR/Cas9 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. We will continue to develop this approach into a practical strategy to suppress psyllid populations and limit spread of bacteria in citrus trees causing Huanglongbing. A new psyllid infecting virus was discovered and the genome sequenced. The virus promoters will be examined for use to develop biopesticides expression system for psyllid pests. Whole plant studies on transgenic citrus expressing antimicrobial peptides in citrus were conducted to evaluate their effect on psyllid development. Experiments showed that plants producing these peptides supported significantly reduced efficacy of psyllid development, as compared to non-transgenic control plants, when adults were placed on these plants and females were allowed to lay eggs. Whole plant studies supported previous leaf assays, and selections of these plants are now being propagated and will be planted in 2023 for field evaluation at our Picos research farm site. A plant-based method was developed for the economic production of biological therapeutic molecules that can be used to treat citrus for HLB and other disease control. This method is based on in vitro culturing of plant cell “symbiont tissue”. During the past year this system was optimized and shown to produce greater than 500 mg of desired protein per liter of culture per week. Furthermore, the system secretes the desired proteins into the liquid media and we have demonstrated continuous production for several months from a single culture system and harvesting the media containing the desired protein weekly. Our USDA ARS propriety Symbiont strategy was further optimized for sustainable development on citrus and for high level of desired protein production. Proprietary methods were developed to increase the desired protein production levels when these symbionts are grown on plants. ARS researcher at Fort Pierce, Florida, continued to manage, as the lead investigator on a 5-year $15 million NIFA 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 at Fort Pierce, Florida, 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 $15,000,000 NIFA grant was developed and submitted for review based on a systems approach to delivery field testable solutions to citrus HLB. 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 gene families annotated for: immunity, development, digestion, salivary gland enzyme, and insecticide resistance. Improved accuracy of gene sequences aided selection of effective biopesticides developed for psyllid control. Patents were submitted and are pending.

Accomplishments
1. Development of “Plant Symbiont System". This system promises a cost-effective delivery of biological therapeutic molecules for control of HLB. Traditionally, scientists use Agrobacterium, a common bacteria, to modify plant genes to generate transgenic plants. Transgenic plants enable farmers to protect their crops against harmful insect pests and pathogens and to enable them to optimally grow and respond to environmental changes. 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 their deregulation. ARS researchers in Fort Pierce, FL and Ithaca, NY worked with a small Florida agribusiness to develop a method (‘SYMBIONT’) that, for the first time, used 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, eliminating the need to generate a transgenic plant entirely. Current results include 1) a demonstration that symbionts on citrus trees lasted over 2 years without producing harmful effects to the trees; 2) improved symbiont transplantation and inoculation methods to produce more uniform and rapid growing symbiont; 3) identifying plant species that can support multiple symbionts, which, in turn, produce harvestable amounts of therapeutic proteins. Plants were selected on their ability to support rapid symbiont growth at multiple locations. The results show that the SYMBIONT system has potential to efficiently deliver therapeutic molecules to the difficult-to-reach vascular system where it is needed most.

2. Use of SYMBIONT technology for in vitro production of therapeutic molecules. Continued activities in the second year of a $15 million 5-year NIFA grant demonstrated that our proprietary SYMBIONT technology can be used for in vitro production of large quantities of therapeutic molecules. The multidisciplinary work conducted on this grant and also as part of a CRADA with an agricultural Biotechnology Company (AgroSource, Inc.) demonstrated that Symbiont tissue can be cultured in vitro to economically produce desired molecules that are secreted into the media and continuously harvested. This was demonstrated using fluorescent marker proteins and also by production of a special class of mammalian antibodies called nanobodies. We studied the ability to make nanobodies in our SYMBIONT system because we are also developing nanobodies against identified effector proteins produced and secreted by the HLB-causing bacterium. These effectors induce the disease symptoms in the infected citrus plants and other lines of research indicate that binding antibodies to these effectors prevents disease development and therefore are producing nanobodies that can block the effector function. While these antibodies are being produced by collaborators, we demonstrated that functional mammalian nanobody can be produced our plant cell SYMBIONTS using a gene encoding a nanobody that targets the spike protein of COVID-19. Functional binding of this nanobody was demonstrated using an assay that measure the ability to inhibit the interaction of the spike protein and the surface antigen on mammalian cells. This work shows that our plant-based SYMBIONTS can be used to produce therapeutic molecules that we can test as therapeutic candidates to be used in treating citrus HLB disease.

Review Publications
Sundar, N.S., Karthi, S., Sivanesh, H., Stanley-Raja, V., Canthini, K.M., Ramasubramanian, R., Ramkumar, G., Ponsandar, A., Narayanan, K., Vasantha-Srinivasan, P., Jawaher, A., Alwahibi, M.B., Hunter, W.B., Senthil-Nathan, S., Patcharin, K. 2021. Efficacy of Precocene I from Desmosstachya bipinnata as an ef-fective bioactive molecules against the Spodoptera litura Fab. and its impact on Eisenia fetida Savigny. Molecules. https://doi.org/10.3390/molecules26216384.
Borovsky, D., Rouge, P., Shatters, R.G. 2022. The Ribosome is the ultimate receptor for Trypsin Modulating Oostatic Factor (TMOF). Biomolecules 12:577. https://doi.org/10.3390/biom12040577.
Shippy, T.D., Miller, S., Blessy, T., Hosmani, P.S., Flores-Gonzalez, M., Mueller, L.A., Hunter, W.B., Brown, S.J., D'Elia, T., Saha, S. 2021. Annotation of chitin biosyentesis genes in Diaphorina citri, the Asian citrus phyllid. GigaByte. https://doi.org/10.46471/gigabyte.23.
Renolds, M., Oliveira, L., Vosburg, C., Paris, T., Massimino, C., Norus, J., Ortiz, Y., Espino, M., Davis, N., Masse, R., Neiman, A., Holcomb, R., Gervais, J., Kemp, M., Hoang, M., Shippy, T.D., Flores-Gonzalez, M., Panitz, N., Mueller, L.A., Hunter, W.B., Benoit, J.B., Brown, S.J., D'Elia, T., Saha, S. 2021. Annotation of putative circadian rhythm-associated genes in Diaphorina citri (Hemiptera: Liviidae). GigaByte. https://doi.org/10.46471/gigabyte.21.