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





- Heck, Michelle
- Milbrath, Lindsey
- Bushley, Kathryn
- Lovett, Brian
- 2023
- 2022
- 2021
- Publications
- Crop Protection & Quarantine (304)

Research Project: Management and Biology of Arthropod Pests and Arthropod-borne Plant Pathogens **Location:
                            Emerging Pests and Pathogens Research** 2022 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) has continued to provide the following services: 1) fungal culture deposition; 2) distribution of isolates; and 3) identification through examining morphological characters and/or sequencing of diagnostic loci or genomes. Between July 1, 2021 and June 30, 2022, 142 new isolates were accessioned to the collection. We shipped out 31 requests (218 isolates) to 10 U.S. states (California, Connecticut, Kansas, Maryland, Massachusetts, Michigan, New Jersey, New Mexico, New York (3), and Texas) and 12 foreign countries (Austria, Belgium, Brazil, Canada, Germany (2), Greece, India, Italy, Slovakia, Tanzania, Thailand, and UK (5). The database software system was brought into compliance with modern operating systems and will enable development of a new web interface to simplify ordering, accession isolates and facilitate stakeholders searches of the collection. The ARSEF catalog was updated and made available to stakeholders through the website. Research experimental activities focused on isolation and identification of new isolates associated with two invasive insect pests: 1) emerald ash borer and 2) Asian giant hornet. Computational research on the collection has analyzed the genomes of species within the entomopathogenic fungal genus Beauveria to both better characterize phylogenetic relationships within the genus and to identify genes involved in production of chitinases, proteases, or secondary metabolites involved in virulence against insects. Analyses of lyophilization experiments and storage times of selected ascomycete fungal accessions were also performed to identify viable isolates with the goal of creating an alternative in-house backup of these strains and facilitating speed of shipment of commonly requested isolates. 

Objective 2: One poleroviruses. Poleroviruses are aphid-transmitted agricultural pathogens that infect a wide array of staple food crops, including cotton and potato. 

Cotton leafroll dwarf virus (CLRDV) is an emerging threat to cotton grown in the United States. This year, we validated CLRDV antibodies that were generated in alpacas using purified CLRDV structural proteins. We established a collaboration with a company to develop these antibodies into diagnostic kits. We hired two new postdoctoral associates, both molecular virologists from the University of Hawaii, on the project, and they are due to start in August. Research has continued on generating the CLRDV infectious clone and all viral genome fragments have been assembled and sequence verified. Research demonstrated that the polerovirus NRTD protein is an inhibitor of virus transmission and NRTD mutant variants that are lethal to aphids. Sequence conservation argues that polerovirus NRTDs will follow the same structural blueprint, which affords a novel approach to block the spread of these agricultural pathogens in a generalizable manner. A provisional patent was submitted. 

The causative agent of citrus greening disease, Candidatus Liberibacter asiaticus (CLas) is transmitted by the Asian citrus psyllid, in a circulative propagative manner. We developed a simple pre-treatment DNase and filtration (hereafter PDF) protocol to remove host DNA and directly sequence CLas and the complete, primarily uncultivable, microbiome from psyllid adults. The PDF protocol yielded CLas abundances upwards of 60% and facilitated direct measurement of CLas and endosymbiont replication rates in psyllids, the first demonstration of CLas replication in psyllids. The PDF protocol confirmed our strains derived from a progenitor Florida CLas strain and accumulated 156 genetic variants, underscoring the utility of this data for bacterial strain tracking. These variants suggest laboratory propagation of CLas may result in different phenotypic trajectories among laboratories, and may confound CLas physiology or therapeutic design and evaluation if these differences remain undocumented. Finally, we obtained genetic signatures affiliated with Citrus nuclear and organellar genomes, entomopathogenic fungal mitochondria, and commensal bacteria from laboratory-reared and field-collected psyllid adults.

We continued to investigate the basic biology of two ambrosia beetle species, Xylosandrus crassiusculus (granulate ambrosia beetle) and Xylosandrus germanus (black stem borer), wood-boring insects that are widespread pests in orchard and ornamental production systems. Artificial overwintering in sawdust-based diet tubes was successful for black stem borer which extends our ability to work with this species by several months, freeing us from an inability to continuously rear this species and from low natural populations. Outdoor mass rearing is not feasible due to a high risk of fungal mite infestations. We documented the development of black stem borer symbiont and brood production in cut beech bolts. Behavioral studies document for the first time social behaviors (mainly hygienic grooming) among gallery members. However, social interactions are limited because we do not observe an overlap in generations within a gallery. 

Two outdoor phenology studies with the black stem borer are in progress. In the first study, we are completing a second year tracking the timing and number of generations produced. Black stem borer is reported to have two generations per summer, but we have observed a partial third generation in central New York that contributes to an extended flight season from late April through mid-September. The second study is a trapping network being conducted in collaboration with a Cornell University extension agent. We are documenting the ambrosia beetle complex attracted to ethanol (that stressed trees produce) and their seasonal preferences over two years for different habitats (woods, wood edge, orchard block) near commercial wholesale and cider apple orchards. Black stem borer populations were very low in 2022, and for the first time granulate ambrosia beetles were noted as present but rare in upstate New York woods. A third species, Anisandrus maiche, is a potential new pest of concern as it was trapped across all habitats. We have further shown that it will infest stressed-apple trees; biological and management studies are planned. Trap data is being contributed to a multi-state ambrosia beetle network. Data will inform optimal timing of management of the different beetle generations and species through chemical or microbial sprays and general patterns of pest population shifts over the summer. 

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. Based on continued inquiries since summer 2021 to the present with researchers and extension agents in New York and other northern states, neither rapid apple decline nor significant ambrosia beetle damage are being reported. Seasonal surveys of microorganisms on the surfaces of live-trapped, dispersing adults and the gallery walls of tunneling beetles are in progress. 

Objective 3:  Research focused on: 1) the identification of natural, plant-derived antimicrobial compounds that could kill CLas, 2) RNA aptamers that block psyllid feeding, and 3) neuropeptide mimics that kill the Asian citrus psyllid. 1. We identified multiple peptides produced by the legume plant, Medicago truncatula, that have antimicrobial activity. This year, we used our previously developed excised-leaf acquisition assay to test whether the peptides that were CLas-inhibitory in plants showed an effect on CLas acquisition by the insect vector. Strikingly, two of the top seven peptide candidates from the leaf screening completely blocked the development of psyllid adults with high CLas titer. 2. Interestingly, a re-analysis of the RNA aptamer dataset revealed additional candidate aptamers that blocked formation of the psyllid’s stylet sheath on artificial diets. Expression of RNA aptamers in plants is proving to be challenging. 3. The neuropeptide screen was also completed this year, and top peptides were selected for their ability to kill the psyllid and move systemically in leaf vascular tissue. Large-scale synthesis (1g) of the top performing peptides that killed the psyllids was completed and shipped to collaborators at the ARS Fort Pierce location for greenhouse trials. We are generating transgenic citrus expressing one neuropeptide under the control of phloem-specific promoters in collaboration with ARS scientists at the Fort Pierce location.  

A substantial focus of research centered around SymbiontTM technology, the novel tree delivery strategy for citrus greening therapeutics, such as those mentioned above. We engineered autonomously growing plant cells, referred to as symbionts, to express a gene of interest. SymbiontsTM facilitate the production of molecules that can be harvested directly in symbiont cell culture or from in planta grown symbionts. Nanobodies are small antibodies derived from camelids or cartilaginous fishes, referred to as VHH and VNAR, respectively. In collaboration with a CRADA partner and ARS scientists in Fort Pierce, we demonstrated production of VHH nanobodies against the SARS-CoV-2 spike proteins in Nicotiana benthamiana through expression from SymbiontTM plasmids and showed that these nanobodies competitively inhibit binding between the SARS-CoV-2 spike protein receptor binding domain and its human receptor protein, angiotensin converting enzyme 2 (ACE2). Plant production of nanobodies is a low-cost way to rapidly respond to therapeutic needs for emerging pathogens, such as the SARS-CoV-2 virus. **Accomplishments**
                                

1. New method for tracking citrus greening pathogen in single insects. Citrus greening disease is the most serious disease of citrus. In the U.S. the disease is endemic in Florida and spreading in California and Texas. Citrus greening disease is ultimately fatal for infected citrus trees, and there is no cure. The disease comes from plant infection by a bacterium, referred to as CLas. CLas is spread by the Asian citrus psyllid that spreads the bacteria during feeding on infected citrus trees. ARS scientists in Ithaca, New York, developed a method to enrich CLas cells from single psyllid insects for genome sequencing called the PDF method. This allowed for tracking the strain of CLas from a single insect, which may help trace the origin of new outbreaks. 2. Transmission of a costly grapevine virus by an insect vector in vineyards. A new virus infecting grapevines in North America called grapevine red blotch virus (GRBV) is disrupting grape production and vineyard productivity. GRBV is spread in vineyards by a tiny insect called the threecornered alfalfa hopper. Free-living grape vines are frequently found in areas surrounding commercial vineyards where cultivated wine grapes are grown. The free-living grape plants can serve as a hiding place for GRBV. In this work, ARS scientists in Ithaca New York, and University partners showed that insects transmit the virus more efficiently into free-living grapes as compared to the cultivated wine grapes and that both types of plants, free-living and wine grape plants can serve as sources of virus for three-cornered alfalfa hopper to spread within a vineyard. The results suggest that management of the virus in free-living grape vines in areas around a commercial vineyard may be one way to reduce virus spread into the vineyard by the insect vector. 3. A new method to block virus transmission by aphids. Many viruses that infect plants rely on an insect vector for plant-to-plant spread, including the new cotton leafroll dwarf virus infecting US cotton. USDA ARS scientists together with university cooperators focused on a group of insect-transmitted viruses, the poleroviruses, which cause economically important diseases of vegetables and other crops, including cotton. The scientists determined the structure of a protein on the surface of these viruses that is crucial for the virus to be picked up and transmitted by its insect vector - aphids. ARS scientists have found a new way to block the transmission and kill insect vectors these economically important viruses. 4. Isolate, identify, and accession and test entomopathogenic fungi associated with the emerald ash borer. (EAB; Agrilus planipennis), an invasive insect pest from Asia that is killing ash trees across North America and related invasive wood-boring beetles. 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, including 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, are investigating the fungi associated with the EAB. This research has isolated over 500 fungi associated with the galleries, larvae, and adults of EAB, including insect-infecting (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 across the eastern U.S. 5. Isolate, identify, and accession potential biological control fungi for soybean cyst nematode and plant pathogens potentially vectored by the SCN. 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. Fusarium virguliforme, causal agent of Soybean Sudden Death Syndrome, often co-occurs with SCN in soybean fields and may be associated with cysts of the SCN or introduced through wounding by the nematode. Scientists analyzed the genetic diversity of F. virguliforme isolates across the midwestern U.S. to identify three major genetic groups, one of which has higher genetic diversity that may enable it to adapt and spread as an invasive pathogen. 6. Isolate, identify, and accession and test entomopathogenic fungi associated with the Asian Giant Hornet (AGH; Vespa mandarinia), an invasive insect pest from Asia that threatens bee populations. Although AGH has thus far been successfully contained in Washington state, it has potential to cause devastating effects on U.S. agriculture through killing of bee pollinators. Very little is known about the biology or microbial communities associated with AGH. ARS scientists in Ithaca, New York and Beltsville, Maryland, are investigating the microbes associated with AGH, proteins in the venom gland, and receptors in the antennae to identify potential approaches to controlling AGH. Research in Ithaca, New York, has isolated over 25 fungi from the guts and mouthparts of AGH and used high-throughput sequencing to identify microbial communities associated with AGH. Improved understanding of the biology of AGH and role of microbes as potential control agents will make tools available to the USDA ARS and its stakeholders to prevent and control the spread of AGH in the U.S. 7. Bringing clarity to Asian citrus psyllid biology: the importance of a high-quality genome. The bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with citrus greening disease and is transmitted by the Asian citrus psyllid. Transmission of CLas by the Asian citrus psyllid involves the movement of the CLas bacteria across different psyllid tissues, such as the gut and the salivary glands, but the psyllid genes involved in these interactions are not known. The bacterial infection attacks all citrus varieties worldwide, causing reduced fruit marketability and tree death, with no cure available. Half a decade of research on interactions between the Asian citrus psyllid tissues and the citrus greening pathogen relied on an incomplete version of the psyllid genome. In the past 5 years, ARS scientists in Ithaca, New York, and university partners provided a vast improvement over the initial genome sequencing effort for the psyllid. Using the newly improved genome, the team revealed the genes expressed in each psyllid tissue involved in CLas transmission. Each tissue had unique gene profiles and responses to CLas infection. The improved genome assembly led to significant and quantifiable differences in data interpretation and is a valuable tool for the research community and stakeholders. The team showed that future studies on circulative, vector-borne pathogens should be conducted at the tissue specific level using complete, chromosomal-length genome assemblies for the most accurate understanding of pathogen-induced changes in the insect vector. **Review Publications**
                            *Olarte, R.A., Hall, R., Tabima, J., Malvick, D., Bushley, K.E. 2021. Genetic diversity and aggressiveness of Fusarium virguliforme isolates across the Midwestern United States. Phytopathology. https://doi.org/10.1094/PHYTO-05-21-0191-R.*
                                *Ugine, T., Krasnoff, S., Losey, J., Behmer, S.T. 2022. Omnivory in predatory lady beetles is widespread and driven by an appetite for sterols. Functional Ecology. 36(2):458-470. https://doi.org/10.1111/1365-2435.13965.*
                                *Pethybridge, S.J., Sharma, S., Murphy, S., Biazzo, J., Milbrath, L.R. 2021. Southern blight of perennial swallowwort (Vincetoxicum spp.) in New York. Invasive Plant Science and Management. 14(4):223–231. https://doi.org/10.1017/inp.2021.30.*
                                *Magidow, L., Ditommaso, A., Westbrook, A.S., Kwok, M.J., Ketterings, Q.M., Milbrath, L.R. 2022. Soil characteristics of North American sites colonized by the non-native, invasive vines black swallow-wort and pale swallow-wort. Northeastern Naturalist. 29(1):108-132. https://doi.org/10.1656/045.029.0111.*
                                *Hoyle, V., Flasco, M., Choi, J., Cieniewicz, E., Mclane, H., Perry, K., Dangl, G., Rwahnih, M., Heck, M.L., Loeb, G., Fuchs, M. 2022. Transmission of grapevine red blotch virus by spissistilus festinus [Say, 1830] (Hemiptera: Membracidae) between free-living vines and vitis vinifera ‘cabernet franc’. Viruses. 14: Article e1156. https://doi.org/10.3390/v14061156.*


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