Location: Foreign Disease-Weed Science Research
2023 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 1, multiple putative plant pathogens were isolated and characterized from invasive weeds. Two fungal isolates collected from Japanese knotweed (Fallopia japonica) and black swallowwort (Vincetoxicum nigrum) were confirmed to be pathogens and advanced for molecular identification. Colletotrichum shisoi, a fungal pathogen of the perilla mint weed (Perilla frutescens) previously identified in Maryland, was collected from Virginia and Washington, D.C. These additional isolates of C. shisoi were tested for virulence on the target weed. This information will support permit applications for interstate movement of the pathogen during field evaluations. The fungal pathogen C. shisoi was found to reduce invasive perilla mint seed production and interspecies competitive ability under controlled conditions. Experiments adding oil-based adjuvants to pathogen inoculum resulted in higher disease incidence, but led to phytotoxicity symptoms on a non-target plant. C. shisoi was further tested for pathogenicity against native and economically important plant species related to the invasive weed perilla mint. No off-target infections were observed. Following this finding of host specificity, C. shisoi is being proposed for use in preliminary field experiments as a biological control against perilla mint. The fungal pathogen Corynespora cassiicola collected from Japanese honeysuckle was determined to be pathogenic to cotton and tomato. The pathogen was a weak antagonist of honeysuckle, but this invasive host may act as a pathogen reservoir in regions with co-occurring invasions and crop acreage. A novel, unidentified fungal pathogen was discovered on a heavily diseased garlic mustard population in Maryland. The isolate was confirmed to be highly pathogenic on garlic mustard, and preliminary genetic analyses suggest it is a novel fungal species. Whole genome sequencing, isolate characterization, secondary metabolite production, and whole plant inoculations are ongoing.
Novel phytopathogenic bacteria characterized from two prolific non-native weeds, garlic mustard (Alliaria petiolata) and fig buttercup (Ficaria verna), are being investigated due to their recent emergence and close genetic relationship to agronomically damaging bacterial crop strains. The genomes of diverse pathogenic Xanthomonas campestris bacteria isolated from garlic mustard populations from across the Midwest are currently being sequenced and characterized to investigate rates of pathogen emergence within weed reservoirs. Two bacterial Pseudomonas species were identified on heavily diseased fig buttercup populations in Maryland. Inoculations of fig buttercup plants with the Pseudomonas species and field ecological experiments demonstrated pathogenicity and disease spread. Additional experiments are ongoing.
The recently approved biological control fungal agent, Ramularia crupinae, was released at several field sites in Idaho and the Nez Perce Reservation for management of the noxious invasive weed common crupina (Crupina vulgaris). Initial field trials demonstrate that the pathogen remains pathogenic in dry U.S. rangeland environments. Moreover, inoculum production, rating protocols, reisolation/diagnostic assays, and disease modeling were improved and optimized to the unique attributes, weather, and terrain of the western U.S. field sites. Research and long-term monitoring of the release is ongoing.
Under Objective 2, over 300 plant tissue samples from garlic mustard and wavyleaf basketgrass weeds have been processed and prepared for microbiome sequencing and microbial community characterization. Simultaneously, 48 bacterial isolates collected from healthy garlic mustard were used for whole genome sequencing to study the diversity of plant pathogenic genes carried by non-pathogenic endophytes. Further collection of plant tissue is scheduled for July and August as these weeds reach reproductive growth stages. Additionally, leaf samples from replicated plots of common crupina, prior to inoculation with the candidate biocontrol agent Ramularia crupinae, were collected for microbiome sequencing, and 160 fungi were isolated from healthy plants for phenotyping and sequence-based identification. Ramularia crupinae genomic data was used to identify and evaluate gene targets for DNA-based diagnostic assays. Primer sensitivity and specificity were demonstrated. Optimizing primer sensitivity with infected plant material is ongoing.
Also under Objective 2, a comparative genomic analysis between Xanthomonas campestris isolated from diverse cruciferous crops and weeds, allowed for the identification of new strain associations, effector variation, and host range potential. Genomic and phenotypic analyses from these diverse X. campestris crop and weed strains improve our understanding of X. campestris evolution, pathogen emergence, and the role of invasive weeds as pathogen reservoirs.
Under Objective 3, in planta investigations using topically-applied double-stranded RNA (dsRNA) continue with new genetic constructs and gene targets for both the model system Nicotiana benthamiana and garlic mustard. Preliminary analyses have demonstrated foliar uptake and gene silencing. Optimization of delivery methods and evaluating dsRNA gene silencing efficacy and specificity is ongoing. Encapsulation research has demonstrated that X. campestris can survive multiple encapsulation processes and remain pathogenic. Moreover, encapsulation chemistries were tailored to the leaf and bacterial surfaces to ensure tight leaf adherence.
Accomplishments
1. Field release of the first biocontrol agent to control the federal noxious weed common crupina. Rangelands and grasslands make up 30% of land use in the U.S. and are vital to livestock production and ecosystem health. Controlling invasive weeds in these rugged environments is challenging and costs the U.S. $6 billion annually. Common crupina is a foreign weed that has invaded thousands of acres of grasslands in the western U.S. This noxious weed crowds out nutritious plants that provide food for grazing livestock and leads to conditions that result in soil erosion. Chemical-based weed control methods are often impractical because of the large size of invaded regions. Therefore, an environmentally friendly approach was sought to stop the spread of common crupina in U.S. grasslands. In the spring of 2023, ARS scientists from Frederick, Maryland, and collaborators at the University of Idaho and Nez Perce Tribe tested a new non-chemical method to control common crupina. This new approach relies on the use of a fungus that causes a leaf spot disease on only common crupina. Several locations in Idaho and the Nez Perce Reservation were tested. Within weeks of being released at the test sites, the fungus successfully infected common crupina. The results of this research suggest that this new biological approach may be a valuable tool to reduce the impact of common crupina invasions in rangelands and grasslands that benefit private, public, and tribal land managers.
Review Publications
Tancos, M.A., Thomas, J.L., Widmer, T.L. 2023. Screening and assessing biological differences among oomycete-parasitic Trichoderma asperellum isolates for biological control development. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-09-22-0094-SC.
Gaskin, J.F., Chapagain, N., Schwarzländer, M., Tancos, M.A., West, N.M. 2023. Genetic diversity and structure of Crupina vulgaris (common crupina): A noxious rangeland weed of the western United States. NeoBiota. 82:57-66. https://doi.org/10.3897/neobiota.82.90229.suppl1.
Dubrow, Z.E., Carpenter, S.S., Carter, M.E., Grinage, A., Gris, C., Lauber, E., Butchachas, J., Jacobs, J.M., Smart, C.D., Tancos, M.A., Noël, L.D., Bogdanove, A.J. 2022. Cruciferous weed isolates of Xanthomonas campestris yield insight into pathovar genomic relationships and genetic determinants of host- and tissue-specificity. Molecular Plant-Microbe Interactions. 35(9):791-802. https://doi.org/10.1094/MPMI-01-22-0024-R.
Fulcher, M.R., Bergstrom, G.C. 2022. Draft genome sequences of 14 fungal species from Alternaria section Infectoriae. Microbiology Resource Announcements. 12(1). https://doi.org/10.1128/mra.01084-22.
Young, S.L., Campbell, J.W., Fulcher, M.R., Grewell, B.J. 2023. Climate and pest interactions pose a cross-landscape management challenge to soil and water conservation. Journal of Soil and Water Conservation. 78(2):39A-44A. https://doi.org/10.2489/jswc.2023.1025A.
Fulcher, M.R., Owen Smith, P.C. 2023. First report of Colletotrichum shisoi causing anthracnose of Perilla frutescens in the United States. Plant Disease. https://doi.org/10.1094/PDIS-09-22-2121-PDN.
