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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 #430048

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

Location: Emerging Pests and Pathogens Research

2018 Annual Report

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.

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 ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) continues to provide the following services: 1) fungal culture deposition; 2) distribution; and 3) identification. Since July 2017 we accessioned to the collection 225 fungal isolates of scientific, ecological and/or economic value that were collected from the U.S. and four other countries. These include deposits of newly described species and new isolations from economically important insect pests. A revised, updated catalog, listing over 13,300 accessions in the collection, was compiled and uploaded to the ARSEF/Mycology website. The website was also updated to list new distribution policies and contact information. Over 500 isolates are ready for shipment to the National Center for Genetic Resources in Ft. Collins, Colorado and will be sent once viability of all new accessions have been verified. A review of our cryopreservation methods is underway to optimize maintenance of frequently requested species and facilitate their distribution. A total of 553 isolates were shipped in response to 53 requests from US and international clientele (representing eight countries) to sustain a range of research and development activities. We also provided fungal identification services by examining morphological characters and/or by sequencing of diagnostic loci, often requiring the isolation and establishment of pure fungal cultures from insect cadavers. Updating the user interface for the ARSEF database is underway to reflect current demands. This is an iterative multi-year process between the (acting) curator and ARS computational biologists. In collaboration with ARS researchers at Ithaca, New York and Hilo, Hawaii, a survey on the genetic diversity of Beauveria bassiana and other fungi associated with coffee berry borer in Hawaii, was completed, Projects on fungal pathogens of insect pests of citrus, cotton and sorghum were initiated with ARS researchers at Fort Pierce, Florida, Byron, Georgia, and Tifton, Georgia, respectively. The hiring of a temporary curator has enabled reaching the service milestones for ARSEF. The hiring freeze has prevented advertising and filling the scientist position, and as a result, research activities associated with the ARSEF collection have been largely curtailed. Objective 2: The viruses in the family Luteoviridae infect many staple food crops. They are exclusively transmitted by aphids in nature, and there is no cure once they infect a plant. Understanding how these viruses move within their aphid vectors and within the vascular system of their plant hosts offers opportunities to develop strategies to block or interfere with virus movement and prevent infection. These viruses make four proteins known to regulate their movement in plants: two structural proteins that comprise the shell of the virus and two non-structural proteins, P3a and P17. Very little is known about how these proteins interact with each other and with host proteins to coordinate virus movement within and between plants cells. Our ongoing work using affinity purification-mass spectrometry showed that the P3a protein, in association with the P17 protein can complex with virus particles. Using methods that enable scientists to visualize the interactions of proteins in living plant cells, P3a and P17 were shown to self-interact as well as directly interact with each other. Further studies showed that P3a traffics and directs P17 to the mitochondrial outer membrane, where cellular respiration occurs, while P17 regulates the localization of the P17-P3a heterodimer to plastids, where the plant makes and stores compounds important for photosynthesis. Trafficking of these organelles to the cell periphery brings these complexes in close but transient proximity to plasmodesmata where dispersal of P17 towards plastids can be observed in real-time. This work is the first to reveal the dynamics of how any virus in this family hijacks plant cell machinery during infection for intracellular movement. A second focus of research is on aphid vector-virus interactions regulating virus transmission. A correlation between serotype and vector specificity has been observed since the early days of studying the Luteoviridae, before molecular information about the structure was available. In collaboration with Cornell University partners, ARS scientists in Ithaca, New York produced the first ever structure of any part of the luteovirid capsid, a region of the capsid required for virus transmission by aphids. Ongoing work is focused on validating the structure and determining its usefulness as a tool for plant bioengineering. The Asian citrus psyllid (ACP), is a major pest in most citrus-growing regions and the vector of the bacterial pathogen that causes Huanglongbing (HLB) or citrus greening disease. ACP harbors at least three beneficial microbial partners, referred to as bacterial endosymbionts, whose roles in ACP biology are not well understood. ARS scientists in Ithaca, New York investigated the levels of the endosymbionts in different ACP tissues, in each sex, and in ACP reared on healthy citrus and HLB-infected citrus. We found that female ACP may benefit from carrying the HLB pathogen whereas the HLB pathogen may be detrimental to male insects. These results may indicate that HLB management strategies to block transmission may have different effects on males and female insects. The transmission of the bacteria responsible for HLB by ACP involves the bacteria getting into and replicating in various ACP tissues, including the blood. High resolution quantitative mass spectrometry was performed to investigate the effect of the bacteria on the proteins in ACP blood. Our results show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction. ARS scientists have been developing a research program to develop ACP that do not transmit the HLB bacterium as part of a new management program to control the spread of HLB. They collected psyllid male and female pairs from throughout the state of Florida and have maintained these lines in the lab for over two years. The results show that different psyllid populations vary in their ability to spread CLas. Some populations spread the pathogen very efficiently and others not at all. The ability or inability to transmit the pathogen is passed on from parents to offspring, proving that psyllid genes regulate tree-to-tree spread of the pathogen. These populations will enable scientists to develop new ways of blocking the spread of citrus greening by preventing psyllid spread of the citrus greening pathogen. ARS scientists have collaborated with a non-profit organization to develop a You Tube video focused on explaining the importance of this research to stakeholders in the citrus industry and the general public. Sublines are being generated from the original parent lines to reduce the genetic complexity of each line for genetic analysis. Strikingly, some of the non-transmitting lines show higher levels of transmission in recent tests. One difference between the previous work and now is the host plant of the insect. In collaboration with ARS scientists in Fort Pierce, Florida, we are initiating studies to investigate the effect of the host plant on CLas transmission by D. citri. Objective 3: Significant progress was made in the selection and expression of protein targets for the development of strategies that block the spread of HLB by ACP. In collaboration with scientists from the Boyce Thompson Institute, Kansas State University, Indian River State College and the USDA-ARS in Fort Pierce, Florida, ARS scientists in Ithaca, New York have generated a very high quality genome sequence for the ACP. The genome sequence data enabled ARS scientists to identify proteins in the ACP salivary sheath, which it uses to establish feeding sites on citrus plants. The ACP genes encoding these proteins are being used to produce large amounts of purified salivary sheath proteins. The purified proteins will be used for the development of small molecules that will bind to the proteins with high specificity and affinity, resulting in blocking their function and preventing psyllids from feeding. In collaboration with USDA-ARS scientists in Florida, we are optimizing methods to deliver these small molecules into citrus trees so that growers may use them in their groves as a novel insecticide to prevent psyllids from feeding and spreading the HLB bacterium.

1. Genetic diversity of bacteria associated with coffee berry borer beetle in Hawaii. This beetle is the most important insect pest of coffee, and current control methods rely on sanitation practices and spraying of a B. bassiana-based mycoinsecticide. Field efficacy studies of this mycoinsecticide in Hawaii by ARS researchers from Ithaca, New York, and Hilo, Hawaii, revealed presence of indigenous populations of B. bassiana, that under optimal environmental conditions can result in over 40% beetle mortality. Multilocus sequencing of over 180 indigenous Hawaiian isolates by ARS researchers at Ithaca, New York, revealed a very diverse population of this fungus. These results are essential in the selection of representatives of indigenous genotypes for their amenability to biocontrol and mass production. The more prevalent indigenous isolates with high virulence to coffee berry borer adults offer alternatives to the commercial strain of B. bassiana currently registered for use in Hawaii.

2. Natural variation in acquisition of citrus greening. A study conducted by ARS researchers in Ithaca, New York and Fort Pierce, Florida successfully identified genetic diversity in Asian citrus psyllid populations in genes regulating transmission of the citrus greening pathogen. Understanding how citrus greening is transmitted is important for the development of management strategies that target efficient vector populations. This work achieved a major goal of the USDA Specialty Crops Program focused on Citrus Greening Disease, which had been outlined in the 2010 National Academy of Sciences Report on Citrus Greening Disease: the development of Asian citrus psyllid lines that do not transmit the citrus greening bacterium.

3. Cracking the genetic code of the Asian citrus psyllid, the insect vector of citrus greening bacterium. In collaboration with a consortium of scientists from multiple institutions, ARS scientists in Ithaca, New York and Fort Pierce, Florida produced the most complete genome sequence information for the Asian citrus psyllid, the insect vector that spreads the Huanglongbing (HLB) bacterium. The high quality data enables scientists to tackle pesticide resistance and block transmission of the HLB bacterium. The team created a free data analysis tool called the Psyllid Expression Network, or PEN for short, which allows researchers studying psyllids to easily visualize genome data and the impact of HLB on the psyllid. The tool will be useful to ARS and other scientists studying other similar insects, for example the potato psyllid which spreads the zebra chip bacterium of potato.

4. The strategies viruses use to infect plants are revealed. Viruses cause some of the world’s most devastating crop diseases. Plant viruses encode very tiny genomes and these genomes are packed with information the virus needs to infect a plant and spread to a new host plant. The genome of most plant viruses are made up of RNA, and this RNA encodes information that is translated into proteins, which are the molecular machines viruses use to infect the plant. In collaboration with scientists at the University of Washington and Iowa State University, ARS scientists discovered how plant viruses use a process called “translational readthrough” – a first for the plant virology field. Translational readthrough is a strategy used by viruses to make different, longer, multifunctional proteins from the same gene sequence. ARS scientists showed how translational readthrough is regulated and cracked the code used by one family of plant viruses. This information is critical to the development of novel forms of virus resistance in plants, as there are no available cures for plant virus infection.

Review Publications
Ammar, E., Hall, D.G., Hosseinzadeh, S., Heck, M.L. 2018. The quest for a non-vector psyllid: Natural variation in acquisition and transmission of the huanglongbing pathogen 'Candidatus Liberibacter asiaticus' by Asian citrus psyllid isofemale lines. PLoS One. 13(4):e0195804.
Mann, M., Fattah-Hosseini, S., Ammar, E., Strange, R., Warrick, E., Sturgeon, K., Shatters, R.G., Heck, M.L. 2018. Diaphorina citri nymphs are resistant to morphological changes induced by “Candidatus Liberibacter asiaticus” in midgut epithelial cells. Infection and Immunity. 86(4):e00889-17.
Morris, E., Stock, S., Castrillo, L.A., Williams, D., Hajek, A. 2018. Characterization of a novel dimorphic Deladenus beddingi n. sp. and its associated woodwasp and fungus. Nematology.
Castrillo, L., Mayfield, A., Griggs, M., Camp, R., Mudder, B., Taylor, A., Vandenberg, J. 2017. Mortality and reduced brood production in walnut twig beetles, Pityophthorus juglandis (Coleoptera: Curculionidae), following exposure to commercial strains of Beauveria bassiana and Metarhizium brunneum. Biological Control. 114:79-86.
Fisher, J.J., Castrillo, L.A., Donzelli, B., Hajek, A.E. 2017. Starvation and imidacloprid exposure influence immune response by Anoplophora glabripennis (Coleoptera: Cerambycidae) to a fungal pathogen. Journal of Economic Entomology. 110(4):1451-1459.
Castrillo, L.A. 2017. The Host Population-Chapter 4. In: Hajek, A.E., Shapiro-Ilan, D.I., editors. Ecology of Invertebrate Diseases. Oxford, UK: John Wiley & Sons Ltd. p. 101-141.
Ngatat, S., Hanna, R., Kumar, P.L., Gray, S.M., Cilia, M., Ghogomu, R.T., Fontem, D.A. 2017. Relative susceptibility of Musa genotypes to banana bunchy top disease in Cameroon and implication for disease management. Crop Protection. 101:116-122.
DeBlasio, S.L., Bereman, M.S., Mahoney, J., Thannhauser, T.W., Gray, S.M., MacCoss, M.J., Heck, M.L. 2017. Evaluation of a bead-free immunoprecipitation technique coupled with tandem mass spectrometry for identification of plant-virus protein interactions. Journal of Biomolecular Techniques. 28(3):111-121.
Xu, Y., Ju, H., DeBlasio, S.L., Carino, E.J., Johnson, R., MacCoss, M., Heck, M.L., Miller, W., Gray, S.M. 2018. A stem-loop structure in potato leafroll virus ORF5 that is essential for readthrough translation of the coat protein ORF stop codon 700 bases upstream. Journal of Virology.
Alexander, M., Mohr, J.P., DeBlasio, S.L., Chavez, J., Ziegler-Graff, V., Brault, V., Bruce, J., Heck, M.L. 2017. Insights in luteovirid structural biology guided by chemical cross-linking and high resolution mass spectrometry. Virus Research. 241:42-52.
Sangireddy, S., Okekeogbu, I., Ye, Z., Zhou, S., Howe, K.J., Fish, T., Thannhauser, T.W. 2017. Effects of Al3+ and La3+ trivalent metal ions on tomato fruit proteomes. Proteomes. 5(1):7. doi:10.3390/proteomoes5010007.
Cook, D., Donzelli, B., Creamer, R., Baucom, D.L., Gardner, D.R., Pan, J., Moore, N., Krasnoff, S., Jaromczyk, J.W., Schardl, C.L. 2017. Swainsonine biosynthesis genes in diverse symbiotic and pathogenic fungi. G3, Genes/Genomes/Genetics. 7(6):1791-1797.
Szebenyi, D., Kriksunov, I., Howe, K.J., Ramsey, J.S., Hall, D.G., Heck, M.L., Krasnoff, S. 2018. Crystal structure of diaphorin methanol monosolvaate isolated from diaphorina citri kuwayama, the insect vector of citrus greening disease. Acta Crystallographica Secion E. 74(Pt 4):445-449.