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

Location: Emerging Pests and Pathogens Research

2020 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
Final Report. New Project under OSQR review. Objective 1: ARSEF continues to provide the following services: 1) fungal culture deposition; 2) distribution; and 3) identification. ARSEF accessioned 199 new fungal isolates. 187 isolates were shipped in response to 36 requests from non-profit institutions in the US and abroad. ARSEF provided fungal identification services by examining morphological characters and/or by sequencing of diagnostic loci, oftentimes requiring establishment of pure fungal cultures. A subset of deposits are being processed for identification to genus or species via DNA sequencing. Data and services are publicly available on the ARSEF website. Catalogs of common fungal genus or insect host order have been compiled for the ARSEF website. Research focuses on fungal pathogens of Diaphorina citri, the Asian citrus psyllid, and vector of citrus greening disease, whitefly Biotype B, mosquitoes and the sugarcane aphid. A new agreement between ARSEF and the Agricultural Genetic Resources Preservation Research was approved to back up critical fungal strains. Sub-Objective 2.1: Identification of pathogen, host, and vector components that regulate uptake and transmission of pathogens by sap-sucking insects. Work focused on aphids that transmit the circulative, plant pathogenic poleroviruses. ARS scientists discovered that the P0 protein from potato leafroll virus (PLRV), a plant virus that is spread by the green peach aphid, suppresses the aphid immune system. The weakened aphid immune system makes the aphid more susceptible to an aphid-infecting virus, called a densovirus. Densoviruses induce the formation of winged aphids, which would enable the aphid to move plant viruses long distances. In host plants, P0 suppresses the silencing machinery by marking the protein argonaut 1 (AGO1) for degradation. P0 forms an E3 ubiquitin ligase complex in planta that ubiquitinates AGO1. Current research is focused on whether E3 ubiquitin ligase activity is involved in the effect of P0 on the aphid antiviral immune system. ARS scientists have shown that P0 can exert its effect on the aphid antiviral immune system in the context of the plant host. It is not known if P0 signals indirectly through the plant and whether the plant is required for this effect. Ongoing experiments are testing between the hypotheses as to whether P0 exerts its effect directly on the aphid or indirectly via the plant. Research has advanced on the viral structural protein that regulates aphid transmission. Data show that the PLRV structural protein that regulates aphid transmission forms a particular structure that is conserved among different poleroviruses, suggesting a conserved function in virus transmission by aphids. Mutant forms of the protein are lethal to aphids when delivered by an artificial diet. Research is focused on developing and optimizing transmission blocking strategies using this form of the virus structural protein, characterizing aphid proteins that bind to this protein and visualization of the virus at the atomic level. This work will generate new fundamental knowledge about aphid transmission of poleroviruses and help our team to optimize a strategy to block virus transmission to transfer to growers. An invasive polerovirus, Cotton leafroll dwarf virus (CLRDV) is an emerging threat to cotton grown in the United States. ARS scientists in Ithaca, NY are collaborating with ARS scientists in Stoneville, MS to translate the Potato leafroll virus management strategies to the new emerging cotton polerovirus. While there is a wealth of data on polerovirus biology as a result of studies on PLRV and the related yellow dwarf viruses, very little is known about the biology of CLRDV, its interactions with host cotton varieties and transmission by the cotton aphid, Aphis gossypii. The impact of this virus on cotton production is currently unknown, and management strategies are nonexistent. Together with a Cornell PhD student, the Lead Scientist traveled to the ARS location in Stoneville, MS to collect A. gossypii genotypes from cotton. Over 40 aphids were collected from various sites in Mississippi and Alabama. Two A. gossypii clones survived and are reproducing parthenogenetically under lab conditions. These clones will have their genomes sequenced next year as a part of the Ag100 Pests Project. Research is focusing on determining whether plants collected from the field are infected with CLRDV for aphid transmission studies and generation of a CLRDV infectious clone for laboratory research. Work on this sub-objective also focused the bacterium associated with citrus greening in the USA, Candidatus Liberibacter asiaticus (CLas). Liberibacter crescens is the only axenically cultured Liberibacter and serves as a surrogate for functional genomic studies of the pathogenic “Ca. Liberibacter” spp. All Liberibacters encode a complete repertoire of genes for flagella and Tad (Tight Adherence) pili. University cooperators and ARS scientists discovered that flagella and Tad pili were observed in L. crescens and functionally corroborated by swimming, twitching motility, DNA uptake and molecular biology. How motility contributes to infection and transmission of CLas is not yet known. Presymptomatic detection of citrus trees infected with CLas is critical to controlling the spread of the disease. Little is known about how tolerant vs. susceptible varieties respond to the infection and how the tree metabolism responds to infection over time. In collaboration with University partners, we compared the transcriptome (RNA-seq), proteome (LC-MS/MS), metabolome (1H NMR), and micronutrient profiles (ICP-MS) of graft-infected, CLas-tolerant Lisbon lemon (Citrus limon) and susceptible Washington navel orange (Citrus sinensis (L.) Osbeck), to understand differences in susceptibility to CLas. The findings highlight differences in response to CLas between two varieties with differing tolerances. This integrated approach to quantifying plant molecular changes in leaves of CLas-infected plants supports the development of diagnostic technology for presymptomatic detection to control HLB. Subobjective 2.2: Functional analysis of genes, proteins and metabolites involved in circulative plant pathogen transmission. Previously, ARS scientists showed that PLRV binds to an aphid protein, C1qBP, potentially involved in acquisition of PLRV by the green peach aphid. In other eukaryotes, C1qBP is known to be involved in anti-viral immunity. Experiments captured in vivo co-localization of PLRV and C1qbp in the aphid midgut epithelial cell membrane and support a role of C1qbp as a negative regulator of transmission and potentially performing an antiviral immune function. Acquisition and transmission of CLas by D. citri is influenced by insect development stage. D. citri nymphs and adults demonstrate distinct cellular and molecular responses to CLas exposure, and the bacterium can only be transmitted by an adult insect which has acquired the insect during its nymphal stage. CLas induces widespread programmed cell death in adult midgut tissue, the site of CLas acquisition, but not in nymphs. Isotope-labeled, protein interaction reporter (PIR) cross-linkers coupled to high resolution mass spectrometry analysis were used to identify and quantify protein interactions within nymph and adult D. citri. Notably, quantitative differences in histone posttranslational modifications and protein interactions regulating chromatin structure were identified between nymph and adult D. citri. Future work will focus on understanding how the changes in histone and chromatin structure in the adult psyllids relates to CLas-induced apoptosis. 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. Research has focused on the identification of natural, plant-derived antimicrobial compounds that could kill CLas. We identified multiple peptides produced by the legume plant, Medicago truncatula, that have antimicrobial activity; and, unexpectedly, we identified an entomopathogenic fungal peptide isolated from a cicada-infecting fungus that exhibited more potent antimicrobial activity than the legume-derived peptides. Our findings could offer growers a new and natural suite of options for preventing or treating citrus greening. 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. Research on the two bioactive neuropeptides that are insecticidal to psyllids is ongoing, including synthesis of biostable analogs, optimization of diet delivery methods, generating of transgenic citrus trees, and screening of additional peptides for insecticidal activity. Research with ARS scientists in Fort Pierce, Florida, and a CRADA partner has focused on development of a novel tree delivery strategy for citrus greening therapeutics. This strategy won an ARSX prize. Subobjective 3.2. Develop RNA aptamers that bind to transmission related compounds and test their ability to interfere with pathogen acquisition and transmission. ARS scientists and University partners discovered an RNA aptamer that inhibits psyllid feeding, deforms and reduces the number of sheaths deposited and binds to stylet components within the psyllid’s salivary gland. A method was published describing the use of RNA aptamers for proteomic analysis of protein complexes. Research is focused on experiments to secure the strongest possible patent. Developing an RNA aptamer biopesticide would be highly specific to sheath forming insects (psyllids, aphids, whiteflies, planthoppers, etc.) and could be delivered as a biopesticide suitable for organic production.

Accomplishments
1. Use of fungal pathogens to control of whitefly population on cotton. The whitefly is an important insect pest of many crop families, including cotton, cucumber, and cabbage. A study conducted in collaboration with scientists from ARS in Byron, Georgia, and the University of Georgia identified the fungus Isaria javanica, which kills whitefly on cotton in high numbers compared to commercial fungus used against whiteflies. This new fungus can be used for biological control whiteflies and other sap-sucking insect pests of agriculture crops.

2. Identification of peptide messengers that control the physiology of the citrus greening insect vector. ARS scientists in Ithaca, New York, published a comprehensive description of the Diaphorina citri peptidome, which included hundreds of molecules with classic signatures of biologically active peptides. This data is a rich resource for not only novel antimicrobials, which might be viable alternatives to antibiotics, but it also uncovered over 100 candidate insect neuropeptides. This work led to an HLB MAC initiative to screen a series of these neuropeptides, some of which have demonstrated insecticidal activity against other hemipteran insects, as new and environmentally-friendly pesticides for the management of D. citri in citrus producing areas.

3. Molecular profiling of citrus leaves for early detection of citrus greening disease. A collaboration involving ARS scientists in Ithaca, New York, and Riverside, California, has identified molecular changes in citrus leaves resulting from infection with the citrus greening bacterium. Leaf samples were collected biweekly from lemon and navel orange trees for a year following graft inoculation with either pathogen-containing or healthy budwood, and analyzed for biomarkers of infection. RNA, protein, and metabolite levels were compared over time between healthy and infected plants, revealing molecular profiles associated with infection months before visual symptoms of disease appeared. Results from this study reveal differences in the response to infection between two distinct varieties of citrus, and provide insight into how the plant response to the pathogen changes with time. These findings support the development of diagnostic tests for citrus greening disease which are designed to detect specific plant changes in response to pathogen infection. This high profile research was featured on the cover of the Journal of Proteome Research.

Review Publications
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. https://doi.org/10.1094/MPMI-12-18-0337-R.
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.
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. https://doi.org/10.1093/database/baz035.
Ghanim, M., Fattah-Hosseini, S., Levy, A., Heck, M.L. 2018. Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus. Scientific Reports. 6:33418. doi:10.1038/srep33418.
Luna, E., Van Eck, L., Campillo, T., Weinroth, M., Metcalf, J., Perez-Quintero, A.L., Botha, A., Thannhauser, T.W., Pappin, D., Tissarat, N.A., Lapitan, N.L., Argueso, C.T., Ode, P.J., Heck, M.L., Leach, J.E. 2018. Bacteria associated with Russian Wheat Aphid (Diuraphis noxia) enhance aphid virulence to wheat. Phytobiomes Journal. https://doi.org/10.1094/PBIOMES-06-18-0027-R.
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. https://doi.org/10.21775/cimb.034.113.
Heck, M.L., Brault, V. 2018. Targeted disruption of aphid transmission: A vision for the management of crop diseases caused by Luteoviridae members. Current Opinion in Virology. 33:24-32. https://doi.org/10.1016/j.coviro.2018.07.007.
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. https://doi.org/10.21775/cimb.034.113.
Yin, D., Sun, D., Norris, A.M., Han, Z., Ni, D., Jiang, C. 2019. PhERF2, an ethylene-responsive element binding factor, plays an essential role in waterlogging tolerance of petunia. Horticulture Research. 6:83. https://doi.org/10.1038/s41438-019-0165-z.
Ugine, T., Krasnoff, S., Grebenok, R., Behmer, S., Losey, J. 2018. Prey nutrient content creates omnivores out of predators. Ecology Letters. 22:275-283.
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. https://doi.org/10.3390/insects10090300.
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. https://doi.org/10.1021/acs.jproteome.9b00509.