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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

2022 Annual Report

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.

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 1, multiple putative plant pathogens were isolated and characterized from invasive weeds, including fig buttercup (Ficaria verna), garlic mustard (Alliaria petiolata), poison hemlock (Conium maculatum), mock strawberry (Potentilla indica), Japanese hop (Humulus japonicus), Japanese knotweed (Fallopia japonica), and Japanese honeysuckle (Lonicera japonica). In total, approximately 150 putative pathogens were isolated from these diverse invasive plant species. Isolate characterization, classification, and whole plant inoculations are ongoing. Following pathogen confirmation, promising endemic pathogens will undergo efficacy and host specificity analyses. The effects of infection with a fungal plant pathogen (Colletotrichum shisoi) on invasive perilla mint (Perilla frutescens) seedling survival, root growth, shoot height, and above ground biomass were evaluated, leading to the advancement of a candidate isolate with superior virulence for further development. An ongoing experiment measured the effect of C. shisoi infection on perilla mint competition with desirable pasture crops and native mint species. Additionally, the impact of leaf spot disease caused by Xanthomonas hederae on English ivy (Hedera helix) was measured and determined to be minimal. Significant progress was made in optimizing the growing conditions of diverse target and non-target plant hosts. New protocols were developed for growing diverse plant species in regulated containment facilities, leading to the identification of optimized conditions favorable for germination, flower initiation, and seed production. These optimized experimental conditions set the foundation for all subsequent experiments, which will streamline experimental processing and ensure a reliable source of plant material for research. Host range studies were expanded for the bacterial garlic mustard pathogen, Xanthomonas campestris strain 18048, for diverse cruciferous crops, weeds, and ornamentals. An initial host range study was performed with the perilla mint fungal pathogen, Colletotrichum shisoi strain 21-067, and cooperative research has been initiated with Hood College (Maryland) to complete an expanded host range study. Collaborations have been developed with federal agencies, university, and state scientists from Idaho, Oregon, California, Washington, and Montana for the release and evaluation of the federally approved fungal pathogen, Ramularia crupinae, to manage the federally noxious weed common crupina. As a result, preliminary field releases are anticipated to begin in the fall of 2022 at common crupina field sites identified in Idaho and the Nez Perce Reservation. Genotyping studies have identified common crupina populations that will be optimal for initial field release and establishment studies. Moreover, culturing, inoculation, and rating protocols were developed for collaborators and stakeholders as part of the project’s technology transfer activity. Under Objective 2, study sites were selected in Maryland for invasive weed microbiome and virome characterization. Additionally, cooperators from 12 states contributed 130 garlic mustard leaf samples for an expanded study. A collection of over 700 fungi and bacteria were produced to screen for microbes with potential weed biocontrol activity. In a second line of inquiry, an agreement with Towson University and cooperation with University of Richmond has enabled the transfer to ARS of over 300 soil samples collected from wavy leaf basketgrass (Oplismenus undulatifolius) invasion sites for microbial community analysis and the impact of plant invasion on resident microbiota. The genome of X. campestris strain 18048 isolated from garlic mustard was sequenced and published. In addition to sequencing the genome of strain 18048, the genomes of an additional 59 X. campestris strains isolated from crops and weeds were sequenced and phenotyped to investigate pathovar specificity, pathogen emergence/evolution, and the role of invasive weeds as pathogen reservoirs. Ramularia crupinae gene targets were identified for DNA-based diagnostic assays for monitoring pathogen establishment in the field. Specificity and sensitivity testing of the DNA-based assays were initiated and are ongoing. Under Objective 3, genes that produce visual phenotypes when silenced were targeted in both the model plant system Nicotiana benthamiana and the invasive weed Alliaria petiolata. Targeted gene fragments were cloned into double-stranded RNA (dsRNA) plasmids and produced in E. coli. DsRNA was extracted and verified. Optimization of plant inoculation protocols and additional gene targets was initiated and is ongoing. In addition, the genome of the invasive weed common tansy (Tanacetum vulgare) is being sequenced as it will serve as a model system for novel genetic biocontrol strategies. Preliminary encapsulation technology was developed for X. campestris strain 18048 isolated from garlic mustard in collaboration with Cornell University scientists. Initial encapsulation experiments demonstrated the ability to successfully encapsulate a bacterial pathogen without inhibiting viability or pathogenicity.


Review Publications
Peritore-Galve, C.F., Tancos, M.A., Smart, C.D. 2020. Bacterial canker of tomato: revisiting a global and economically damaging seedborne pathogen. Plant Disease. 105:1581-1595.
Lange, H.W., Tancos, M.A., Smart, C.D. 2021. Cruciferous weeds do not act as major reservoirs of inoculum for black rot outbreaks in New York State. Plant Disease. 106:174-181.
Frederick, R.D., Cavin, C.A., Thomas, J.L., Bruckart, W.L., Tancos, M.A. 2021. Colletotrichum fioriniae infecting invasive Japanese hop (Humulus scandens) in the United States. Plant Disease. 106:330-330.
Tancos, M.A., Dubrow, Z.E., Carpenter, S.S., Bogdanove, A.J. 2022. Genome sequence of Xanthomonas campestris strain FDWSRU 18048, an emerging pathogen of non-native, invasive garlic mustard (Alliaria petiolata). Microbiology Resource Announcements. 11:e00942-21.
Fulcher, M.R., Owen Smith, P.C. 2022. First report of Erysiphe cruciferarum causing powdery mildew of Alliaria petiolata in Maryland. Plant Disease. 106(5):1532.
Fulcher, M.R., Law, E.P., Wayman, S., Ryan, M.R., Bergstrom, G.C. 2022. Fungal plant pathogens observed on perennial cereal crops in New York during 2017-2018. Renewable Agriculture and Food Systems. 37:279-291.