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

Location: Emerging Pests and Pathogens Research

2023 Annual Report

Objectives
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.

Approach
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: ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) provided the following services: fungal culture deposition; distribution of isolates; and identification by examining morphological characters and/or DNA sequencing. A total of 29 requests were shipped (171 isolates) to 10 US institutions, 6 ARS facilities, and 13 foreign institutions. Two shipments of 500 isolates were sent as backup stocks to the Agricultural Genetic Resources Preservation Research Center in Colorado. The database housing records of all ARSEF accessions and associated metadata (ALICE) was updated to version V19.5 to keep current with operating systems and functionalities for adding DNA sequence and image data were added. A new catalogue was created and made available. Research activities included analysis of 14 genomes in genus Beauveria for taxonomic relationships and secondary metabolite gene content, isolation and identification new fungi from significant invertebrate pathogens: 1) emerald ash borer (EAB; ~100 isolates), 2) fall army worm (FAW; ~100 isolates), soybean cyst nematode (SCN;~50 isolates), and Asian giant hornet (AGH;~25 isolates), and screening fungi for parasitism and other biocontrol activity. Approximately 20% of fungi from EAB galleries or frass (~20 isolates) were identified as known entomopathogens. A manuscript evaluating pathogenicity of Beauveria sp., Lecanicillium sp., and Akanthomyces sp., among others, in parasitism assays against EAB eggs is in preparation and an initial field study spraying fungal spore suspensions onto ash bark to evaluate their effect on egg hatch, larval colonization, and larval and adult mortality was conducted. Microbiome sequencing was also completed for several of these invertebrate pathogens to characterize symbionts that may not be easily cultured but could be targeted with specialized media. With a new grant from ARS-Federal-State Potato Partnership, nematode parasitic fungi isolated from SCN that strongly antagonized SCN were screened against the potato cyst nematode. This research provides a better understanding of entomopathogenic fungi naturally associated with these pathogens that could be harnessed for biological control. Objective 2: Ambrosia beetles: We continued to investigate the basic biology of two ambrosia beetle species, Xylosandrus germanus (black stem borer) and Xylosandrus crassiusculus (granulate ambrosia beetle), wood-boring insects that are widespread pests in orchard and ornamental production systems, although currently little to no apple decline or associated ambrosia beetle damage in apple are being reported. Black stem borer can be reared in sawdust-based diet tubes for 4 or more generations, but dormancy of beetles rapidly increases after 1 generation. Mass rearing in wood bolts is feasible but emerging beetles often attack the original bolt; beetle collection techniques are being explored. A two-year phenology study of the black stem borer is complete. We consistently observed two and a partial third summer generation (all overlapping) with beetle flight into late September. Some dormancy of black stem borer may begin in the first summer generation. We documented the development of granulate ambrosia beetle symbiont and brood production in cut beech bolts. Behavioral studies of this species document social behaviors (such as hygienic grooming) among gallery members, similar to black stem borer. However, social interactions are limited because we do not observe an overlap in generations within a gallery. A trapping study continues in collaboration with a Cornell University extension agent. Additional studies to better define the effectiveness and applications of entomopathogenic fungal products, including Beauveria bassiana, Metarhizium brunneum, Isaria fumosoroseus, and Trichoderma spp. have begun and will be carried out by a new post-doctoral research associate. Entomopathogenic fungi: We have redoubled our genome sequencing of insect-killing fungi, with sequences generated from species spanning the fungal tree of life. These genomes will enhance our understanding of fungal evolution and provide a foundation for their genetic modification using modern molecular techniques, such as CRISPR-Cas9. The generated genome sequence of Conoideocrella luteorostrata, a pathogen of the Christmas tree pest elongate hemlock scale, and deposition of this strain in ARSEF will facilitate its rapid development as a biopesticide for this industry. We are interrogating the cross-kingdom interactions between insect-associated microbes. For example, our team found mosquitoes infected with the symbiotic bacterium Wolbachia were less likely to carry the human malaria parasite in field populations. The microbiome of insect hosts is an essential consideration for insect pathogens, including biocontrol fungi and insect-vectored diseases. Sap-feeding insects, the primary vectors of plant disease, can domesticate their fungal pathogens: an arrangement in which a pathogen becomes a supportive partner living in specialized symbiotic organs. Since pest insects rely on such symbioses to survive, these events represent a broad opportunity to disrupt insect health. We sequenced the genome of a yeast-like symbiont of leafhoppers, which is a domesticated pathogen. Unlike most identified strains, this fungus can be grown in the lab, allowing further interrogation of this system. These genomes support our investigation of host-pathogen interactions using direct observational techniques, such as thin slicing and staining of insect tissue to describe infection. We reported recently on the development of Massospora cicadina infection in periodical cicadas using these approaches. Poleroviruses: Research has focused on the structure of the RTD (readthrough domain) on the virion and the basis of its interaction with presumed receptors in the aphid that facilitate transmission. ARS scientists found that the RTD is the evolutionary predecessor of tombusvirus coat proteins. The RTD contains a novel “cap” domain which they demonstrated regulates the aphid transmission function unique to poleroviruses, and the related enamo and luteoviruses and is absent in tombusvirids. Research is ongoing to identify the RTD aphid receptors for potato leafroll virus (PLRV). We are evaluating the ability of RTD transgenic potato to block aphid transmission of PLRV in large-scale polyhouse experiments. The immediate translational applications of this work resulted in a patent filing and attracted major stakeholders like the Cotton Board, which has invested funds to expand these technologies to mitigate the emerging cotton leafroll dwarf virus (CLRDV) in cotton. Research has focused on developing reliable diagnostic RT-PCR assays and serological diagnostic assays for CLRDV. The team constructed an infectious clone of the Argentinian isolate and construction of a wild type clone of the Mississippi isolate is in progress. A total of five cotton aphid genotypes from different geographic locations were collected and are in culture. Vector competence of the clones is being tested. Characterization of virus populations in CLRDV-infected plants is underway. A new caulimovirus, CotV-A, has been discovered in upland cotton with integrations into the cotton genome. As a result of this finding, funds have been obtained through the Department of Homeland Security PANTHR program for additional sequencing in cotton. Objective 3: Insect-killing fungi are ideal candidates for expression of compounds that interfere with host health and transmission of pathogens. Biocontrol fungi can be modified as research tools and as applied biotechnology, expressing proteins, metabolites or nucleotides to alter host health and gene expression. Our team is engaged in the construction of plasmids for fungal genetic modification, the refinement of fungal modification techniques and the development of candidate effectors designed to target insect hosts. We developed an excised leaf assay to quantify acquisition of the citrus greening bacterium. This assay was used to discover five plant-derived peptides that inhibit the insect spread of the citrus greening bacterium. A paper and a pre-print were published and a provisional patent was submitted. To discover additional bioactive peptides, we extracted protein from several Asian Giant Hornet (AGH) venom glands to characterize the venom proteome via mass spectrometry. A filtration method was developed to characterize the small peptides, referred to as the peptidome, of the AGH venom gland. These data have been analyzed and a manuscript is in preparation. The AGH venom peptidome was rich source of new bioactive peptides, including putative antimicrobial peptides. We synthesized and screened a subset of these peptides against the culturable relative of the citrus greening bacterium and found several that strongly inhibited the growth of Liberibacter. An invention disclosure has been submitted. Therapeutic peptides may prove to be too expensive for growers, especially in Florida where the economic impacts of citrus greening are severe. ARS scientists conceptualized a new way to cheaply deliver peptides to trees via the use Agrobacterium. The team hypothesized that adding back the plant growth regulator genes into the T-DNA together with a gene of interest would result in a transgenic, gall-like structure, referred to as a Symbiont, a technology that is now patented and undergoing licensing with a CRADA partner. Excitingly, in a proof-of-concept experiments, greenhouse evaluations of Symbionts that express antimicrobial peptides eliminate citrus greening symptoms and the bacteria in potted citrus trees. An ARS scientist in Ithaca, New York, is leading field trials to evaluate the technology for controlling citrus greening in Florida.

Accomplishments
1. Isolated and identified entomopathogenic fungi from the highly destructive and globally invasive insect, the fall army worm (Spodoptera frugiperda). Fall army worms are highly destructive invasives, yet little is known about their microbial symbionts that could be harnessed or targeted for biological control. In collaboration with University researchers, ARS scientists at Ithaca, New York, characterized the fungal microbiome of S. frugiperda. Fungi cultured from this insect were accessioned into the ARS entomopathogenic fungi collection. Identification of new fungi provides options for biological control and management of fall army worms.

2. Identified several isolates producing filtrates highly bioactive against potato cyst nematode. Cyst nematodes are persistent and damaging pests of many agricultural crops. Methods of control remain limited, with genetic sources of resistance losing efficacy, and crop rotations being economically nonviable. We obtained funding from ARS (ARS State Partnership Potato Program) to screen fungi previously isolated from the soybean cyst nematode against the potato cyst nematode and were awarded a new contract with stakeholder groups in New York, (NY Corn and Soybean Growers) for FY 2023 to identify new fungi native to NY from soybean cyst nematode. The research on potato cyst nematode identified two fungi producing filtrates with high toxicity to the potato cyst nematode that can be explored as soil drench or seed coat applications to control this pest.

3. Herbicidal control of black swallow-wort. Black swallow-wort (Vincetoxicum nigrum) is a European twining vine that was introduced into eastern North America. Herbicides can be an effective management tool for invasive plants but had not been studied for black swallow-wort. Scientists from ARS in Ithaca, New York, and Cornell University evaluated glyphosate (two products) and triclopyr, with or without mowing. Both glyphosate products greatly reduced black swallow-wort biomass as well as cover and stem densities, but mowing several weeks before spraying did not always increase the effectiveness of herbicide treatments. Triclopyr was ineffective. Repeated, single applications of glyphosate can be useful for the management of black swallow-wort.

4. Knapweed population model helps guide control efforts. The European perennial plants spotted knapweed (Centaurea stoebe subsp. micranthos) and the hybrid meadow knapweed (C. x moncktonii) are invasive in grasslands and pastures across North America, including in the Northeast. The two species may develop similarly and therefore be controlled by targeting the same life stages. Scientists from ARS in Ithaca, New York, and the University of Vermont developed northeastern plant population models of the two species that identified key points of the plants’ life cycles to be targeted. Both species have similar life histories and therefore both species will likely be controlled through increased mortality of older seedlings and juvenile plants as well as delaying the maturation of vegetative individuals. Using this information, specific chemical, mechanical and biological control tactics can be developed for these invasive knapweeds.

5. Development of innovative delivery methods for insect-killing fungi. Consistent delivery of spores in laboratory and field conditions is a persistent challenge for successful evaluation and application of insect-killing fungi. ARS sientists at Ithaca, New York, developed a laboratory bioassay to briefly expose and maintain ambrosia beetles on artificial medium for screening virulence of potential biocontrol fungi. These methods provide a pathway for collaboration with university and ARS partners to rapidly screen entomopathogenic fungi against emerging invasive insect pests.

6. Discovery of a new virus infecting upland cotton. The United States is the world’s third largest producer and the leader in cotton exports. Cotton leafroll dwarf virus (CLRDV) is an emerging RNA viral pathogen of cotton transmitted in the field by the cotton aphid. Viral symptoms in the field have been challenging to understand and pinpoint to CLRDV alone. ARS scientists in Ithaca, New York, hypothesized that other plant viruses may be co-infecting cotton with CLRDV. Using different molecular and computational tools, ARS scientists in partnership with university colleagues discovered a new DNA virus infecting cotton growing in Mississippi and tentatively named the virus cotton virus A (CotV-A). Similar to other DNA plant viruses, they discovered that copies of the CotV-A genome are inserted into the genome of upland cotton, which may give rise to virus infection. Importantly, the team developed a method to distinguish between the real CotV-A infection and copies of CotV-A in the cotton genome by specifically degrading the virus copies in the cotton genome prior to a diagnostic test. The cotton industry was informed about the new virus and a paper has been published. A new grant was funded by the Department of Homeland Security to determine how widespread CotV-A is in upland cotton production in the United States. Additional research is needed to determine whether CotV-A can cause detrimental effects in cotton production, if the virus copies inserted in the upland cotton genome can initiate a real virus infection or whether the virus is transmitted by insects.

7. Plant-based peptides block insect transmission of the citrus greening disease bacterium. Citrus growers need effective strategies for managing citrus greening disease, also known as Huanglongbing (HLB). The presence and, therefore, the likely spread, of HLB within California are a major concern for growers because of the mobility of the Asian citrus psyllid (ACP), the insect vector of citrus greening in the United States. In Florida, the disease is endemic and growers need treatments to mitigate infection in existing groves and prevent transmission into new plantings. Organic growers, in particular, seek HLB management strategies that do not rely on synthetic chemicals. USDA ARS scientists discovered plant-based peptides that have the ability to thwart the spread of the HLB bacterium by the ACP. The work represents a collaboration among a team of USDA, University and industry partners and was funded by a CRADA with a small agri-businesss in Florida and a grant from the California Citrus Research Board, a consortium of growers in California. A patent has been filed and a preprint describing the peptides is available. These plant-based peptides have different killing modes of action that could effectively control citrus greening and/or prevent ACP from transmitting the pathogen to citrus trees.

8. Plants to the rescue from the next global pandemic. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pathogenic virus that causes severe respiratory syndrome in humans. SARS-CoV-2 is related to SARS-CoV-1 and Middle Eastern Respiratory Syndrome (MERS)-CoV, which emerged in humans in 2003 and 2012, respectively. SARS-CoV-2 is responsible for the 2019 pandemic and COVID-19 disease. COVID-19 disease results in a range of outcomes, ranging from asymptomatic infection to patient death. To date, global vaccinations for SARS-CoV-2 protections are underway, but additional treatments are needed to prevent infection among naïve and even vaccinated individuals. ARS scientists from Ithaca, New York, and Fort Pierce, Florida, together with a CRADA partner, demonstrated plant production of small, functional molecules, referred to as nanobodies, interfere with the molecular interactions required for virus spread in humans. Production of plant-based nanobody therapies to control virus spread represents a promising new development in the mitigation of the COVID-19 pandemic.

Review Publications
Adams, M., Schiltz, C., Heck, M.L., Chappie, J. 2021. Crystal structure of the potato leafroll virus coat protein and implications for viral assembly. Journal of Structural Biology. 214(1):107811. https://doi.org/10.1016/j.jsb.2021.107811.
Higgins, S., Mann, M., Heck, M.L. 2022. Strain tracking of ‘candidatus liberibacter asiaticus’, citrus greening disease pathogen, enabled by high-resolution microbiome analysis of the Asian citrus psyllid. Phytopathology. 112(11):2273-2287. https://doi.org/10.1094/PHYTO-02-22-0067-R.
Larrea-Sarmiento, A., Olmedo-Velarde, A., Preising, S., West-Ortiz, M., Fei, Z., Heck, M.L. 2023. Potato leafroll virus modulates aphid infection with a newly described insect flavivirus. Phytopathology. 125:102015. https://doi.org/10.1016/j.pmpp.2023.102015.
Olmedo-Velarde, A., Wilson, J.R., Stallone, M., Deblasio, S.L., Chappie, J.S., Heck, M.L. 2023. Potato leafroll virus molecular interactions with plants and aphids: gaining a new tactical advantage on an old foe. Physiological and Molecular Plant Pathology. https://doi.org/10.1016/j.pmpp.2023.102015.
Mann, M., Saha, S., Pitino, M., Moulton, K.M., Cano, L., Hunter, W.B., Mueller, L.A., Heck, M.L. 2022. Lessons learned about the biology and genomics of Diaphorina citri infection with “Candidatus Liberibacter asiaticus” by integrating new and archived organ-specific transcriptome data. Gigascience. 11:1-16. https://doi.org/10.1093/gigascience/giac035.
Kennedy, J.P., Wood, K., Pitino, M., Mandadi, K., Igwe, D., Shatters, R.G., Widmer, T.L., Niedz, R.P., Heck, M.L. 2023. A perspective on current therapeutic molecule screening methods against ‘candidatus liberibacter asiaticus’, the presumed causative agent of citrus Huanglongbing. Phytopathology. https://doi.org/10.1094/PHYTO-12-22-0455-PER.
Ramsey, J.S., Ammar, D., Mahoney, J.E., Rivera, K., Johnson, R., Igwe, D.O., Thannhauser, T.W., Maccoss, M.J., Hall, D.G., Heck, M.L. 2022. Host plant adaptation drives changes in Diaphorina citri proteome regulation, proteoform expression and transmission of Candidatus Liberibacter asiaticus, the citrus greening pathogen. Phytopathology. 112:101-115. https://doi.org/10.1094/phyto-06-21-0275-r.
Ramsey, J.S., Zhong, X., Saha, S., Chavez, J., Johnson, R., Mahoney, J., Keller, A., Moulton, K., Mueller, L., Hall, D.G., Maccoss, M., Bruce, J., Heck, M.L. 2022. Quantitative isotope-labeled cross-linker proteomics reveals developmental variation in protein interactions and post-translational modifications in diaphorina citri, the citrus greening insect vector. ACS Agricultural Science and Technology. 2(3):486-500. https://doi.org/10.1021/acsagscitech.1c00264.
Milbrath, L.R., Biazzo, J., Morris, S.H., Ditommaso, A. 2023. Response of black swallowwort (Vincetoxicum nigrum) to herbicides plus mowing. Invasive Plant Science and Management. 15:161-167. https://doi.org/10.1017/inp.2022.27.
Westbrook, A.S., Milbrath, L.R., Weinberg, J., Ditommaso, A. 2023. Biology of invasive plants 3: Vincetoxicum nigrum (L.) Moench and Vincetoxicum rossicum (Kleopow) Barbarich. Journal of Invasive Plant Science Management. 16:3-26. https://doi.org/10.1017/inp.2023.7.
Molofsky, J., Thom, D., Keller, S.R., Milbrath, L.R. 2023. Closely related invasive species may be controlled by the same demographic life stages. NeoBiota. 82:189-207. https://doi.org/10.3897/neobiota.82.95127.
Choi, J., Pakbaz, S., Yepes, L., Cieniewicz, E., Schmitt-Keichinger, C., Labarile, R., Minutillo, S., Heck, M.L., Hua, J., Fuchs, M. 2023. Grapevine fanleaf virus RNA1-encoded proteins 1A and 1BHel individually or cooperatively suppress RNA silencing. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-01-23-0008-R.