Research Project: Management and Biology of Arthropod Pests and Arthropod-borne Plant Pathogens

Location: Emerging Pests and Pathogens Research

2019 Annual Report

Objectives
Objective 1. Using and developing genetic resources and associated information of the ARS Collection of Entomopathogenic Fungal Cultures (ARSEF), conserve, characterize (including taxonomic revision), and exchange insect pathogenic fungi such as Beauveria, Metarhizium, and Hirsutella species complexes to facilitate use of these fungi as biocontrol agents of key arthropod pests and disease vectors. Subobjective 1.1. Continue the curation, operation, and expansion of the ARSEF culture collection and associated information resources. Subobjective 1.2. Improve methods to isolate, culture, and preserve fungal entomopathogens. Subobjective 1.3. Conduct research on the systematics, taxonomy, and organismal biology of these fungi. Objective 2. Identify genetic, environmental and behavioral mechanisms that regulate circulative transmission of insect-borne plant pathogens. Subobjective 2.1. Identification of pathogen, host, and vector components that regulate uptake and transmission of pathogens by sap-sucking insects. Subobjective 2.2. Functional analysis of genes, proteins and metabolites involved in circulative plant pathogen transmission. Objective 3. Explore the utility of novel interdiction molecules that could interfere with plant pathogen acquisition and transmission. Subobjective 3.1. Continue efforts to define the chemistry of fungal secondary metabolites and characterize their effects on phloem-feeding insects, their endosymbionts, and on plant pathogen transmission. Subobjective 3.2. Develop RNA aptamers that bind to transmission related compounds and test their ability to interfere with pathogen acquisition and transmission.

Approach
Control of arthropods that transmit pathogens is arguably one of the biggest challenges to human health and agriculture. Many serious plant and animal pathogens are dependent upon arthropod vectors for transmission between hosts. Nearly all arthropod-transmitted animal pathogens are internalized and circulate in their insect vectors, while plant pathogens are divided between those that circulate in their vectors and those that are carried on the cuticle linings of mouthparts and foreguts. The mechanisms of circulative transmission are only beginning to be dissected, but already commonalities among transmission of both circulative plant and animal pathogens have been discovered. Our experimental systems offer innovative approaches to manage circulative-transmitted plant pathogens that have been recalcitrant to the development of host resistance and for which the economic and environmental costs of vector control has been prohibitive, unsustainable and/or ineffective. Scientists' incomplete understanding of interactions among insect vectors, plant pathogens and plant hosts limits the development of new tools to block or interfere with pathogen transmission by insects in the field. We address this problem by attempting to discover genes and products that mediate the associations among insect vectors, circulative plant pathogens and plant hosts. The new technologies and knowledge are expected to be extended and applied to the study of other circulative pathogens and will greatly impact growers, industry stakeholders, and other research communities. The project will also focus on maintaining the extensive ARS Collection of Entomopathogenic Fungal Cultures (ARSEF). ARSEF contains 12,500 isolates representing 700 fungal taxa from 1,300 hosts and 2,400 locations worldwide, and will be managed to ensure ongoing accession, preservation, identification, and distribution of fungal isolates for development and deployment as biocontrol agents and for research purposes. The ARSEF collection also plays a central role in revising taxonomies of fungi using the state-of-the-art systematic methods.

Progress Report
Objective 1: The Curator position is still vacant due to serious delays with the hiring process. ARSEF is managed by an Acting Curator. ARSEF continues to provide: 1) fungal culture deposition; 2) distribution; and 3) identification. Since July 2018 ARSEF accessioned 630 new fungal isolates of scientific, ecological and/or economic value from around the world. Over 50 unidentified isolates or new deposits have been processed for identification to genus or species via DNA sequencing. Data, policies, and services are made publically available on the Emerging Pests and Pathogens ARSEF/Mycology website. A review of our cryopreservation methods is ongoing to optimize maintenance and facilitate isolate distribution. A total of 140 isolates were shipped in response to 26 requests from the United States (representing seven states) and international clientele (representing eight countries). ARSEF provided fungal identification services by examining morphological characters and/or by sequencing of diagnostic loci, oftentimes requiring isolation and establishment of pure fungal cultures from insect cadavers. Projects on fungal pathogens of insect pests of citrus, cotton and sorghum, in collaboration with ARS researchers at Fort Pierce, FL, Byron, GA, and Tifton, GA, are ongoing. A new project on fungal pathogens of medically important mosquitoes, with an ARS scientist in Peoria, IL, was initiated. Sub-Objective 2.1: Identify genetic, environmental and behavioral mechanisms that regulate circulative transmission of insect-borne plant pathogens. Work on this objective focused on aphids and circulative plant viruses. Many plant viruses are spread in a crop and between crops by aphids. Plant viruses are known to manipulate aphids to promote their own spread among plants, but the mechanisms viruses use for manipulation are largely unexplored. Understanding how viruses manipulate aphids for their own spread provides a novel approach to block virus transmission within and between crops. ARS scientists discovered that potato leafroll virus, a plant virus that is spread by the green peach aphid, suppresses the immune system of the aphid. The weakened aphid immune system makes the aphid more susceptible to an aphid-infecting virus, called a densovirus. The effect on the aphid immune system is mediated by a single potato leafroll virus protein called P0. In plants, P0 is known to suppress the plant immune system but a function for P0 in suppressing the aphid immune system was not known before. In other aphids, densoviruses induce the formation of winged forms of the insect, which would enable the aphid to move long distances. In this study, aphids with wings also had higher levels of the densovirus. Thus, the results lead to a new hypothesis on how potato leafroll virus manipulates aphids for spreading within a crop: the increase in densovirus in aphids caused by the suppression of the aphid’s immune system by potato leafroll virus may serve to enhance the spread of both viruses. Potato leafroll virus also caused other changes in aphids at the molecular level, including the regulation of gene expression and interactions with the beneficial bacterial partner in aphids, suggesting that potato leafroll virus can no longer be considered a silent hitchhiker in its aphid vector, but must now be considered a noisy back seat driver. This work has been highlighted in the popular press (https://btiscience.org/explore-bti/news/post/bti-researchers-discover-interactions-between-plant-and-insect-infecting-viruses/) and as part of a video series, Science in Seconds (https://youtu.be/yymEPmyAOS4). Sub-Objective 2.2: Functional analysis of genes, proteins, and metabolites involved in circulative plant pathogen transmission. Work on this objective focused on citrus greening disease, including the Asian citrus psyllid insect vector, ‘Candidatus’ Liberibacter asiaticus (abbreviated CLas) and citrus host plants. The molecular interactions between the Asian citrus psyllid and CLas is a critical area of citrus greening research. ARS scientists and university partners used high resolution mass spectrometry to measure the collection of small, native proteins found in the psyllid (the peptidome), and discovered evidence for multiple classes of bioactive peptides that are up or down-regulated in CLas-infected psyllids as compared to non-infected psyllids. Psyllid peptides with strong sequence and structural homology to insect neuropeptides were identified, and remarkably, 10 of the 13 neuropeptide precursors identified were down-regulated in CLas-infected insects. Oral delivery of two neuropeptide analogs to the psyllid from artificial diets induced wing deformities and high levels of mortality in adult psyllids. A paper describing these results is in preparation. To understand the molecular interactions between the psyllid and CLas, a proteome analysis of the psyllid has been completed comparing blue to non-blue color morphs as well as males and females. Previous work has shown that CLas infection has distinct effects on the psyllid sexes and color morphs. Vectors of Citrus tristeza virus expressing antimicrobial proteins and silencing constructs have been generated. A total of seven mother plants are infected with these constructs, which serve as a source of graft tissue for inoculation of new budwood. All but one of these mother plants have been propagated for future experimentation on CLas transmission. The Asian citrus psyllid harbors a unique endosymbiotic bacterium called Profftella. This highly specific relationship is a key point of leverage in our effort to find specific strategies to control citrus greening. An unusually large proportion of the Profftella genome is devoted to producing the small molecule diaphorin suggesting it plays an important role in the survival of Profftella and its insect host. ARS scientists studied the movement of diaphorin from the producing endosymbiont through the ACP host and onto/into the host plant. Diaphorin was detected in psyllid honeydew secretions, which are deposited on leaf surfaces, and in an artificial sucrose-solution diet fed upon by infected psyllid indicating that it is present in the insect’s saliva. Ongoing experiments are focused on increasing diaphorin yields from saliva with a focus on the influence of the host plant. In a different study, ARS scientists and university partners showed strong relationships among the relative abundances all the beneficial bacterial species in the Asian citrus psyllid as well as CLas in all psyllid tissues where they infect, suggesting direct inter-species interactions using a possible uncharacterized quorum sensing mechanism. Sub-Objective 3.1: Continue efforts to define the chemistry of fungal secondary metabolites and characterize their effects on phloem-feeding insects, their endosymbionts, and on plant pathogen transmission. Entomopathogenic fungi are a rich source of biologically active compounds, including siderophores, iron scavenging molecules used by microbes to sequester environmental iron. Pathogenic microorganisms deploy siderophores with iron affinities that are high enough to abstract iron from the hosts' specialized mobilization and storage proteins (transferrins and ferritins). ARS scientists hypothesized that plant-pathogen-insect interactions in citrus greening disease are iron-dependent and may be perturbed by the introduction of exogenous iron-chelating agents such as siderophores. Significant progress was made on isolation and structural characterization of siderophores and related compounds from entomopathogenic fungi Beauveria bassiana and Hirsutella species from ARSEF. Isolated siderophores demonstrated significant antibacterial activity and ongoing work is focused on defining the structural features responsible for this activity and on determining the mode of action of the active compounds. Sub-Objective 3.2: Develop RNA aptamers that bind to transmission related compounds and test their ability to interfere with pathogen acquisition and transmission. RNA aptamers are single stranded RNAs that bind to a target with specificity similar to an antibody. Aptamers can be selected to virtually any target using a process called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). ARS scientists worked with university partners to identify three abundant proteins in the mouth parts, referred to as stylet sheaths, of the Asian citrus psyllid, a vector of citrus greening disease. These proteins are being purified and studied to further develop them as candidates for aptamer binding. In a parallel approach, SELEX was applied to dissected stylet sheaths from the Asian citrus psyllid. Preliminary tests with a selected RNA aptamer displays inhibition of sheath formation when psyllids feed in artificial diets. Experiments are planned to test whether the aptamer can be orally delivered to psyllids via citrus trees. With University partners, ARS scientists developed a method to use RNA aptamers to isolate specific protein complexes from cells and characterize their composition by high resolution mass spectrometry. Understanding the protein complexes that function with aptamer targets is critical for its safety as an interdiction molecule to block transmission. A manuscript is in preparation describing this method. In addition to the RNA aptamer approach, ARS scientists are developing two additional peptide-based approaches to the development of interdiction molecules that will help to control citrus greening disease under a CRADA. The first approach builds on the discovery of the psyllid neuropeptides, as described under Sub-objective 2.1. The second approach involves screening antimicrobial proteins produced by legumes. These peptide therapeutics will integrate into existing integrated pest management programs and ongoing bactericide research in ARS labs to strengthen tree protection against CLas transmission and psyllid feeding.

Accomplishments
1. Identification of two fungal pathogens attacking the invasive spotted lanternfly. A study conducted in collaboration with Cornell University scientists at Ithaca, New York, revealed two native fungal pathogens causing a decline in populations of this newly invasive insect pest. The spotted lanternfly has a wide host range, including grape vines, apple trees and hops, and its susceptibility to these native fungal pathogens provide another option of controlling field populations by using natural enemies.

2. Connecting body color and immunity in the Asian citrus psyllid to block the spread of citrus greening disease. A collaboration between ARS scientists in Ithaca, New York, and Fort Pierce, Florida, has illuminated the relationship between insect color and immunity in the Asian citrus psyllid, the insect vector that spreads the Huanglongbing (HLB) bacterium. The copper-containing insect protein hemocyanin is expressed at different levels within an insect colony, where a significant fraction of insects have blue abdomens correlated with high hemocyanin levels. The team used gene silencing to decrease expression of hemocyanin, resulting in reduced levels of the HLB bacterium in the insect vector. The research resulted in a better understanding of the Asian citrus psyllid vectoring characteristics, which will aid growers and state regulators in disease management practices and epidemiological modeling.

3. Strange bedfellows: Evidence for cooperation between a plant and insect virus. Aphids are tiny plant-feeding insects that transmit hundreds of viruses to plants. ARS and university scientists in Ithaca, New York, discovered that the potato leafroll virus encodes a protein, P0, which alters the immune system of its vector, the green peach aphid. The virus P0 protein is known to suppress the immune system of the plant, but its role in suppressing the immune system of the aphid was a surprise to the researchers. As a result of suppressed immunity, the aphids became more susceptible to an aphid-infecting virus called a densovirus, which promotes the formation of wings on adult aphids. Winged adults can fly plant-to-plant, which would promote the spread of both viruses. Understanding the role of P0 in altering the aphid’s immune system is an important new area of research for developing novel ways to block the transmission of potato leafroll viruses and aphid-transmitted viruses related to potato leafroll virus.

4. Fruitful thinking: The fight against citrus greening goes corporate. USDA ARS scientists in Ithaca, New York, and Fort Pierce, Florida, have partnered with a United States agricultural company in a Cooperative Research and Development Agreement (CRADA) to help citrus growers in the fight against citrus greening, an achievement borne out of the USDA ARS Citrus Greening Grand Challenge Synergies program to accelerate a comprehensive, nationwide citrus greening management and readiness plan. A CRADA is a special agreement between a United States government agency and a private company to help advance research and development to produce a commercial product. The partnership will lead to the discovery of new molecules that kill the citrus greening bacterium, prophylactic treatments to prevent citrus greening infection in healthy trees and strategies to deliver therapeutic treatments to help citrus trees with existing infections recover from the damaging effects of greening.

Review Publications
Kruse, A., Ramsey, J.S., Johnson, R., Hall, D.G., MacCoss, M., Heck, M.L. 2018. ‘Candidatus Liberibacter asiaticus’ minimally alters expression of immunity and metabolism proteins in the hemolymph of Diaphorina citri, the insect vector of Huanglongbing. Journal of Proteome Research. 17(9):2995-3011. https://doi.org/10.1021/acs.jproteome.8b00183.
Heck, M.L. 2018. Insect transmission of plant pathogens: a systems biology perspective. mSystems. 3(2). https://doi.org/10.1128/mSystems.00168-17.
DeBlasio, S.L., Xu, Y., Johnson, R., Rebelo, A., MacCoss, M., Gray, S.M., Heck, M.L. 2018. The interaction dynamics of two potato leafroll virus movement proteins affects their localization to the outer membranes of mitochondria and plastids. Viruses. 10(11):585. https://doi.org/10.3390/v10110585.
DeBlasio, S.L., Rebelo, A., Parks, K., Gray, S.M., Heck, M.L. 2018. Disruption of chloroplast function through downregulation of phytoene desaturase enhances the systemic accumulation of an aphid-borne, phloem-restricted virus. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-03-18-0057-R.
Hajek, A.E., Steinkraus, D.C., Castrillo, L.A. 2018. Sleeping beauties: Horizontal transmission by Entomophthoralean Fungi via resting spores. Insects. 9(3). https://doi.org/10.3390/insects9030102.
Wraight, S.P., Wraight-Galaini, S., Castrillo, L.A., Griggs, M., Keith, L.M., Matsumoto Brower, T.K. 2018. Collection, isolation, in vitro culture, and laboratory transmission of Hirsutella eleutheratorum (Hypocreales: Ophiocordycipitaceae) from coffee berry borer on Hawaii Island. Journal of Invertebrate Pathology. 157:53-66. https://doi.org/10.1016/j.jip.2018.08.002.
Pinheiro, P.V., Wilson, J.R., Xu, Y., Zheng, Y., Rebelo, A., Fattah-Houseini, S., Kruse, A., Santos Dos Silva, R., Xu, Y., Kramer, M.H., Giovannoni, J.J., Fei, Z., Gray, S.M., Heck, M.L. 2019. Plant viruses transmitted in two different modes produce differing effects on small rna-mediated processes in their aphid vector. Phytobiomes Journal. 3(1):71-81. https://doi.org/10.1094/PBIOMES-10-18-0045-R.
Hosseinzadeh, S., Shams-Bakhsh, M., Mann, M., Fattah-Hosseini, S., Bagheri, A., Heck, M.L. 2018. Distribution and variation of bacterial endosymbiont and “candidatus liberibacter asiaticus” titer in the huanglongbing insect vector, diaphorina citri kuwayama. Microbial Ecology. 78(1):206-222. https://doi.org/10.1007/s00248-018-1290-1.
Clifton, E., Castrillo, L.A., Gryganski, A., Hajek, A.E. 2019. Native fungal pathogens drive collapse of a new invasive herbivore. Proceedings of the National Academy of Sciences. 116:9178-9180. https://doi.org/10.1073/pnas.1903579116.
Hosseinzadeh, S., Ramsey, J.S., Mann, M., Bennett, L., Hunter, W.B., Shams-Bakhsh, M., Hall, D.G., Heck, M.L. 2019. Hemocyanin protein expression is correlated with color morphology and immunity to ‘Candidatus Liberibacter asiaticus’ in the Huanglongbing insect vector, Diaphorina citri. PLoS One. 14(5):e0216599. https://doi.org/10.1371/journal.pone.0216599.