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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Emerging Pests and Pathogens Research » Research » Research Project #439292

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

Location: Emerging Pests and Pathogens Research

2021 Annual Report

Objective 1. Curate and expand the ARSEF for research, industrial and commercial uses. [NP304, C1, PS 1C; C3, PS 3B] Sub-objective 1.1 Continue the curation, operation, and expansion of the ARSEF culture collection and associated information resources. Sub-objective 1.2 Improve methods to isolate, culture, and preserve fungal entomopathogens. Sub-objective 1.3 Conduct research on the taxonomy, systematics, organismal biology, and population genetics of entomopathogenic fungi. Objective 2. Identify genetic, molecular, ecological, and environmental factors that are associated with a) plant maladies such as rapid apple decline, citrus greening and cotton blue disease; b) arthropod-host interactions, such as ambrosia beetles, psyllids and aphids; and c) arthropod-borne plant pathogen-vector interactions such as Liberibacter and poleroviruses using advanced molecular approaches. [NP304, C3, PS 3A and 3B] Sub-objective 2.1 Identify pathogen, host, and vector components that regulate uptake and transmission of plant pathogens by sap-sucking insects. Sub-objective 2.2 Carry out functional analysis of genes, proteins and metabolites involved in plant pathogen transmission. Sub-objective 2.3 Identify pathogen and host components that regulate entomopathogen infection. Sub-objective 2.4 Document biology and phenology of ambrosia beetles. Sub-objective 2.5 Test for an association of insects and plant pathogens with rapid apple decline. Objective 3. Develop methods using novel interdiction molecules (RNAi, RNA aptamers, siderophores, antimicrobial peptides, modified insect neuropeptides, entomopathogenic fungi) that may interfere with vector-pathogen-host interactions. [NP304, C3, PS 3B] Sub-objective 3.1 Develop a new tool to block aphid transmission of poleroviruses. Sub-objective 3.2 Develop RNA aptamers that bind to transmission-related compounds and test their ability to interfere with pathogen acquisition and transmission. Sub-objective 3.3 Test the utility of plant, insect and microbial derived proteins, peptides and metabolites for control of vector borne diseases. Sub-objective 3.4 Test entomopathogens against the ambrosia beetle. Sub-objective 3.5 Identify RNAi targets for ambrosia beetle control.

Symbiotic interactions between arthropods and microbes span a continuum where mutualism and pathogenesis represent the extremes. Microbial associations with arthropods can be extracellular or intracellular. A subset of arthropod-associated microbes is pathogenic to plants and animals. Many serious plant and animal pathogens are dependent upon arthropod vectors for transmission between hosts. Targeting the relationships between arthropods and microbes is a major focus of research to manage arthropods and arthropod-borne plant diseases. Control of arthropods and arthropod vectors that transmit pathogens is arguably one of the biggest challenges to human health and agriculture. Our experimental systems offer innovative approaches to study and manage arthropods and arthropod-borne plant diseases that have been recalcitrant to the development of host resistance and for which the economic and environmental costs of control has been prohibitive, unsustainable and/or ineffective. Scientists' incomplete understanding of interactions among arthropods, plant associated microbes (including plant pathogens) and plant hosts limits the development of new tools to block or interfere with pathogen transmission by arthropods in the field. We address this problem by investigating the ecological and molecular interactions that mediate these associations. New technologies and knowledge from the planned research are expected to be extended to the study of other arthropod-microbe interactions 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 is a central tool for research in the project and the entire scientific community. It contains 14,342 isolates representing 721 fungal taxa from over 1,300 arthropod hosts (representing major insect orders) in 112 countries. It will be managed to ensure ongoing accession, preservation, identification, and distribution of fungal isolates for the development and deployment as biocontrol agents and for research purposes. The ARSEF also plays a central role in revising taxonomies of fungi using state-of-the-art systematic methods.

Progress Report
Objective 1: A new curator was hired to lead and modernize ARS Collection of Entompathogenic Fungal Cultures (ARSEF). ARSEF continues to provide the following services: 1) fungal culture deposition; 2) distribution; and 3) identification. ARSEF accessioned 105 new fungal isolates. 115 isolates were shipped in response to 12 requests from non-profit institutions in the U.S. and abroad. ARSEF provided fungal identification services by examining morphological characters and/or sequencing of diagnostic loci, often requiring the establishment of pure fungal cultures. We completed an update of the software used for the ARSEF database. This is the first step in overhauling the software to continue to function with modern operating systems, which will be completed in FY 2022. The ARSEF catalog was updated, and a current version is searchable through the ARSEF website. Research also focused on lyophilization methods to allow more rapid shipping of commonly requested viable isolates to users. Objective 2: Poleroviruses. Poleroviruses are aphid-transmitted agricultural pathogens that infect a wide array of staple food crops, including cotton and potato. Previous cryo-electron microscopy studies of virus-like particles indicate that polerovirus capsids are built from a structural coat protein that organizes with T=3 icosahedral symmetry. In collaboration with University partners, we solved the crystal structure of a truncated version of the coat protein (CP) monomer from potato leafroll virus at 1.5-Å resolution. The results have important implications in viral assembly and maturation, suggesting that interactions between the coat protein and viral RNA drive the assembly of infectious virus particles. The PLRV CP assemblies described and compared in this work will help in modelling virion maturation pathways in plants. Cotton leafroll dwarf virus (CLRDV) is an emerging polerovirus threat to cotton grown in the United States. This year, we validated that field-collected material from the previous year was infected with CLRDV, established two A. gossypii colonies in culture, established a leaf disk transmission assay in the lab to study CLRDV acquisition and transmission by aphids, established parameters for systemic plant infection in cotton, established and validated a qPCR assay for CLRDV titer estimation, generated CLRDV structural protein constructs to express in E. coli for structural studies, and established a collaboration with Codex DNA, a synthetic biology company, to generate an infectious CLRDV clone. The purified CLRDV structural proteins were sent to an antibody facility for antibody production. Candidatus Liberibacter asiaticus (CLas) and citrus greening disease. CLas is transmitted by Diaphorina citri, the Asian citrus psyllid, in a circulative propagative manner. Circulative propagative transmission is a complex process comprising at least three steps: acquisition of the pathogen into vector tissues, translocation and replication of the pathogen within the vector host, and pathogen inoculation of a new host by the vector. We described an excised leaf CLas acquisition assay, which enabled precise measurements of CLas acquisition by D. citri in a streamlined lab assay. This simple assay could be used to detect CLas acquisition phenotypes and their underlying genotypes, facilitate the assessment of plant factors that impact acquisition, and screen for compounds that interfere with CLas acquisition by delivering these compounds through the excised leaf, critical for research being done under Objective 3. D. citri exhibit at least two color morphotypes, blue and non-blue, the latter including gray and yellow morphs. Blue morphs have a greater capacity for long-distance flight and transmit CLas less efficiently as compared to non-blue morphs. By studying the proteome profiles of the different color morphs, we showed that differences in physiology and immunity between color morphs of the insect vector may influence disease epidemiology and biological control strategies. In areas where citrus is grown, citrus relatives serve as host plants to the insect vector. We conducted research to show that movement between host plant species impacts transmission CLas. This study further elucidates the complex interactions affecting D. citri vector competence and may lead to the development of novel strategies to control the spread of citrus greening disease. Ambrosia beetles and rapid apple decline. Foundational studies were initiated to improve rearing and investigate the basic biology of two ambrosia beetle species, Xylosandrus crassiusculus and X. germanus, wood-boring insects that are widespread pests in orchard and ornamental production systems. Mass rearing can now proceed with predictable insect production to populate various experiments. Behavioral studies of black stem borer are in progress to document the potential presence of social behaviors among gallery members, such as cooperative brood care by adult female offspring. Two outdoor phenology studies with the black stem borer are in progress. The first is a trapping network being conducted in collaboration with Cornell University personnel, one to serve as an alert to different apple growers in New York State and a second to test hypotheses on the dispersal of beetles from galleries formed by overwintered foundresses. Novel emergence traps have been created to capture dispersing beetles. Data will inform optimal timing of management of the different beetle generations through chemical or microbial sprays and general patterns of population increase over the summer. Outdoor mass rearing of black stem borer in wood bolts is underway to produce diapausing beetles. Studies of artificial overwintering with these beetles are being developed to allow for earlier initiation of experiments in the spring, as we are currently constrained by natural beetle emergence that begins in May and an inability to rear black stem borer continuously. These techniques are also crucial to future cold tolerance studies. Rapid apple decline (RAD) is a potential syndrome of causes that result in the death of trees within weeks of initial symptoms of decline, typically later in summer. A possible relationship may exist between RAD and ambrosia beetles. Based on inquiries with researchers and extension agents in New York and Pennsylvania over the last 1 1/2 years, RAD and significant, direct ambrosia beetle damage, are not being reported by growers. In preparation for future studies, pilot surveys of microorganisms on the surfaces of live-trapped, dispersing adults of black stem borer are in progress with a Cornell University pathologist. Completing research from the previous research project, the soilborne fungus Athelia rolfsii showed promise in killing the invasive plant pale swallow-wort in greenhouse studies but failed to control populations in a county park in upstate New York where it was originally discovered. Disease spread was limited and infected plants appeared aggregated. Objective 3: Outstanding progress has been made on all sub-objectives of Objective 3 and includes potentially transformative research on a plant therapy delivery strategy that will have broad impacts across many national programs. Research has also focused on the identification of natural, plant-derived antimicrobial compounds that could kill CLas, RNA aptamers that block psyllid feeding, and neuropeptide mimics that kill the Asian citrus psyllid. We identified multiple peptides produced by the legume plant, Medicago truncatula, that have antimicrobial activity. This year, screening of these molecules has been completed, and a pool of candidate peptides that either kill or inhibit CLas growth in excised leaf assays will be advanced to in planta testing. A screening of RNA aptamers that block psyllid feeding is complete, and excitingly; four unique aptamer sequences that interfere with the formation of the psyllid mouthparts were identified. Strategies to deliver stable RNA aptamers into citrus trees is ongoing. ARS scientists and University partners previously used high-resolution mass spectrometry to measure the collection of small, native proteins found in the psyllid (the peptidome). Psyllid peptides with strong sequence and structural homology to insect neuropeptides were identified. A pilot screen using artificial diets reveals a subset of 20 peptides that have mortality effects on the psyllid. Research is ongoing to determine the ability of these peptides to move within the citrus phloem and remain stable while exerting mortality effects on the psyllid using in planta delivery. A substantial focus of research centered around a novel tree delivery strategy for citrus greening therapeutics, such as those mentioned above. This work was conducted in collaboration with a CRADA partner and ARS scientists at Fort Pierce. We engineered autonomously growing plant cells, referred to as symbionts, to express a gene of interest. When transplanted onto a plant, symbionts impart desirable phenotypic traits to plants by real-time, in vivo delivery of genetically-encoded molecules to a plant without generation of a transgenic plant. The symbiont technology may be transformational for plant disease management and rural agriculture because benefits include: 1) allowing selection of cultured symbiont cells that highly express the gene of interest; 2) symbiont cells cannot survive away from plants and may be cured of Agrobacterium; thus, mitigating concerns over environmental contamination with modified genetic material and the environmental release of genetically modified organisms; 3) rapid phenotypic evaluations; and 4) production of molecules that can be harvested directly in symbiont cell culture or from in planta grown symbionts.

1. Seed germination traits documented for meadow knapweed. The introduced hybrid meadow knapweed (Centaurea × moncktonii) is an increasingly common weed in pastures and meadows across many U.S. states, but little is known about its biology. Scientists from ARS in Ithaca, New York, and Cornell University evaluated the effects of temperature, light, cold-wet storage, scratching of the seed coat, and source population on seed germination. Greater seed germination occurred at higher temperatures (up to 30°C), when seeds were exposed to 14 hours of light daily, and when stored cold-wet. Cool temperatures down to 15°C) and continuous dark reduced but did not prevent germination. These results indicate that meadow knapweed can germinate under a broad range of environments, including suboptimal conditions, which may have facilitated range expansion of this species in recent decades.

2. Isolate, identify, and accession and test entomopathogenic fungi associated with galleries of the emerald ash borer. The emerald ash borer (EAB; Agrilus planipennis) is an invasive insect pest from Asia that is killing ash trees across North America. EAB has caused substantial economic losses to wood related industries and homeowners, municipalities, and state and national forests. Other invasive wood-boring beetles are known to spread microbes, in particular fungi, that may cause tree disease, but little is known about the microbial communities associated with the EAB. ARS scientists in Ithaca, New York, in collaboration with researchers at the University of Minnesota, investigated the fungi associated with the EAB. This research uncovered many plant disease-causing fungi (soft rot and canker) and 93 insect-infecting fungi, referred to as entomopathogenic fungi, with potential as biocontrol agents. The insect-infecting fungi expand the arsenal of tools available to the USDA ARS and its stakeholders to fight the EAB, a severe pest in the Northeast U.S.

3. Isolate, identify, and accession potential biological control fungi for soybean cyst nematode. The soybean cyst nematode (SCN; Heterodera glycines) causes the largest yield losses in soybean both in the U.S. and worldwide. This pathogen a particularly difficult to manage as most nematicides that were historically effective are highly toxic to both humans and wildlife and have been banned. Scientists from ARS in Ithaca, New York, have obtained funding to isolate and characterize the fungi in soybean cyst nematode cysts and suppressive soils. Results of greenhouse testing of egg-parasitic fungi showed that several isolates were as effective as commercial fungal biocontrol products (DiTera ® and MeloCon® WG) products at lower application doses and more effective than bacterial products (CLARIVA® pn and Poncho/VOTiVO®). These isolates show potential for developing new biocontrol agents or biopesticides for more sustainable integrated pest management of SCN.

4. Modifying plant traits without modifying plant genes. ARS researchers in Fort Pierce, Florida, and Ithaca, New York, in collaboration with a small agribusiness in Florida developed a method to use 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 without the generation of a transgenic plant or modifying plant genes in any way. Benefits include: 1) eliminating concerns over environmental contamination with transgenic plants or the transgenes that can escape in pollen, seed or vegetative propagules; 2) rapid analysis of plant traits of interest; and 3) new ways to produce and farm biomolecules in plants. The symbiont technology is transformational for plant disease management and potentially rural agriculture. The technology is being delivered to citrus growers as part of the solution to citrus greening disease.

5. A rapid assay to measure uptake of the citrus greening bacterium by psyllids from citrus leaves. Citrus greening disease attacks all citrus varieties worldwide, causing reduced fruit marketability and tree death, with no cure available. The disease has decimated Florida's annual 9-billion-dollar citrus industry and spreads in California and other citrus-growing states. The bacterium associated with citrus greening disease, referred to as Candidatus Liberibacter asiaticus (CLas), is spread from tree to tree by the Asian citrus psyllid, a process called transmission. Transmission of CLas by the Asian citrus psyllid is a three-step process. The first step is called acquisition and involves the movement and uptake of the bacteria into the psyllid's body following ingestion from an infected citrus tree. ARS scientists in Ithaca, New York, described a method to study CLas acquisition which allows scientists to study this step in detail for the first time. The method reduces the time needed to study CLas acquisition from months or years to three weeks.

6. A punch in the gut: Transmission mode of grapevine red blotch virus by the three-cornered alfalfa hopper revealed. In California, grapevine red blotch disease was recognized as a new threat to viticulture in the mid to late 2000s. Grapevine red blotch virus was identified as the causative agent in diseased vines. The virus causes red and discolored blotches on the leaves, reduces grape quality, wine quality, and lowers yield. The virus is spread within a vineyard by a tiny sap-sucking insect called the three-cornered alfalfa hopper. ARS researchers in Ithaca, New York, in collaboration with partners from Cornell University revealed how the three-cornered alfalfa hopper transmits the virus. Grapevine red blotch virus was discovered to circulate throughout and persist in the body of the insect prior to transmission to a new host. Extended periods of time were required for virus uptake into the hopper gut. Different plant species other than grapes had impacts on the ability of the hopper to spread the virus, underscoring the importance of further research on the transmission of this virus in vineyards.

7. Not all whiteflies pose equal threats as vectors of plant viruses. Since first described over 100 years ago, the whitefly Bemisia tabaci has become an agricultural pest distributed worldwide. Its importance stems from its extreme invasiveness. B. tabaci causes direct cosmetic damage to various crops during feeding and by the growth of sooty mold fungus in its sugar-rich honeydew secretions on plants. However, the most severe damage caused by B. tabaci is due to its ability to spread plant viruses. B. tabaci is a vector for >100 different plant viruses, primarily the plant-infecting begomoviruses. ARS scientists in Ithaca, New York, in collaboration with collaborators at the University of Washington and the Volcani Center in Israel discovered that nine different B. tabaci populations collected in Croatia and Israel vary in their ability to transmit or spread, the begomovirus Tomato yellow leaf curl virus. Some populations spread the virus very efficiently, while other populations did not spread the virus readily between plants. Differences in B. tabaci proteins involved in virus transmission were found among these populations. Understanding the B. tabaci proteins that regulate virus transmission will lead to the development of novel strategies that block virus spread within a crop.

Review Publications
Heck, M.L., Neely, B. 2020. Proteomics in non-model organisms: A new analytical frontier. Journal of Proteome Research. 19:3595-3606.
Ammar, E., Heck, M.L., Shatters, R.G. 2020. Asian citrus psyllid: biology, ecology and management of the huanglongbing vector. Book Chapter. 127-155.
Krasnoff, S., Howe, K.J., Heck, M.L., Donzelli, B. 2020. Siderophores from the Entomopathogenic Fungus Beauveria bassiana. Journal of Natural Products. 83(2):296-304.
Igwe, D.O., Higgins, S.A., Heck, M.L. 2021. An excised leaf assay to measure acquisition of “candidatus liberibacter asiaticus” by psyllids associated with citrus Huanglongbing disease. Phytopathology. in press.
Kruse, A., Fleites, L.A., Heck, M.L. 2019. Lessons from one fastidious bacterium to another: What can we learn about Liberibacter species from Xylella fastidiosa. Insects. 10(9):300.
Wilson, J.R., Deblasio, S.L., Alexander, M.M., Heck, M.L. 2019. Looking through the lens of ‘omics technologies: Insights into the transmission of insect vector-borne plant viruses. Insect Molecular Biology. 6(34):113-144.
Wilson, J.R., Deblasio, S.L., Alexandr, M.M., Heck, M.L. 2019. Looking through the lens of ‘omics technologies: Insights into the transmission of insect vector-borne plant viruses. Current Issues in Molecular Biology. 34:113-144.
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.
Osterbann, L.J., Hoyle, V., Curtis, M., Deblasio, S.L., Rivera, K., Heck, M.L., Fuchs, M. 2021. Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of Nicotiana benthamiana by affinity purification and tandem mass spectrometry. Journal of General Virology. 102:001607.
Fleites, L.A., Johnson, R., Kruse, A.R., Nachman, R.J., Hall, D.G., Maccoss, M., Heck, M.L. 2020. Peptidomics approaches for the identification of bioactive molecules from Diaphorina citri . Journal of Proteome Research. 19/4; 1392-1408.
Kliot, A., Johnson, R., Maccoss, M., Kontsedalov, S., Lebedev, G., Czosnek, H., Heck, M.L., Ghanim, M. 2020. A proteomic approach reveals possible molecular mechanisms and roles for endosymbiotic bacteria in begomovirus transmission by whiteflies. Gigascience. 9:1-10.
Hosseinzadeh, S., Higgins, S.A., Ramsey, J.S., Howe, K.J., Griggs, M., Castrillo, L.A., Heck, M.L. 2021. Proteomic polyphenism in color morphotypes of diaphorina citri, insect vector of citrus greening disease. Journal of Proteome Research. 20(5):2851-2866.
Osterbaan, L., Choi, J., Kenney, J., Flasco, M., Vigne, E., Schmitt-Keichinger, C., Rebelo, A., Heck, M.L., Fuchs, M. 2019. The identify of a Single Residue of the RNA-dependent RNA Polymerase of Grapevine Fanleaf Virus modulates vein clearing symptoms in Nicotiana benthamiana. Molecular Plant-Microbe Interactions. 32:7.
Huang, W., Reyes-Caldas, P., Mann, M., Seifbarghi, S., Kahn, A., Almeida, R., Beven, L., Heck, M.L., Hogenhout, S., Coaker, G. 2020. Bacterial vector-borne plant diseases: unanswered questions and future directions. Molecular Plant.
Flasco, M., Hoyle, V., Cieniewicz, E., Roy, B., Mclane, H., Perry, K., Loeb, G., Nault, B., Heck, M.L., Fuchs, M. 2021. Grapevine red blotch virus is transmitted by the three-cornered alfalfa hopper in a circulative, nonpropagative mode with unique attributes. Phytopathology.
Chin, E., Ramsey, J.S., Mishchuk, D.O., Saha, S., Mitrovic, E., Chavez, J.D., Howe, K.J., Zhong, X., Polek, M., Godfrey, K.E., Mueller, L.A., Bruce, J.E., Heck, M.L., Slupsky, C.M. 2019. Longitudinal transcriptomic, proteomic, and metabolomic analyses of Citrus sinensis (L.) Osbeck graft-inoculated with ‘Candidatus Liberibacter asiaticus’. Journal of Proteome Research. 19(2):719-732.
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.
Saha, S., Hosmani, P.S., Villalobos-Ayala, K., Miller, S., Shippy, T., Flores, M., Rosendale, A., Shatters, R.G., D'Elia, T.D., Brown, S.J., Hunter, W.B., Heck, M.L. 2019. Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community. Database: The Journal of Biological Databases and Curation. 2019.
Haarith, D., Kim, D., Chen, S., Bushley, K.E. 2021. Growth chamber and greenhouse screening of promising in vitro fungal biological control candidates for the soybean cyst nematode (Heterodera glycines). Biological Control. 160:104635.
Milbrath, L.R., Dolgovskaya, M., Volkovitsh, M., Sforza, R.F., Biazzo, J. 2019. Photoperiodic response of Abrostola asclepiadis (Lepidoptera: Noctuidae), a candidate biological control agent for swallow-worts (Vincetoxicum, Apocynaceae). Great Lakes Entomologist. 52(2).
Milbrath, L.R., Biazzo, J. 2020. Demography of meadow and spotted Knapweed populations in New York. Northeastern Naturalist. 27(3):485-501.
Biazzo, J., Milbrath, L.R. 2019. Response of pale swallowwort (Vincetoxicum rossicum) to multiple years of mowing. Invasive Plant Science and Management. 12(3):169-175.
Ditommaso, A., Milbrath, L.R., Marschner, C.A., Morris, S.H., Westbrook, A.S. 2020. Seed germination ecology of meadow knapweed (Centaurea x moncktonii) populations in New York State, USA. Weed Science. 69(1):111-118.