Project : USDA ARS

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Research Project: Discovery and Development of Microbial-Based Biological Control Agents for Use Against Invasive Weeds in the United States

Location: Foreign Disease-Weed Science Research

2024 Annual Report

Objectives
Objective 1: Isolate, identify, and evaluate endemic plant pathogens that can be utilized as biological control agents of invasive weeds, such as swallow-wort, garlic mustard, and Japanese hop. [NP304, Component 1, Problem Statement 1C; Component 2, Problem Statement 2B] Sub-objective 1.A – Isolation and identification. Sub-objective 1.B – Evaluation of pathogen efficacy. Sub-objective 1.C – Evaluation of disease reaction among non-target and target species. Sub-objective 1.D – Develop and submit a proposal for release, and if approved, participate with cooperators in release and post-release monitoring of the pathogen(s). Objective 2: Determine the diversity and population dynamics of microbes associated with invasive weeds. [NP304, Component 1, Problem Statement 1C; Component 2, Problem Statements 2A and 2B] Sub-objective 2.A – Characterize the microbiome/virome of invasive weed species. Sub-objective 2.B – Evaluate the emergence of endemic phytopathogens on non-native, invasive weed species. Sub-objective 2.C – Develop accurate and rapid means for identification and detection of microbes permitted for field release. Objective 3: Develop innovative technologies to enhance or complement biological control agents and suppress weed health, such as RNA interference and encapsulation matrices. [NP304, Component 2, Problem Statement 2A] Sub-objective 3.A – Evaluate the efficacy and applicability of exogenous double-stranded RNA applications for plant health suppression. Sub-objective 3.B – Evaluate encapsulation matrices that support microbe survival and disease development.

Approach
Plant pathogens and plant-associated microorganisms will be collected from target invasive weeds in the U.S.A, and evaluated for their potential use as biological control agents using conventional, molecular, and technology driven approaches. The conventional approach is a cyclical method that identifies and evaluates promising candidate plant pathogens as biological control agents of invasive weeds. The molecular approach leverages advances in genomics to characterize and exploit weed microbiomes and viromes for insights into pathogen emergence and novel microbial candidates for host suppression. The technology driven approach will investigate technological advances to augment microbial-based biological control agents and to provide value-added synthetic properties to increase disease development under diverse environmental conditions. Microorganisms will be evaluated for the risk associated with intended release into ecosystems containing economically and ecologically important North American plant species. Risk will be evaluated based on disease reaction of species related to the target weed from a test-plant list reviewed and modified according to recommendations of regulators at the USDA Animal and Plant Health Inspection Service. Microorganisms determined to have an adequately narrow host range will be proposed for release in the U.S.A. Proposals for release of the microorganism will be developed for review by the Technical Advisory Group for Biological Control Agents of Weeds, and subsequent development of an Environmental Assessment, declaration of Finding of No Significant Impact and issuance of federal and state permits for release. Inoculum of the microorganism will be prepared in sufficient quantity for release, and target weeds will be inoculated in the field under conditions that favor disease development and establishment. Establishment and spread of microorganisms will be monitored in the field by recording disease symptoms on the target weed and re-isolating the microorganism. Damage to target weed populations and environmental factors important in microorganism establishment, efficacy and spread, will be measured.

Progress Report
Under Objective 1C, the environmental safety of multiple candidate biocontrol agents continues to be evaluated through greenhouse and laboratory experiments. A flowering population of the endangered species California seablite (Suaeda californica) was established under controlled growth conditions in Maryland to allow for supplemental host range assessment of a proposed Russian thistle biocontrol agent, the fungus Uromyces salsolae, in response to stakeholder feedback from the U.S. Fish and Wildlife Service and to support a petition for field releases. Previously released biocontrol agents of common crupina and perilla mint were monitored for off-target impacts under natural field conditions, with the results confirming that these agents pose no risk of harm to local plant communities. Under Objective 1D, multi-year field release trials for use of the biological control agent, Ramularia crupinae, against the Federal noxious weed common crupina (Crupina vulgaris) were expanded substantially. In addition to existing release sites in Idaho and the Nez Perce Reservation, new experiments were established in collaboration with the Confederated Tribes of the Umatilla Indian Reservation in Oregon. Portable weather stations and seed addition plots have been incorporated at these sites to study plant demographic changes and disease epidemiology in cooperation with researchers from Sidney, Montana. Fungal inoculum was prepared in Frederick, Maryland, and shipped to university collaborators for both autumn and spring field releases. Sites were inoculated with the pathogen under favorable plant and disease conditions, resulting in successful disease development. Pathogen establishment was confirmed through physical re-isolation of the fungus and with two DNA-based methods of detection. Incorporating this biocontrol agent into the landscape will assist private, public, and tribal stakeholders in managing large infestations of the federally noxious weed across remote, rugged rangeland. Decreasing common crupina populations will directly benefit U.S. and tribal agriculture by increasing rangeland forage productivity and livestock carrying capacity, preserving native and beneficial plant communities, preventing the displacement of rare and threatened species, and reducing the funds and resources necessary to maintain long-term common crupina management operations. Separately, preparations began for new field releases of a previously approved biocontrol agent, Puccinia jaceae, to control of yellow starthistle (Centaurea solstitialis). Permits were secured for movement of the pathogen out of containment in Maryland and into the environment in Idaho, Washington, Utah, and Nevada. Mass production of the pathogen has begun in Maryland for future inoculum distributions and experiments on integrated management practices. Under Objective 2A, microbial communities associated with invasive weeds were characterized through conventional culture-based approaches as well as high-throughput DNA sequencing methods. Bulk microbiomes from over 400 samples of garlic mustard (Alliaria petiolata), wavyleaf basketgrass (Oplismenus undulatifolius), and Japanese stiltgrass (Microstegium vimineum) were characterized using DNA sequencing. These sequences are being used to identify conserved weed-microbe associations for consideration as targets for biocontrol research and to understand how weed microbiomes are structured across space, time, plant tissue, and host species. To complement this DNA focused research, more than 300 individual fungal endophyte isolates were collected from basketgrass leaves and identified using DNA sequences. These cultures were compared to the predicted core microbiome communities and are currently being screened for weed tissue degradation potential and herbicidal metabolite production. Under Objective 2B, new plant pathogens posing a risk to agronomic crops were identified from diseased weed populations. Identifying the origins of these novel weed-associated pathogens is critical to monitoring crop risks and improving our understanding of pathogen evolution, emergence, and the role of invasive weeds as plant pathogen reservoirs. A novel fungal species damaging to garlic mustard (Alliaria petiolata) was recently characterized and genome sequenced. It is capable of causing disease on diverse brassica species and may pose a threat to crop production. Similarly, new alligator weed (Alternanthera philoxeroides) pathogens were identified in Maryland that are capable of damaging related Amaranthaceae crops, like table beet and spinach; a previously undescribed fungal pathogen affecting knotweed in Rhode Island and Maryland was found to infect rhubarb; and a fungal pathogen recovered from black swallowwort was capable of causing disease on tomato. Additional host range studies, toxin production, and genome analysis studies are ongoing. Lastly, bacterial pathogens (Xanthomonas campestris) of garlic mustard that displayed broad host ranges were collected and sequenced from across several Midwestern states. Comparative bacterial genomic analyses will allow for improved characterization of virulence genes, pathogen evolution, and recombination potential with Brassica-infecting crop strains. Genomic analyses are ongoing. Under Objective 2C, Ramularia crupinae specific diagnostic primers and protocols were optimized for screening diseased field material using a quick and efficient DNA-based method. Primers are R. crupinae-specific and highly sensitive, which allows for rapid pathogen detection and field monitoring of the agent following release. Separately, a bulk microbiome DNA analysis was used to confirm the presence of the pathogen in a disease-symptomatic leaf sample from the field. Under Objective 3, gene drive research is being investigated as a novel weed management tool using common tansy (Tanacetum vulgare) as a model weed system. Tansy is a non-native, invasive plant that invades diverse agroecosystems impacting timberland regeneration, forage production and livestock health, and threatens endangered and threatened native plant species. Collaborative research is developing an effective tansy transformation system focusing on optimizing genetic modification and plant regeneration. Research is ongoing with university colleagues. Similarly, research is continuing to investigate the efficacy of exogenous double-stranded RNA (dsRNA) foliar applications as a genetic biocontrol weed management tool that exploits RNA interference and plant defense pathways. Approximately 30 dsRNA constructs targeting three conserved plant genes were synthesized for the model plant system, Nicotiana benthamiana, and the invasive weed garlic mustard. Select genes were targeted based on severe N. benthamiana phenotypes when silenced with a virus induced gene silencing (VIGS) approach. Optimization of delivery methods, dsRNA fragment size, and evaluating gene silencing efficacy and specificity is ongoing

Accomplishments
1. Redistribution of a fungal biological control agent for invasive perilla mint. Perilla mint is a non-native weed species that excludes desirable pasture and native plant communities while posing a toxicity risk to livestock. Because perilla persists in ecologically sensitive environments, a biologically-based management solution is needed. ARS scientists in Frederick, Maryland, have evaluated and redistributed a biological control agent of invasive perilla mint in cooperation with National Park Service staff from Maryland, Virginia, and Washington D.C. A naturally occurring plant pathogenic fungus (Colletotrichum shisoi) was collected, determined to be environmentally safe, and shown to be effective at reducing the survival and reproduction of perilla plants. The development and redistribution of this biological control agent benefits land managers and livestock producers in the 22 states where perilla infestations can be found.

U.S. DEPARTMENT OF AGRICULTURE

Foreign Disease-Weed Science Research: Frederick, MD