Location: Subtropical Insects and Horticulture Research
2020 Annual Report
Objectives
Objective 1: Discover, develop and implement semiochemical-based control and monitoring methods for key pests of orchard crops including, but not limited to, Asian citrus psyllid, citrus leafminer, citrus canker disease, and the Diaprepes root weevil.
Sub-objective 1a: Identify physiologically active odorants and attractant blends for detection and monitoring of ACP.
Sub-objective 1b: Complete large scale tests and promote adoption of CLM mating disruption.
Sub-objective 1c: Identify attractants for DRW and Sri Lankan weevil.
Objective 2: Identify sources of resistance and characterize traits and mechanisms conferring plant resistance to the Asian citrus psyllid in Citrus and near-Citrus relatives.
Sub-objective 2a: Identify and determine the underlying mechanism of resistance in Poncirus trifoliata to oviposition by Asian citrus psyllid (ACP).
Sub-objective 2b: Describe feeding behavior of ACP on susceptible and resistant citrus and near-citrus germplasm.
Objective 3: Develop and implement new and improved biological control strategies for key pests of citrus, including Asian citrus psyllid, using existing and new natural enemies.
Sub-objective 3a: Biological control of Asian citrus psyllid by Hirsutella citriformis.
Sub-objective 3b: Development of an autodisseminator of entomopathogens to suppress ACP populations.
Objective 4: Develop and implement control of key pests and vectors including, but not limited to, Asian citrus psyllid by 1) identifying interdiction points in key biological processes through genomics, proteomics and metabolomics, 2) identifying inhibitors (dsRNA, peptides, chemicals), and 3) developing delivery methods, e.g., transgenic plants and topical applications of exogenous compounds.
Sub-objective 4a: Combining molecular/cellular biology (including targeted and omics level research) with bioassays to identify interdiction molecules including but not limited to dsRNAs (as RNAi inducers), peptides, peptidomimetics and RNA aptamers that block key molecular events in targeted processes such as, but not limited to, salivary sheath formation, specific digestive processes, and/or disease transmission.
Sub-objective 4b: Develop delivery strategies for interdiction molecules.
Approach
Insect-plant interactions are varied and complex. The processes of host location, selection, feeding, and oviposition are only broadly understood, and for relatively few species. In the case of recent arrivals of invasive pests of orchard crops, these aspects of pest biology are not understood in the detail required to design appropriate, novel, and environmentally sound management strategies, such as the following examples. Information-transmitting odors (semiochemicals) can often be inexpensively synthesized and used to interfere with insect pest behavior. Also, understanding the physical or biochemical basis for plant resistance to insects allows engineering or selection of crop varieties with endogenous resistance. In the case of invasive vectors of plant pathogens, lack of understanding of the mechanisms of pathogen transmission (i.e., acquisition, retention and inoculation) further impedes progress in pest management. These mechanisms are also complicated, and are layered onto the complex biological processes described above. The objectives of this project focus on both vector and non- vector pests in orchards. They address discovery, study and utilization of: 1) semiochemicals and other physical or chemical bases of host plant resistance, 2) mechanisms involved in host plant resistance in compatible near-Citrus germplasm, 3) new biological control agents and novel utilization of known ones, and 4) key biological processes that represent opportunities for interdiction of insect-host interactions. Together, these projects aim to design all-new biological control and non-pesticidal management strategies. An advantage of these approaches is their compatibility with existing, especially pesticidal, methods in citriculture. Several of the approaches are broadly applicable to a range of subtropical orchard crops.
Progress Report
This serves as the final report for Project No. 6034-22320-004-00D. Summary of last five years: An ‘Attract-and-Kill’ device (AKD) was developed to control Asian citrus psyllid (ACP) 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, scent, and taste stimuli that are attractive to the psyllid but not to beneficial insects such as bees. The AKD is coated with a waxy substance that transfers a lethal dose of an insecticide to the psyllid. The device was tested in field cages and in small scale field trials. ACP mortality was consistently high. The device does not become fouled with dead psyllids and nontarget insects as do traditional yellow sticky card traps. Efficacy tests are ongoing.
Development of a commercially marketed product for mating disruption of citrus leafminer was essentially completed. The scientist in charge of the project retired and the project has been terminated.
Electroantennogram and olfactometer studies indicated that Sri Lankan weevils responded to hexanol, a common plant odor. The scientist in charge of the project retired and the project has been terminated.
Poncirus trifoliata (trifoliate orange) is resistant to oviposition by Asian citrus psyllid (ACP). Laboratory assays showed that Poncirus leaves emit volatiles that discourage oviposition, and candidate chemicals responsible were identified. Field surveys of 24 citranges (hybrids between Poncirus and sweet oranges) revealed none displayed reduced Asian citrus psyllid (ACP) infestations. The scientist in charge of the project retired and the project has been terminated.
In vivo and in vitro cultures of Hirsutella citriformis were established in collaboration with another ARS location out of state to elucidate the biology of the fungus and to phenotype five isolates. The scientist in charge of the project retired and the project has been terminated.
Field trials are identifying which plant species can be used to attract and sustain the natural enemies of the Asian citrus psyllid, such as ladybugs. 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 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. 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.
ARS scientist worked with a 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 BAPtofect kit, in the United States and seven countries Norway, Israel, Taiwan, China, Canada, Ireland and United Kingdom. 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.
An innovative method was developed to allow economic production and delivery of biological defense molecules, such as peptides and Ribonucleic acid (RNAs), to crop plants. This production and delivery system is based on creating isolated host plant cells that produce desired defense molecules and which are connected to the vascular system of the crop plant. This approach permits the introduction of transgenes into crop plants without producing a transgenic whole plant. A patent was submitted, and demonstration of the effectiveness of the method was completed. This novel strategy will allow within-tree production of antimicrobial peptides that will kill Asian citrus psyllids on citrus trees.
Experiments were finalized demonstrating that transgenic plants expressing either of two antimicrobial peptides can induce psyllid mortality. Highest expressing lines have been identified and are being propagated for both further greenhouse and field evaluation. Further evaluations will include analysis of acquisition and transmission of Candidatus Liberibacter asiaticus (CLas).
We develop devices that can be attached to citrus trees and used to deliver bioactive defense molecules to whole citrus trees for the purpose of controlling Huanglongbing (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.
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 $15,000,000 National Institute of Food and Agriculture (NIFA) grant was developed and submitted for review based on a systems approach to delivery field testable solutions to citrus Huanglongbing (HLB).
An improved genome and official gene set of the Asian citrus psyllid were completed. The Open Source Datasets are hosted at
Accomplishments
1. New method of delivering genetic engineering solutions to crops could revolutionize U.S. agriculture. There is an urgent need to deliver solutions for the control of citrus Huanglongbing (HLB) disease. Delivery of genetic engineering solutions to problems in crop production have a costly and time-consuming regulatory approval process and consumer acceptance can be an issue. Regulatory and consumer concerns include: 1) mobility of recombinant genetic material through pollen/seed or escape of engineered plant; 2) creating a weedy/invasive engineered plant; and 3) consumption of genetically engineered food commodities. ARS researchers at Fort Pierce, Florida, in collaboration with private industry developed a method of delivering genetic engineering solutions through engineering only a group of cells that can be attached to plants (essentially as a new organ) to produce desired molecules that are secreted into the plant vascular tissue and move throughout the plant. This “new organ” cannot survive away from the plant and does not move from the location where it is attached, thus the harvested commodity (i.e. fruit, nut etc.) is not genetically engineered. It also cannot form whole plants, seed or pollen; thus, there is no escape of genetic material. ARS scientists are evaluating the ability of this strategy to cure HLB-infected trees in existing groves by engineering these organs to produce natural peptides and Ribonucleic acid (RNAs) that kill the HLB-causing bacterium and attaching these organs to the trees. If successful, this strategy could be adapted as a means to rapidly deliver genetic engineering solutions in an environmentally sustainable and consumer acceptable method.
2. Developing a ‘Conservation Biological Control’ strategy for Asian citrus psyllid. Growers rely on insecticides to control Asian citrus psyllid (ACP), but insecticide-resistant psyllid populations are emerging & control costs are high. As an alternative to insecticide control, ARS researchers,Ft. 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. They 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 ACP. Following mixtures of plant species were tested: Expt 1: crown-of-thorns, lima beans, & wild poinsettia; Expt 2: crown-of-thorns, flowering buckwheat, & partridge pea &, Expt 3: crown-of-thorns, buckwheat, & ornamental portulaca. RSM analysis showed 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 & both linear mixture effects & 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 proportion of each plant species included in the mixture.
Review Publications
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. https://doi.org/10.1016/j.asd.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. https://doi.org/10.3390/insects11010048.
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. https://doi.org/10.1111/jen.12747.
Borovksy, D., Nauwelaers, S., Shatters, R.G. 2020. Biochemical and molecular characterization of Pichia pastoris cells expressing multiple TMOF genes (tmfA) for mosquito larval control. Frontiers in Physiology. 11:527-541. https://doi.org/10.3389/fphys.2020.00527.
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. https://doi.org/10.1016/j.plaphy.2020.01.013.
Ammar, E., Heck, M.L., Shatters, R.G. 2020. Asian citrus psyllid: biology, ecology and management of the huanglongbing vector. Book Chapter. 127-155. https://doi.org/10.1079/9781786394088.0113.
