Progress 02/01/22 to 01/31/24
Outputs
Target Audience:The target audiences of this project include fellow bacteriologists and virologists in the scientific community, plant science reserachers interested in plant-microbe interactions, stakeholders including peach growers in the Southeast US, and undergraduate and graduate students. The results generated from this study are of direct relevance to the scientific community, particularly those interested in bacteria-phage interactions and plant-microbe intereactions, including how these intereactions contribute to the evolution and ecology of bacterial plant pathogens. Applied results from this project are highly relevant to stakeholders including peach growers managing bacterial spot. This study served graduate and undergraduate students through training opportunities in all aspects of the research conducted, espeically through undergraduate reserach opportunities offered by the university and through internal and external grants. This project also focused on recruiting and retaining Latino and other underrepresented students in STEM disciplines. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Over the past two years, this project has provided numerous opportunities for professional development and training for PD D'Amico-Willman as well as the students and technicians she has mentored. In total, PD D'Amico-Willman has mentored 11 undergraduate students, 2 lab technicians, and 1 graduate student conducting work on this project including through bacterial and phage isolations, DNA extractions, sequencing, and bioinformatic analysis. PD D'Amico-Willman has also given several oral and poster presentations at local, national, and international conferences including two oral presentations at meetings in Assisi, Italy and Lyon, France to experts in her field. Additonally, PD D'Amico-Willman has worked to establish collaborations with USDA ARS Scientists and contributed to writing multiple federal grants including a USDA NIFA and NSF proposal. How have the results been disseminated to communities of interest?In the two-year duration of this project, PD D'Amico-Willman has disseminated information primarily through peer-reviewed publications and presentations at regional, national, and international conferences. She has co-authored threepublished manuscripts with another manuscript accepted with revisions in the journals: Microbology Resource Announements, Plant Disease, PhytoFrontiers, and Phytobiome Journal. She has presented 4 poster and 7 oral presentations to date, and has been accepted to present three oral presentations and one poster in summer 2024 on results from this project. Finally, Katherine has participated in the Juntos summer academy at NC State in summer 2023 and will particpate again in summer 2024 where she demonstrated DNA extraction methodoloy and biology to primarily Hispanic and Latino highschool students. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In the final year of this project, progress was made by PD Katherine D'Amico-Willman on each of the original, proposed objectives in order to complete the project. In addition to completing these objectives, PD D'Amico-Willman participated in a number of professional development opportunities, mentored four undergraduate students, one graduate student, and one lab technician, published two co-author, peer-review publications and submitted one peer-reivewed publicaiton that was accepted pending revisions, attended and presented at two conferences (including an international meeting in France), and has an additional four publications in progress. 1. Characterize the phenotypic diversity and bacterial host range of phageXanthomonas arboricolapv.pruni. a.Standardize phage inoculum for host-range studies and phenotypic diversity assays. This objective was completed in YR1 of the project. b.Assess phenotypic diversity of phage-bacterial host interactions. Phage-bacteria host range assays were performed in YR1 of this project utilizing bacterial isoaltes and phages collected in 2021 from the Sandhills Research Station in North Carolina. Additional samples collected in 2022 and 2023 from both the Sandhills Research Station and from locations throughout the Southeast have been tested for virulence to a panel of bacterial strains. Additionally, bacterial strains have been isolated from samples collected in 2022 and 2023 from NC and throughout the Southeast to test phage-bacteria interactions. In total, the collection of phage isoaltes contains ~250 phages collected by PD D'Amico-Willman that will continue to be tested for virulence against additionalXanthomonasstrains. c.Select phage and bacterial hosts for whole genome sequencing. Both phages and bacterial hosts were selected for whole genome sequencing based on host-range assays performed by PD D'Amico-Willman and by collaborators including Dr. David Ritchie. These isolates represent a range of geographic and temporal variation in both the phages and bacteria as well as response to phage infections. In total, ~100 bacterial strains wand ~150 phage isoaltes were chosen for sequencing. 2.Explore genetic components contributing to phage - bacterial interactions. a.Whole-genome sequencing of selected phage isolates representative of phenotypic and host range diversity. All identified bacterial strains were sequenced by PD D'Amico-Willman during the final year of this project. In total, PD D'Amico-Willman genearted genomes for ~110 bacterial strains using the Illumina or PacBio/Illumina hybrid platform. b.Whole-genome sequencing ofXanthomonasbacterial strains representing a range in phenotypic and host range diversity based on phageassays. All identified phage isolates were sequenced by PD D'Amico-Willman during the final year of this project. In total, PD D'Amico-Willman genearted genomes for ~150 phage isoaltes using the Illumina platform. c.Comparative genomic analyses to identify genes and/or gene clusters associated with host specificity in phage isolates. Comparative genomic analysis performed to date by PD D'Amico-Willman has revealed several regions within phage genomes that exhibit variability based on spatiotemporal distribution. The terminase large subunit gene was identified and found to be highly variable in more recently collected phage isoaltes compared to those collected in the decades prior from the same research station in NC. Additonally, a single amino acid subsitiution was identified in the gene for the tail fiber protein and associated with year of phage isoaltion. These provide excellent targets for future work on phage genomics and phage evolution. Studies are already in progress to further characterize these gene targets. 3. Characterize spatiotemporal dynamics of phage-bacteria interactions a.Profile changes in phage genomes over time and geography. Publications are in progress and one is accepted with revisions demonstrating the variation in phage genomes across a 35-year time span. This work shows that regions of the phage genomes remain highly conserved across time wihle others show high levels of variability. These regions of variation tend to be associated with specific genes. Additional work will be needed to determine the activity of these genes through transcriptomics and proteomic approaches. b.Examine patterns of horizontal gene transfer in bacteria resulting from interactions between phage isolates and theirXanthomonasspp. hosts. Comparative genomic analysis using the 110 genomes generated from Xap strains has revealed several regions of interest related to phage-bacteria interactions including a single gene, a gene cluster comprised of eleven genes, and a pathogencity island (~25 Kb). These all provide targets for future study to determine their activity and function in relation to phage susceptibitliy. c.Effect of phage-bacteria interaction on bacterial host pathogenicity and virulence. PD D'Amico-Willman established a virulence assay protocol in the lab to test virulence of Xap strains on peach. Two peach cultivars (O'Henry and Winblo) were used for proof-of-concept studies using the strain Xcp1, which PD D'Amico-Willman published on in YR1 of this project. This experiment demonstrated that the deatched leaf assay will be suitable to complete experiments testing the impact of phag applications on virulence and pathogenicity of Xap strains.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
DAmico-Willman, K., Ritchie, D.F., Huerta, A.I. (2022). Building an ecological model of bacteria-bacteriophage interactions using the Xanthomonas arboricola pv. pruni-peach pathosystem. Phytopathology 112(11S), 189. https://doi.org/10.1094/PHYTO-112-11-S3.1.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
DAmico-Willman, K., Ritchie, D.F., Huerta, A.I. (2023). Bacteriophage, Xanthomonas, and peach (Prunus persica): A model for tripartite host-microbe interactions on disease outcomes. Phytopathology 113(11S), 126. https://doi.org/10.1094/PHYTO-113-11-S3.1.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
DAmico-Willman, K.M., Montalban, K.M., Joglekar, P., Duin, I.M., Ritchie, D.F., Fagen, J., Huerta, A.I. (202X). Whole genome sequence resources for Pseudomonas amygdali pv. sesami and Xanthomonas arboricola isolated from sesame (Sesamum indicum) in North Carolina in 2022. PhytoFrontiers.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
DAmico-Willman, K., Joglekar, P., Heiberger, H., Smith, A.M., Ritchie, D.F., Huerta, A.I. (202X). Genetically similar Xanthomonas arboricola pv. pruni strains and associated phage display phenotypic and genotypic variation across 35 years. Phytobiomes. Accepted with revisions.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
D'Amico-Willman, K.M., Ritchie, D.F., Huerat, A.I. 2024. Xanthomonas arboricola pv. pruni-associated phage diversity across one geographic location over four decades. International Conference on Plant Pathogenic Bacteria.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
D'Amico-Willman, K.M., Ritchie, D.F., Huerta, A.I. 2024. Five decades of genetic and phenotypic diversity of Xanthomonas arboricola pv. pruni strains isolated from peach in the Eastern US. International Conference on Plant Pathogenic Bacteria.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
D'Amico-Willman, K.M., Ritchie, D.F., Huerat, A.I. 2024. Xanthomonas arboricola pv. pruni-associated phage diversity across one geographic location over four decades. Phytopathology.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
D'Amico-Willman, K.M., Ritchie, D.F., Huerta, A.I. 2024. Five decades of genetic and phenotypic diversity of Xanthomonas arboricola pv. pruni strains isolated from peach in the Eastern US. Phytopathology
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
D'Amico-Willman, K.M., Ritchie, D.F., Huerta, A.I. 2023. Biogeographic distribution impacts Xanthomonas arboricola pv. pruni susceptibility to bacteriophage. International Committee on Plant Pathology.
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Progress 02/01/22 to 01/31/23
Outputs
Target Audience:The target audiences of this project include fellow bacteriologists and virologists in the scientific community, plant science reserachers interested in plant-microbe interactions, stakeholders including peach growers in the Southeast US, undergraduate and graduate students, and underserved highschool students. The results generated from this study will be of direct relevance to the scientific community, particularly those interested in bacteria-phage interactions and plant-microbe intereactions, including how these intereactions contribute to the evolution and ecology of bacterial plant pathogens. Applied results from this project will be highly relevant to stakeholders including peach growers managing bacterial spot. Through this study, phage will be assessed as potential treatments for managing this important bacterial disease of Prunus species which includes almond, peach, plum, and nectarine. This study will also serve graduate and undergraduate students through training opportunities in all aspects of the research conducted, espeically through undergraduate reserach opportunities offered by the university and through internal and external grants. Finally, Latino high school students will be served through outreach programs developed during this project in collaboration with the Juntos program which aims to provide these students and their families resources and information to gain access to college. Specifically, this project will focus on recruiting and retaining Latino and other underrepresentedstudents in STEM disciplines. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In year one of this project, PD D'Amico-Willman participated in numberous training and professional development activities. Most importantly, she trained and mentored six undergraduate students and one lab technician, all who worked on aspects of this project including phage isolations, DNA extractions, bacterial strain isolation, phage purificiation, host range assays, and bioinformatic analysis. This experience will help PD D'Amico-Willman as she pursues her career goals of working as a faculty member at a research university. In addition to mentoring, PD D'Amico-Willman participated in a 12-week leadership training program for agricultural professionals host by NC State University in summer 2022. She earned a leadership certificated as part of this effort where she gained skills in leadership valuable for her future career. PD D'Amico-Willman also attended and presented at three national and international meetings that provided networking opportunity and through which she was able to make connections with a USDA ARS scientist who is now a collaborator on this project. How have the results been disseminated to communities of interest?In year one of this project, PD D'Amico-Willman co-first authored a peer-reviewed maunscript published in Microbiology Resource Annoucements. She also presented at three conferences in 2022: the 7th Xanthomonas Genomics Conference (Florida), the International Conference on Plant Pathogenic Bacteria (Assisi, Italy), and the American Phytopathologial Society Plant Health Conference (Pittsburgh). Finally, Katherine participated in a JUNTOS summer academy day where she provided a protocol and samples for a DNA extraction demonstration to primarily Hispanic and Latiomiddle school students. What do you plan to do during the next reporting period to accomplish the goals?In year two of this project, PD D'Amico-Willman will continue working to complete analyses for objective two of this project as well as perform necessary experiments to complete objective three. To do this, she will work with additional undergraduate and technical support staff to not only train and mentor students, but also to ensure the work is completed efficiently and effectivel. This work includes sequencing an additional fifty phage isolates and at least ten additional bacterial strains for analysis, completing comparative genomic anslysis using bioinformatic methods, and performing a leaf inoculation expeirment to test the ability of bacteriophage isolates to control bacterial disease in planta. PD D'Amico-Willmanis also in the process of submitting three additional manuscripts for peer-review (two first author, one coauthor), and has been invited to give an oral presentation at the Southern Division American Phytopathological Soceity meeting in February.
Impacts
What was accomplished under these goals?
Like many living organisms, plants can get sick with bacterial, viral, and fungal infections. Plant disease can have major impacts on our access to healthy foods like vegetables, fruits, and nuts as well as negatively impact farmers who produce these crops. To overcome the impacts of plant disease, farmers need improved strategies that protect crops but do not affect the safety of the environment and consumers. Bacterial plant pathogens are particularly devastating because they are able to evolve resistance to many of the management strategies available to treat infections such as antibiotics and copper treatments. One such pathogen is Xanthomonas arboricola pv. pruni (Xap) which causes bacterial spot in peach and can be devastating to peach growers when resistance emerges in disease-causing bacterial populations. Bacterial pathogens are not alone in the plant, however; they are constantly interacting with other microbes present on and within plant tissues. One such microbe is bacteriophage (phage) which are viruses that infects and can kill bacterial cells. The interaction between bacteria and phage can be important in driving bacterial evolution, including impacting the bacteria's ability to infect its host (the plant). The ultimate goal of this project is to further our understanding of phage and how they might play a role in combating antibiotic resistance in bacterial pathogens. Antibiotic resistance is an issue in all bacterial infections, including in humans. Research on phage is not limited to agriculture; however, results from work in plants can be extended to other organisms such as humans when trying to understand how bacteria evolve and how their interaction with phage could drive that evolution in these disease-causing agents. Developing treatment methods for bacterial infections that do not rely on antibiotics or can be used in conjunction with antibiotics is necessary to overcome bacterial resistance. This project will contribute to our understanding of the complex interaction between bacteria and phage and lead to greater knowledge on how phage might be used in managing bacterial diseases. This study is to investigate the interaction between Xap and its associated phage to determine how this interaction contributes to plant disease and to assess whether phage could serve as an alternative treatment for bacterial spot . To address the aim of this project, work will be conducted in both the field and in the laboratory focused on collecting and isolating Xap and associated phage from peach leaf tissue. Following collection and isolation, host range assays will be completed to determine how well the phage are able to infect the Xap isolates and how resistant the Xap isolates are the phage. Finally, analysis will be performed to investigate the genetic components contributing to the host range of phage that infect Xap to determine how phage might be used as a treatment for Xap infection in peach orchards. Tests will also be conducted using peach seedlings to see how well phage do at treating Xap infection to assess their potential as a treatment. In this first year of this project, progress was made by PD Katherine D'Amico-Willman on each of the original, proposed objectives. In addition to developing the research project, PD D'Amico-Willman also participated in a number of professional development activities, mentored six undergraduate students and one lab technician, published a co-first author, peer-reviewed manuscript, attended and presented at three conferences (including an international meeting in Italy), and has an additional three publications in progress. Below the progress in under each objective and subobjective is outlined. 1. Characterize the phenotypic diversity and bacterial host range of phageXanthomonas arboricolapv.pruni. a.Standardize phage inoculum for host-range studies and phenotypic diversity assays. A standard method was established by PD D'Amico-Willman for quantifying and normalizing phage inoculum. This method has been employed for all host range and phenotypic assays including for DNA isolations and phage imaging. b.Assess phenotypic diversity of phage-bacterial host interactions. Phage-bacteria host range assays have been performed using a panel of 21 bacterial isolates and 12 phage isolates that were collected in 2021 for the Sandhills Research Station in North Carolina. These host range assays were performed by first quantifying and normalizing the phage inoculum for each of the isolates. Following this, each phage-bacteria combination was tested in a minimum of three replicate assays. Results from these initial host range assays already revealed substantial variation in both the ability of the phage to lyse the bacterial cells in the panel and the ability of the bacteria to resist phage infections, providing candidates for DNA isolations and whole genome sequencing. c.Select phage and bacterial hosts for whole genome sequencing. Based on host range and lysis phenotypes, to date, 55 phage isolates have been sequenced using Illumina sequencing platforms to generate complete, whole genome assemblies. Twenty-one bacterial strains have been sequenced using the Illumina platform and four have been sequenced using the PacBio sequencing platform to generate 17 and 4 draft and complete genome assemblies, respectively. 2.Explore genetic components contributing to phage - bacterial interactions. a.Whole-genome sequencing of selected phage isolates representative of phenotypic and host range diversity. Please see the outcomes for Objective 1C stating the number of phages selected for sequencing in year one of the project. b.Whole-genome sequencing ofXanthomonasbacterial strains representing a range in phenotypic and host range diversity based on phageassays. Please see the outcomes for Objective 1C stating the number of bacteria selected for sequencing in year one of the project. c.Comparative genomic analyses to identify genes and/or gene clusters associated with host specificity in phage isolates. Comparative genomic analysis has been conducted using phage genome assemblies to identify genes associated with host specificity. This analysis, using only a subset of the phage sequenced thus far, is based on the host range assays performed on 12 phage isolates. A gene has been identified with substantial variation in a highly virulent phage compared to the less virulent phage. Characterization of this gene has revealed its potential involvement in host infection in phage. Additional studies are underway to further characterize this gene in the phage population. 3. Characterize spatiotemporal dynamics of phage-bacteria interactions. a.Profile changes in phage genomes over time and geography. Phage genomes of phage isolates collected in 2012 and in 1987 from the Sandhills Research Station in North Carolina have already been sequenced and assembled and are currently being used for comparative genomic analysis to better understand how phage genomes changes over time. A collaboration was recently established between PD D'Amico-Willman and USDA ARS scientist Dr. Tami Collum to collect phage isolates from infected peach in West Virginia. This collection will be used to assess geographic differences in phage populations in year two of this project. b.Examine patterns of horizontal gene transfer in bacteria resulting from interactions between phage isolates and theirXanthomonasspp. hosts. This analysis will be completed using the genomes already sequenced and assembled from both the phage and bacterial isolates as well as from additional phage and bacteria yet to be sequenced in year two of this project. c.Effect of phage-bacteria interaction on bacterial host pathogenicity and virulence. This experiment will be completed in year two of the project using phage isolates and bacterial strains collected in 2021, 2022, and from the historical collection dating back to 1987.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2022
Citation:
Building an ecological model of bacteria-bacteriophage interactions using the Xanthomonas arboricola pv. pruni-peach pathosystem. 14th International Conference on Plant Pathogenic Bacteria, July 3 8th 2022, Assisi, Italy.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
DAmico-Willman, K.M., Prasanna Joglekar*, Emily K. Luna, David F. Ritchie, Jennie Fagen, and Alejandra I Huerta. (2022). Complete genome sequence of Xanthomonas arboricola pv. pruni strain Xcp1 isolated in 1984 from a bacterial spot canker on Prunus persica var. nucipersica cv. Redgold . Microbiology Resource Announcements 11. https://doi.org/10.1128/mra.00209-22.
*co-first authors
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