Location: Emerging Pests and Pathogens Research
2024 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 and operation, of the ARSEF culture collection and associated metadata and information resources.
Sub-objective 1.2 Expand the ARSEF collection through isolation and accession o new fungal strains, emphasizing emerging invasive pests and invertebrate hosts or fungal taxa under represented in the collection, and improve methods to isolate, culture, and preserve fungal entomopathogens.
Sub-objective 1.3 Conduct research on the ecology, host-interactions, chemistry, systematics, organismal biology, and population and comparative genomics 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 to support development of biotechnology.
Sub-objective 2.4 Document biology and phenology of ambrosia beetles.
Sub-objective 2.5 Test for an association of insects and plant pathogens that may be relevant to rapid apple decline.
Subobjective 2.6 Test for associations of emerald ash borer with plant or insect-pathogenic fungi that may be relevant for biocontrol or ash death due to emerald ash borer.
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 and emerald as borer.
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: Preserving entomopathogenic fungi from diverse habitats is vital for the biosecurity of U.S. agriculture. ARS Collection of Entomopathogenic Fungai Cultures ARSEF processed 179 isolates and shipped them to U.S. and foreign institutions and 122 new isolates were added. Identification services were performed using ITS barcode sequencing and sequencing of an intergenic locus used to study genetic variation within the genus Beauveria bassiana. A shipment of 500 isolates were sent as backup to the Agricultural Genetic Resources Preservation Research Center. A public web interface for the collection will be available in FY 25, with additional search features added to the database (ALICE). Research included isolating and identifying new fungi and bacteria from emerald ash borer (EAB), entomopathogens from specimens sent by citizen scientists and New York soils infested with soybean cyst nematode to develop biocontrol strains. Among newly accessioned fungi were 63 isolates of pathogens of spotted lanternfly (Lycorma delicatula), an emerging pest. Additional fungi included new isolates of underrepresented fungi (Entomophthorales; Furia, Pandora) and 46 nematode-associated strains. We assessed viability for cryopreservation methods and experimented with new protocols for Fusarium isolates. Research on interactions between fungal pathogens, invertebrate, and plant hosts generated 26 publicly available genomes of nematode parasitic fungi. In-vitro bioassays collected transcriptomic and metabolomic samples for these fungi during parasitism of nematodes. A SCINet postdoctoral fellow mined sequence data and genomes for ARSEF and is initiating phylogenetic analyses of entomopathogenic fungi. Other research examined microbiomes of invasive pests to understand their role in antagonism, suppression, or beneficial effects: metagenomic and metabolomic analysis of EAB larvae infesting ash hosts and microbiomes of cysts/eggs of soybean cyst nematode in suppressive soils. Phylogenetic analysis of Beauveria bassiana isolates is underway to understand genetic diversity. Another project traced the evolution of a mycotoxin-producing gene found in Fusarium verticillioides and entomopathogen Tolypocladium inflatum.
Objective 2: Entomopathogenic fungi - Genomic resources and fungal strains provide a foundation for development of fungal biotechnology. We developed a protocol for using CRISPR-Cas9 for efficient and specific modification of entomopathogenic fungi. We are investigating a library of sequences and suitable genomic locations for CRISPR-Cas9 modification to allow rapid introduction or removal of genes in entomopathogenic fungi for applied and research purposes. Wood-boring beetles - We continued to investigate the basic biology of the ambrosia beetles Xylosandrus germanus (black stem borer), X. crassiusculus (granulate ambrosia beetle) and Anisandrus maiche, pests in orchard and ornamental production systems. Currently little to no apple decline or associated ambrosia beetle damage are being reported. The second year of a phenology study for the emerging pest A. maiche indicates it has two generations each summer. The black stem borer is common in the Lake Ontario region whereas A. maiche is abundant in the Finger Lakes region. As expected, beetle numbers were highest in the woods and lowest at the orchard edge. Trap data are shared with a multi-state network to alert growers. Research on the microbial communities associated with wood-boring insects, including ambrosia beetles and EAB is underway and may reveal mechanisms of tree pathogenesis or novel biological control agents. Metagenomic analysis of ash trees in various stages of decline identified soft-rot fungi (Peniophora, Phaeoacremonium, Fusarium) and potential plant pathogens (Didymella, Ophiostoma) in EAB galleries of trees with high-dieback/decline, suggesting that these microorganisms could play a role in ash-decline. We isolated cultures of many of these fungi and will test their activity in lignin or cellulose degradation in bioassays. Vascular-plant pathogens and their insect vectors - Research on insect-vector borne plant pathogens has focused on both poleroviruses transmitted by aphids, including potato leafroll virus (PLRV), cotton leafroll dwarf virus (CLRDV), and Candidatus Liberibacter asiaticus (CLas), presumed causative agent of citrus greening disease, transmitted by the Asian citrus psyllid, Diaphorina citri. 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. In a two-year study conducted in a large-scale polyhouse, we demonstrated that transgenic potato expressing the RTD blocks aphid transmission of PLRV and reduces percent infection of tubers. Only a small subset of transgenic plants is needed to protect non-transgenic plants from infection, changing the paradigm for how transgenic plants may be for plant protection. We developed reliable diagnostic RT-PCR and serological diagnostic assays for CLRDV using nanobodies and filed a provisional patent to make these tools available. We showed minimal differences in transmission among 5 cotton aphid clones collected from the southeastern US. Unexpectedly, we demonstrated that Myzus persicae, the green peach aphid, is competent to transmit CLRDV into cotton. We completed a 1-year grant from the Department of Homeland Security, allowing us to survey cotton for new, emerging viruses. Our group was the first to report the new virus in Arizona and California. Data mining showed CLRDV has been in the United States since at least 2005, redefining the timeline and opening new research questions on this emerging virus. Extension agents have been sending us cotton samples demonstrating terminal abortion and stem splitting, but HTS has not yet identified a causative pathogen. During an High throughput Sequencing (HTS) survey of our aphid colonies, a positive, single-stranded RNA virus within the Flavivirus genus was identified and characterized by infecting M. persicae. Insect-specific viruses (ISV) have been reported to play a role in host behavior manipulation and morphology. This new ISV, Myzus persicae flavivirus (MpFV), has 23,236 nucleotides in length and encodes a large polyprotein from a single ORF. Analysis of conserved domains showed canonical flavivirus proteins are present in the polyprotein. Small RNA (sRNA) profile analysis of M. persicae demonstrated that PLRV suppressed aphid antiviral immunity against MpFV. Titers of MpFV and PLRV are correlated in viruliferous insects, suggesting synergistic interactions in the aphid. These findings build on our previous work showing PLRV suppresses aphid immunity against insect-infecting viruses. Citrus greening – In partnership with Telesis Bio, a total of 10 specific nanobodies were developed targeting CLas effectors from in vitro panning and alpaca injection. These will be evaluated for their ability to bind CLas effectors in vivo. A psyllid nymph bioassay enabled identification of antimicrobial peptides (AMPs) from plants (NCR peptides) and wasp venom (Mastoparans) for their ability to kill the CLas bacterium. A patent and provisional patent have been filed.
Objective 3: Entomopathogenic fungi – We developed rapid screening protocols to assess commercially available entomopathogenic fungi against ambrosia beetle species. We characterized these interactions by allowing beetles to develop near-natural galleries in cut wooden bolts. These studies provide a comprehensive view of the opportunities and challenges for biocontrol fungi in ambrosia beetle systems. This work provided opportunities to refine ambrosia beetle rearing methods for RNAi studies. Sequencing the fungi the beetles farm will provide resources for development of RNAi targeting the fungal symbiont. Mortality of beetles within petri dishes that are directly exposed to insect-killing fungi can be 100% within a few days but only 25% in cut wood bolts, suggesting galleries may protect the beetles from infection. Pilot field tests with wood bolts are unreliable due to premature drying of the wood; potted trees will be used instead. Citrus greening - Florida growers are now using injectable oxytetracycline (OTC) to individually treat HLB-infected trees. The swift and efficient adoption of trunk injection challenges the notion treating individual trees is unfeasible. However, growers need a broader array of treatments to further improve economic yield, to enhance OTC efficacy, and to hedge against the emergence of OTC-resistant CLas. To meet this pressing need in the shortest possible time frame, we built upon the growers’ advancements in grove-scale trunk injection practices to develop the ‘Grove-First’ framework as part of a NIFA project. Grove-First is built on three principles: assess whole tree health in the field, efficiently screen for strong effects, and prioritize commercially available candidates with a favorable regulatory path. Field trials are in progress across Florida. To enable inclusion of antimicrobial peptides (AMPs) in the Grove-First framework, we expanded research on optimization and field deployment of “Symbiont” technology, a new method to produce and deliver AMPs and other biomolecules to the citrus tree vascular tissues. Field evaluations of Symbionts are occurring at a field site in FL. Over the past year, we inoculated field trees with Symbionts and developed methods to grow Symbionts on field trees that express green fluorescent protein (GFP) and AMPs. In hundreds of DNA samples collected, no movement of Symbiont genetic material has been detected within the inoculated trees (leaves, roots, trunk, flowers, pollen), into non-inoculated trees or soil using qPCR.
Accomplishments
1. Updated synthesis of the invasive emerald ash borer. Healthy forests provide a myriad of ecosystem, economic, and social benefits. Invasive forest insects, such as the emerald ash borer, the most damaging forest pest in U.S. history, threaten both native forests and urban trees across North America and continue to spread. These pests are difficult to control, and the last comprehensive review of the invasive emerald ash borer was published in 2014. Much has changed over the past 10 years in terms of knowledge of these beetles and the evolving management strategies. An update was needed to synthesize developments over the past 10 years and guide current research and management. We published a synthetic review in Annual Review of Entomology that evaluates the successes and failures of different control measures that have been deployed over the past decade and provides updates on new research on the insect’s biology, with a focus on promising new technologies for early detection, roles of microbial symbionts, and novel molecular control strategies.
2. SYMBIOTA portal for the ARS Collection of Entomopathogenic Fungai Cultures ARSEF collection. Microbial culture collections provide a valuable resource for control of agricultural pests and pathogens through biocontrol. Public access and synthetic ecology aid in these efforts, but limited funding for collections in general have hampered those efforts. Scientist participated in a USDA effort to develop a USDA Collections SYMBIOTA portal that will provide a search interface for isolates and associated metadata in the ARSEF collection to meet this need. Access to metadata on ARSEF isolates and ability to search interactively will improve stakeholder access and use of the ARSEF collection.
3. Beetle pest is social. The black stem borer (Xylosandrus germanus), an introduced ambrosia beetle that is a pest of ornamental and orchard trees, bores tunnels or galleries into the sapwood of stressed trees. It grows a fungus that it brings into the galleries to feed its offspring. A few other species of ambrosia beetles display cooperative social behaviors inside their galleries. If these social behaviors could be disrupted, presumably this could cause beetle colonies to fail. ARS researchers at Ithaca, New York, used a novel arena with an artificial diet to document the hidden behaviors of the adult female and her offspring. The adult female constructed the gallery and initially tended the fungal garden and her brood, but eventually it spent almost all its time blocking the gallery entrance (a protective behavior). In contrast, her offspring helped to varying degrees with keeping the gallery and each other clean. Cooperative social behaviors exist in the black stem borer, which opens opportunities for management of this and related pest species.
4. Hidden lives revealed for pest ambrosia beetles. Various ambrosia beetles are introduced pests of ornamental and orchard trees, including the black stem borer (Xylosandrus germanus) and granulate ambrosia beetle (X. crassiusculus). They bore tunnels or galleries into the sapwood of stressed trees where they grow symbiotic fungi to feed their offspring; this can lead to tree decline or death. Preventing attacks is preferred by growers but difficult to achieve. Biological studies can help identify different avenues or timing of control once attacks have already occurred. ARS researchers at Ithaca, New York conducted outdoor rearing and laboratory studies to better understand the life cycle of the two beetle species. The black stem borer produces two and an unexpected partial third summer generation every year, with adults flying from mid-April to late September to start new galleries. For both species, the symbiotic fungus grows within a few days after the gallery is started. Eggs are laid soon after and as long as the fungus is present. Although the lag between beetle colonization and reproduction is too brief to be exploited for management, disruption of other stages in gallery development may be useful for minimizing damage if attacks cannot be prevented.
5. Characterization of effectors that mediate cross-kingdom microbial interactions. Effectors can manipulate the gene expression of host organisms. These important proteins have been studied primarily in plant pathogens, where they can modulate immune responses to benefit pathogenic bacteria. We have focused on bacteria that live within fungal hypha, which serves as a resource and model for understanding and investigating these proteins. We found a structurally diverse repertoire of small effector proteins that represent an emerging strategy for engineering and disrupting cross-kingdom interactions.
6. Development of genomic resources on entomopathogenic fungi. Fungi from the group Entomophthorales are difficult to work with in laboratory conditions. These fungi have among the largest fungal genomes ever sequenced: a further challenge for assembling their genomes. Despite these challenges, we worked with an international team to produce the most comprehensive comparative genomics investigation of entomophthoralean fungi, centered on the widespread fly pathogen Entomophthora muscae. These genomic resources will streamline evaluation and development of entomopathogenic fungi.
7. Isolation and description of a potential biocontrol fungus for scale insects. Scale insects present a persistent challenge in Christmas tree orchards. Elongate hemlock scale is a particularly concerning and widespread invasive scale insect, which limits tree export. We surveyed Christmas tree farms for fungi that may provide biocontrol of elongate hemlock scale. We isolated and confirmed elongate hemlock scale was susceptible to the scale pathogen Conoideocrella luteorostrata. We sequenced the genome of this fungus and deposited the fungal isolate in the ARS collection of entomopathogenic fungi. This fungus represents a novel biocontrol option for Chirstmas tree farmers.
8. A Grove-First approach to solving citrus greening disease. ARS scientists in Ithaca, New York, and Fort Pierce, Florida, developed an efficient design-of-experiments approach to screen citrus greening treatments directly in the grove. Their 'Grove-First' approach bypasses expensive and questionably useful lab assays and screens treatments directly on sick trees in the grove. The approach was developed based on the observation that no solutions had been found for Huanlongbing (HLB) in citrus despite spending over $1 billion on research. The Grove-First approach enabled the scientists to identify 20 treatments in the first year that rejuvenate sick trees. The Grove-First principles of active inclusion and engagement with subject matter experts resulted in the removal of the 3rd year restriction on oxytetracycline injection. The citrus industry, registrant, and state and federal regulatory personnel contributed to the many discussions that resulted in this ruling. The decision is an important step in restoring economic citrus production in Florida.
9. Data mining is a powerful tool to understand plant disease epidemiology. Cotton leafroll dwarf virus (CLRDV), a threat to the cotton industry, was first reported in the United States (US) as an emergent pathogen in 2017. Phylogenetic analysis supports the hypothesis that U.S. CLRDV strains are genetically distinct from strains in South America and elsewhere, which is not consistent with the hypothesis that the virus is newly introduced into the country. Therefore, using database mining, scientists in the USDA ARS in Ithaca, New York, in partnership with university colleagues evaluated the timeline and geographic distribution of CLRDV in the country. The team uncovered evidence that shows that CLRDV had been in the US for over a decade before its official first report. Retrospective analysis shows CLRDV presence in the US since at least 2006 in Mississippi. These findings necessitate a re-evaluation of spread patterns, inoculum sources, symptomology variations, and control strategies. Our findings challenge the current understanding of the arrival and spread of CLRDV in the US, highlight the power of data mining for virus discovery, and underscore the need for further investigation into CLRDV’s impact on US cotton
Review Publications
Milbrath, L.R., Biazzo, J., Mudrak, E. 2024. In-gallery social behaviors of the ambrosia beetle Xylosandrus germanus (Coleoptera: Curculionidae). Environmental Entomology. 53(1):85-93. https://doi.org/10.1093/ee/nvae003.
Gryganskyi, A.P., Hajek, A.E., Voloschuk, N., Idnurm, A., Eilenberg, J., Manrino, R.G., Bushley, K.E., Kava, L., Kutovenko, V.B., Nie, Y., Anike, F. 2024. Potential for Use of Species in the Subfamily Erynioideae for Biological Control and Biotechnology. Microorganisms. 12(1):168. https://doi.org/10.3390/microorganisms12010168.
Watson, M., May, G., Bushley, K.E. 2022. Sources of fungal symbionts in the microbiome of a mobile insect host, spodoptera frugiperda. Microbial Ecology. 86:900-913. https://doi.org/10.1007/s00248-022-02140-3.