Progress 09/01/16 to 08/31/23
Outputs
Target Audience:During this project, we reached several target audiences. We reached fellow scientists by conducting a workshop at the annual meeting of the Mycological Society of America, releasing software online, by delivering presentations at conferences, and by publishing papers in peer-reviewed journals. We reached undergraduate and graduate students through classroom teaching, as well as guest lectures and panels at other universities. We mentored a high school student through a summer internship. We reached high school students and their teachers via virtual classroom visits, field trips and lab tours. We also reached K-12 teachers through a workshop to develop classroom activities based on our research. Via outreach, we reached several other target audiences. We reached people in the social media network of the UNC Microbiome Club. We reached recipients of the Duke Forest newsletter, a general audience of about 1600 people, including recreational users of the Forest, neighbors, and local government officials. Finally, we reached the general public through a major outreach exhibit at the Morehead Planetarium and Science Center. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project trained 2 postdocs, 5 graduate students, 10 undergraduate students, 1 high school student, 3 technical IT staff, 3 lab managers and 8 other technicians. Each high school student, undergraduate student, graduate student, and postdoc received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students and postdoc additionally received training in publishing research in scientific journals. Additionally, all students, postdocs and research technicians received mentoring in career development. This project supported presentation of project results at 41 conferences and universities. Seventeen of those presentations were invited talks. We gave seven presentations in the final project year alone. At the 2023 Helminthological Society of Washington meeting, Natalie Harper and Evan Morgan jointly won the award for Best Undergraduate Presentation. Project products were integrated with classroom teaching by faculty and postdocs in guest lectures at three universities and three full-semester classes at both UNC and North Carolina State University. Project team members lead or participated in 33 workshops. Workshop topics were wide-ranging, including data science, genomics, bioinformatics, evolutionary medicine, teaching, mentoring, proposal development, data visualization, and science communication. Three graduate students on the project completed internships funded by UNC's Training Initiatives in Biomedical and Biological Sciences, with one interning at an academic institution and two interning at private companies in medical or agricultural biotechnology. Two graduate students served as the UNC Department of Biology's Research Assistant for Diversity, Equity, and Inclusion. One other graduate student served as a Research Assistant on Predictive Intelligence for Pandemic Prevention. One graduate student served as the Social Media and Outreach Coordinator for the UNC Microbiome Club. This included helping to coordinate and advertise a joint microbiome seminar series with UNC and MIT. One postdoc served on the education committee for the American Society of Parasitologists. How have the results been disseminated to communities of interest?The exhibit that we proposed as this project's major outreach activity continues to be open and visited by the public at UNC's Morehead Planetarium and Science Center. From its opening in November 2020, it is anticipated to remain open for at least 3-5 years. During the project, we conducted outreach through over twenty activities, of which we here highlight a few. Most of our activities were focused on K-12 students: In February 2021, we worked with the Museum of Life and Science in Durham, for their Field Trip Friday series, to create a video tour (16 minutes plus live Q&A) of the lab and field experiments that was available to Durham Public Schools classrooms with over 600 students. We gave a 2021 virtual lab tour and activity organized with UNC's WinSPIRE (Women in Science Promoting Inclusion in Research Experiences). A graduate student spoke in person to a club for females interested in STEM (FemStem) at Chapel Hill High School about her research on this project, her career path, and what it's like to be a scientist. We twice participated in person in the SciREN (Scientific Research and Education Network) Lesson Plan Workshop, bringing together educators and researchers to turn research into lesson plans that fit within the North Carolina science education curriculum. As part of Parasite Week 2020, organized by the American Society of Parasitologists, we gave a two-day online presentation to a group of high school students, introducing them to the world of parasites in general, and to our study system and research more specifically Through Skype a Scientist, from 2016 through 2020 we conducted video chats about ecology of infectious disease and what it's like to be a scientist with elementary, middle, and high school students as far away as Los Angeles. As part of Skype a Scientist Live in 2019, we also spoke with a national audience of students about our research on within-host microbial interactions and infectious disease. Through the SciMatch program run through the NC Science Festival, a graduate student visited three middle school classes in Gastonia, North Carolina (in person, a 2.5 hour trip each way). She taught students about epidemics with a lesson plan she designed, and also discussed her research. We also reached the general public through two activities with our field site, Duke Forest. First, Duke Forest Teaching and Research Laboratory (site of our research) published a Research Update on this project in their newsletter on 1 August 2019. Their newsletter goes out to a general audience of about 1600 people, including recreational users of the Forest, neighbors, and local government officials. Then in 2020, we participated in Duke Forest's online Ask A Scientist. Duke Forest solicited questions about our research project via their website and newsletter. Then, we answered these questions in a half-hour discussion with Duke Forest staff. Video of the discussion was posted online. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project investigated how transmission of microbial parasites across a population of hosts is influenced by the host individuals' microbiomes. To do this, using the grass tall fescue grown for hay as a model system, we conducted surveys in the field, and experiments in the field and lab, and extended our empirical results using a mathematical model. In a field experiment, initial inoculation with different parasite species altered parasite community assembly over time within individual plants, which could impact transmission to other plants. A ubiquitous component of the tall fescue microbiome, which may interact with parasites and influence disease, is a non-parasitic fungus ("endophyte") transmitted in the plant's seed. A lab experiment demonstrated that excluding the endophyte from the host microbiome ameliorated the combined impact of two parasite species on host growth and survival. In a field experiment, inoculation with the endophyte promoted growth of individual plants, which facilitated parasite transmission across the plant population. Building on these empirical results, our mathematical model predicted that, when a parasite that establishes in the host population early in the season competes within leaves with a parasite that establishes later in the season, it can reduce the epidemic of the later arriving parasite. To understand the genetic basis of effects on parasite transmission, we developed new tools to analyze genetic shifts across microbial populations and species. These approaches revealed that epidemics of brown patch can include genetically diverse cryptic lineages of the parasite Rhizoctonia solani, and that most of these lineages appear to be the product of recent recombination events. To test the effects of shifts in the level of expression of microbial genes on parasite transmission, we identified a previously overlooked problem that may have undermined previous studies, and found a way for future studies to avoid that problem. Ultimately, this project contributed fundamental knowledge that can be used to develop targeted approaches that suppress parasite epidemics by leveraging the plant microbiome. Question 1 Of eleven papers produced, we focus here on three that are key. 1-ACTIVITIES A-To test effects of two parasite species on parasite community assembly, we inoculated individual plants, then deployed them in the field as sentinels for transmission. B-To test whether an endophyte can modify interactions between two parasites, we conducted a growth chamber experiment that factorially inoculated plants with the endophyte Epichloë and the parasite species Colletotrichum and Rhizoctonia. C-To test effects of Epichloë on Rhizoctonia epidemics, we grew either Epichloë-inoculated or Epichloë-free plants in kiddie pools in the field, then inoculated with Rhizoctonia. 2-DATA A-Longitudinal biweekly surveys (13 total) of symptomatic parasite infections. B-Plant biomass and infection with Epichloë, Colletotrichum and Rhizoctonia. C-Plant biomass and infection with Epichloë and Rhizoctonia. 3-RESULTS A-Parasite community trajectories generally diverged over time between treatment groups and the magnitude of divergence depended on the parasite species inoculated. Co-inoculation with both Colletotrichum and Rhizoctonia resulted in faster rates of divergence and greater temporal change in parasite communities relative to hosts inoculated with only the parasite Colletotrichum. B-Epichloë-infected plants that were coinfected with both parasites had lower survival and biomass. C-Epichloë-inoculated plants grew larger and together supported larger prevalence of Rhizoctonia. 4-OUTCOMES A-Initial infection by different parasites can alter parasite community assembly within an individual plant, which could impact transmission to other plants. This paper was published: https://doi.org/10.1371/journal.pone.0285129. B-An endophyte can modify interactions between two parasites, increasing disease severity for the host. This paper was published: https://doi.org/10.1098/rspb.2021.1313. C-The effect of an endophyte on host individuals can influence disease transmission in the host population. This paper was published: https://doi.org/10.3389/fmicb.2022.824211. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetic structure of Rhizoctonia solani, we cultured and isolated fungi from brown patch lesions on tall fescue leaves in the field. We then examined population genetic structure among isolates of anastomosis group (AG) AG1-IB. First, we performed next-generation multilocus sequence typing (NGMLST) to confirm AG and to distinguish single alleles in monokaryotic and dikaryotic strains. NGMLST was based on 95 SNPs spanning 400 bp segments of five loci. Second, we examined genome-wide variation (single-nucleotide polymorphisms, or SNPs) using ddRADseq genotyping-by-sequencing. We also used PCR to determine each strain's number of mating type homeodomain (HD) proteins. 2-DATA In total, we detected 7,778 SNPs genome-wide across 58 strains of Rhizoctonia solani anastomosis group AG1-IB. We detected five different homeodomain profiles. 3-RESULTS A SplitsTree analysis of SNPs revealed four distinct genetic clusters. These four clusters were also supported by multi-locus sequence typing. There was evidence of recombination and genetic substructure within field plots as well as migration among plots. All strains in two genetic clusters harbored a single putative mating type homeodomain (W-HD1) whereas the other two clusters were mixtures of strains having either one, two, three or up to four distinct homeodomains (W-HD1, W-HD2, E-HD1, E-HD2). The frequency distribution of strains according to their homeodomain profile was W-HD1 (83%), W-HD1/E-HD1 (2%), W-HD1/W-HD2 (5%), W-HD1/E-HD1/E-HD2 (9%), and W-HD1/W-HD2/E-HD1/E-HD2 (1%). The one singleton strain (S1W6) in our sample harboring all four putative HD domains was used in our inoculation experiments. We developed a multiplex PCR diagnostic test to identify this strain. 4-OUTCOMES Our analyses of Rhizoctonia solani suggest that brown patch epidemics in our system are the product of multiple genetically distinct lineages. Furthermore, while Rhizoctonia populations are thought to typically persist without sexual recombination, many of these lineages are the product of recombination. Question 3 1-ACTIVITIES To test whether RNA-seq can be used for symbiotic species without a reference genome, we computationally simulated RNA-seq of a fungus-infected plant, and mapped the reads to reference genomes of four species varying in evolutionary distance, using four different aligners. 2-DATA Data was collected on the proportion of simulated RNA-seq reads and contigs that mapped to each reference genome. 3-RESULTS Mapping RNA-seq reads to a congeneric reference genome instead of a conspecific reference genome resulted in either a failure to map the reads (with two aligners) or (with the other two aligners) mismapping of reads from plant RNA to the fungal reference genome. Mismapping was prevented by first performing a de novo assembly of the reads into contigs. 4-OUTCOMES The RNA-seq simulations revealed a previously unappreciated problem: mapped RNA-seq reads from fungal infections of plants can be from plant RNA rather than fungal RNA, compromising the integrity of the data. Further simulations and analysis demonstrated a solution to that problem. This paper was published: https://doi.org/10.1111/2041-210X.13135.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Grunberg, R.L., B.N. Joyner, C.E. Mitchell, 2023. Historical contingency in parasite community assembly: Community divergence results from early host exposure to symbionts and ecological drift. PLOS-ONE 18(5): e0285129. (https://doi.org/10.1371/journal.pone.0285129).
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Grunberg, R.L., F.W. Halliday, R.W. Heckman, B.N. Joyner, K.R. OKeeffe, C.E. Mitchell, 2023. Disease decreases variation in host community structure in an old-field grassland. PLOS-ONE 18(10): e0293495. (https://doi.org/10.1371/journal.pone.0293495)
|
Progress 09/01/21 to 08/31/22
Outputs
Target Audience:During this reporting period, we reached several target audiences. We reached fellow scientists by releasing software online, by delivering presentations at conferences, and by publishing papers in peer-reviewed journals. We reached undergraduate and graduate students through classroom teaching. Via outreach, we reached several other target audiences. We mentored a high school student through a summer internship. Finally, we reached the general public through a major outreach exhibit at the Morehead Planetarium and Science Center, as well as through blog posts and extending our research to parks and other local public spaces. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, this project trained 1 postdoc, 3 graduate students, 6 undergraduate students, 1 high school student, 1 technical IT staff, and 1 lab manager. Each high school student, undergraduate student, graduate student, and postdoc received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students and postdoc additionally received training in publishing research in scientific journals. Additionally, all students, postdocs and research technicians received mentoring in career development. INTEGRATION OF PROJECT PRODUCTS WITH TEACHING: DeCIFR tools that were developed in part through this project were incorporated into the classroom in a Lecture and Computer Laboratory hands-on course (PP 715 Applied Evolutionary Analysis of Population Genetic Data) taught at NCSU by Carbone in Fall 2021. This course introduces students to nonparametric and model-based methods for making inferences on population processes (i.e., mutation, migration, drift, recombination, and selection). The goal is to provide a conceptual overview of these methods and hands-on training on how to analyze sample data sets and interpret the results. Specifically, the course provides training on the DeCIFR system developed by the Carbone lab. DeCIFR is a comprehensive suite of biodiversity informatics pipelines and visualization tools to discover, evaluate, and describe microbial taxa at multiple spatial and phylogenetic scales. T-BAS and SNAP Workbench are tools under the DeCIFR umbrella that make core analysis functions in evolutionary and ecological studies more efficient. Students in the course apply these tools for the assembly of multi-locus datasets for small and large-scale phylogenetic studies, the estimation of population parameters, the placement of novel biodiversity in a phylogenetic context, and the rapid visualization of metadata for evolutionary and ecological inferences. CONTRIBUTED PRESENTATIONS: Grunberg, R., F.W. Halliday, K.R. O'Keeffe, B. Joyner, and C.E. Mitchell, 2022. Between-year variation in parasite community assembly within host populations is driven by climate. Ecology & Evolution of Infectious Diseases Conference, virtual, Emory University (Poster presentation). Coomber, A., Saville, A. C., Carbone, I. and J. B. Ristaino. 2022. An open access T-BAS phylogeny for emerging Phytophthora species. Plant Health 2022 Online, Pittsburgh, Pennsylvania, Aug 6-10. Medeiros, I. D., Flakus, A., Magain, N., Rodriguez-Flakus, P., Miadlikowska, J., Arnold, A. E., Carbone, I. and F. Lutzoni. 2022. Phylogenomic and comparative genomic analyses shed light on fungus-photoautotroph symbioses in Eurotiomycetes. MSA Annual Meeting, Gainesville, Florida, Jul 10-14. ADDITIONAL PROFESSIONAL DEVELOPMENT ACTIVITIES: Geyer completed the Basic Assembly and Annotation of Genomes (BAAGs) workshop at NCSU (May 23-28, 2022). Geyer completed the QIIME2 Data Processing Workshop at UNC (March 22, 2022) Geyer completed at internship, funded by UNC's Training Initiatives in Biomedical and Biological Sciences, at VAST Therapeutics in Durham NC, developing further skills in microbiology (May-July 2022). Geyer (Fall 2021) and Troy (Spring 2022 and Fall 2022) each worked as the UNC Department of Biology's Graduate Assistant in diversity, equity, and inclusion. Geyer, Green, Mitchell and Stiver completed Mental Health First Aid training from UNC (October 19, 2021). Green, Grunberg, and Mitchell attended (virtually) the 2022 Evolution and Ecology of Infectious Disease Conference. Grunberg organized a parasite ecology journal club in which multiple participants at early career stages from around the US met weekly via Zoom to discuss recent scientific papers. Grunberg attended the 2022 annual meeting of the Helminthological Society of Washington. Mitchell completed Coach Approach training in academic coaching from UNC (June 6, 2022). Stiver took the UNC class BIOL 568 Disease Ecology and Evolution, taught by Mitchell, in Spring 2022. Troy participated in the 2022 Evolutionary Medicine Summer Institute run by the Triangle Center for Evolutionary Medicine. How have the results been disseminated to communities of interest?The exhibit that was proposed as this project's major outreach activity continues to be open and visited by the public at UNC's Morehead Planetarium and Science Center. From its opening in November 2020, it is anticipated to remain open for at least 3-5 years. In June and July 2022, Green, Grunberg, and Stiver jointly mentored a local high school student, Carden Osborne, through WinSPIRE (Women in Science Promoting Inclusion in Research Experiences). Carden designed and completed a research project, quantifying interactions between Rhizoctonia and Colletotrichum in culture. The program culminated in a research symposium, in which Carden presented their project to an audience including other high school students, members of the UNC community, and the high schooler students' parents. From January through April 2022, Troy participated in the Science Writing and Communication Club at UNC. She conceptualized and edited articles for DNA Day and for the PipettePen, a blog of science-themed articles written for the general public by graduate students and postdocs. Topics of her contributions ranged from Diversity, Equity, and Inclusion in STEM to vaccine efficacy and antimicrobial resistance. From May through August 2022, Troy surveyed fungal diseases on tall fescue planted in parks and other local public spaces, raising awareness of science and our research in the community. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test our overarching hypothesis that epidemics are commonly driven (and controlled) by microbial interactions, specifically testing the roles of population and community genetics, as well as community structure. More specifically, as we move into our final project year, we will focus on completing experiments, analyzing data, and submitting manuscripts to journals for publication. To test interactions between disease and community trajectories in the leaf microbiome, we will complete the sequencing of samples from the growth chamber experiments and analyze the results. To test effects of soil fertility on microbial interactions and epidemics, we will continue our field plot experiment into its third and planned final year. We will work to publish our analysis of spatial and seasonal variation in the population genetics of Rhizoctonia and Colletotrichum. To test the effects of one key component of the seasonal environment on microbial interactions, we will complete the planned repetitions of the growth-room experiment manipulating temperature. We will integrate and extend these results using our mathematical model, conducting simulations to quantify how within-leaf parasite interactions influence the epidemics of two parasite species, particularly when transmission of one parasite species begins earlier in the growing season than the other. Together, these activities will provide further insight into how microbial communities may be manipulated to slow or prevent parasite epidemics.
Impacts
What was accomplished under these goals?
This project investigated how transmission of microbial parasites across a population of hosts is influenced by the host individuals' microbiomes. To do this, we conducted surveys in the field, and experiments in the field and lab, with the grass tall fescue, and extended our empirical results using a mathematical model. In a field survey, microbiome diversity increased with leaf age, which could influence interactions with parasites. Sequencing revealed that the Colletotrichum population appears to be highly clonal. A field experiment revealed the potential for disease to not only decrease biomass production, but to regulate variation in host community structure, which has important management implications. Ultimately, this project contributed fundamental knowledge that can be used to develop targeted approaches that suppress parasite epidemics by leveraging the plant microbiome. Question 1 1-ACTIVITIES A-To test effects of soil fertility on microbial interactions and parasite epidemics, Green continued her field experiment into a second year, adding NPK fertilizer at three levels to replicated plots of intact vegetation dominated by tall fescue. She surveyed all diseases every two weeks and in 2022 collected leaf tissue and extracted DNA every six weeks to characterize the leaf microbiome. To quantify long-term effects, e.g. mediated by shifts in plant community composition, the experiment is planned to run for three years. B-To test whether disease alters community trajectories in the leaf microbiome, Geyer conducted a growth chamber experiment inoculating plants with Rhizoctonia (vs. mock-inoculation) and characterizing community trajectories over time before and after inoculation. To characterize microbiome trajectories under field conditions, Geyer also conducted a field survey, sampling leaves of different ages. Finally, to investigate the role of the plant immune hormone salicylic acid (SA) in microbiome-parasite interactions, Geyer conducted a growth chamber experiment factorially applying SA to plants and co-inoculating them with Rhizoctonia and Colletotrichum. C-To test effects of temperature and plant age on disease and coinfections, Troy began a growth room experiment. Temperature effects will be tested by repeating the experiment multiple times, each time randomly assigning high or normal temperature. Within each temperature manipulation, individual plants were inoculated with Colletotrichum and/or Rhizoctonia, in a factorial design. D-To test effects of soil fertility on parasite coinfections, Green conducted a growth chamber experiment factorially manipulating NPK fertilizer addition and leaf order of inoculation with Rhizoctonia and Colletotrichum. E-To test effects of disease on plant community structure and biomass production, we treated plots of intact vegetation with four different fungicide treatments, including a control. In this reporting period, Grunberg submitted a manuscript to a journal. 2-DATA A-Plot fertilizer experiment: disease prevalence and severity every two weeks. Plant community composition at end of the season. Leaf samples have been submitted for nitrogen concentration, and have been prepped for microbiome analysis. B-Microbiome trajectories: Leaf relative age, length, and 16s amplicon sequences from Illumina MiSeq from the field survey. Data is forthcoming from the growth chamber experiments. C-Temperature and coinfections: Disease severity of Rhizoctonia and Colletotrichum. D-Fertilizer and coinfections: Growth of Rhizoctonia and Colletotrichum lesions over time. E-Disease impacts on plant communities: Plant community structure and biomass over three years. 3-RESULTS A-Plot fertilizer experiment: Fertilizer addition may have decreased prevalence and severity of brown patch, and increased severity of crown rust. B-Microbiome trajectories: Faith's phylogenetic diversity increased with leaf relative age. C-Temperature and coinfections: In the first repetition, at high temperature, older plants were generally more resistant to infection by both Colletotrichum and Rhizoctonia than younger plants. D-Fertilizer and coinfections: Colletotrichum facilitated growth of Rhizoctonia, and this effect was stronger when Colletotrichum was inoculated first. When Rhizoctonia was inoculated first, it inhibited infection and growth of Colletotrichum. Both effects were robust to fertilizer addition. E-Disease impacts on plant communities: Disease decreased plant biomass and variation in structure among communities. 4-OUTCOMES A-Plot fertilizer experiment: Fertilizer applications can have contrasting effects on different diseases. B-Microbiome trajectories: Microbiome diversity increased with leaf age, which could influence interactions with parasites. C-Temperature and coinfections: None yet. D-Fertilizer and coinfections: Interactions between Rhizoctonia and Colletotrichum were consistent across fertilizer treatments, suggesting that the interactions are mediated by mechanisms not involving nutrients. E-Disease impacts on plant communities: Our experiment revealed the potential for disease to regulate variation in host community structure. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetic structure of Rhizoctonia solani, Stiver continued to culture and isolate fungi from brown patch lesions on tall fescue leaves at Widener Farm, Tidewater Field Station and Upper Mountain Field Station. The Carbone lab examined population genetic structure and vegetative compatibility (VC) reactions within 90 isolates of anastomosis group (AG) AG1-IB. First, we performed next-generation multilocus sequence typing (NGMLST) to confirm AG and to distinguish single alleles in monokaryotic and dikaryotic strains. NGMLST was based on 95 SNPs spanning 400 bp segments of five loci. Second, we examined genome-wide variation (7,585 SNPs) using ddRADseq genotyping-by-sequencing. To investigate effects of microbial interactions on population genetic structure of Colletotrichum cereale, Stiver cultured and isolated fungi from anthracnose lesions on tall fescue leaves at Widener Farm. The Carbone lab PCR-amplified and sequenced four loci for four representative isolates. 2-DATA For each isolate of Rhizoctonia, we obtained data on morphotype and ITS sequence. For a subset of isolates, we obtained multi-locus haplotype, and SNPs from ddRADseq. For each isolate of Colletotrichum, we obtained data on morphotype and multi-locus haplotype. For each fungus, similar sequencing of all isolates is in progress. 3-RESULTS Rhizoctonia isolates grouped into four distinct clusters in principal component space, based both on multi-locus sequence typing, and a separate analysis of genome-wide SNPs. Three clusters were associated with three different field plots and the fourth cluster was widely distributed. An examination of mating type distribution suggests that the widely distributed cluster harbors a greater number of mating type homeodomain proteins and also includes putatively dikaryotic isolates. In pairwise VC tests, non-self pairings were incompatible and microscopic examination of incompatible pairings showed hyphal deterioration in the interaction zone. Two of the four Colletotrichum isolates originating from the same field were identical at all four loci even though they were isolated four years apart. 4-OUTCOMES Our analyses of Rhizoctonia suggest that brown patch epidemics are the product of multiple genetically distinct lineages. Our preliminary results with Colletotrichum confirm the utility of these four loci for species identification. Moreover, they suggest a high degree of clonality, which is consistent with the reported existence of only one MAT1-2 mating type idiomorph in C. cereale and allied species. Question 3 1-ACTIVITIES, 2-DATA, 3-RESULTS, 4-OUTCOMES None new. We completed work on Question 3 with a journal publication in a previous reporting period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
OKeeffe, K.R., B.T. Wheeler, C.E. Mitchell, 2022. A microbial mutualist within host individuals increases parasite transmission between host individuals: Evidence from a field mesocosm experiment. Frontiers in Microbiology 13:824211. (https://doi.org/10.3389/fmicb.2022.824211)
|
Progress 09/01/20 to 08/31/21
Outputs
Target Audience:During this reporting period, we reached several target audiences. We reached fellow scientists by releasing software online, by delivering presentations at symposia, conferences and universities, and by publishing papers in peer-reviewed journals. We reached undergraduate and graduate students through classroom teaching, including guest lectures and panels at other universities. Additionally, via outreach, we reached several other target audiences. We reached high school students and their teachers via virtual field trips and lab tours. Finally, we reached the general public by opening a major outreach exhibit at the Morehead Planetarium and Science Center. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, this project trained 1 postdoc, 3 graduate students, 2 undergraduate students, 1 technical IT staff, and 2 lab managers. Each undergraduate student, graduate student, and postdoc received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students and postdoc additionally received training in publishing research in scientific journals. Additionally, all students, postdocs and research technicians received mentoring in career development. GUEST LECTURES: Incorporation of project tools into the classroom (Lecture and hands-on tutorial Nov 2020). In response to the COVID pandemic, BCH453/553 (upper-level undergrad/grad biochemistry course) was delivered virtually and Dr. Carbone provided a pre-recorded guest lecture introducing students to NGS methods and a brief overview on how to analyze NGS data in Galaxy, a local instance deployed at NC State with partial support from this project. INVITED PRESENTATIONS: Mitchell, C.E. 2021: Washington University in St. Louis (Department of Biology). Mitchell, C.E. 2021: Oregon State University (Department of Botany and Plant Pathology). One additional invited presentation was impacted by COVID-19 by being postponed until after this reporting period. CONTRIBUTED PRESENTATIONS: Grunberg, R., B. Joyner, and C.E. Mitchell, 2021. Symbiont Infection History Alters the Trajectory of Parasite Community Assembly. Ecology & Evolution of Infectious Diseases Conference, virtual, Montpellier France (Poster presentation). Grunberg, R., B. Joyner, and C.E. Mitchell, 2021. Infection History Alters the Trajectory of Parasite Community Assembly. American Society of Naturalists, Virtual Asilomar 2021 (Oral presentation). Kloppe, T. Cowger, C., Whetten, R. and I. Carbone. 2021. Phylogenetic history and global population dynamics of wheat powdery mildew. Plant Health 2021 Online, Aug 2-6. Guo,F., I. Carbone, and D. Rasmussen. 2021.The structured coalescent with ancestral recombination model and its application to the fungal pathogen Aspergillus flavus. Plant Health 2021 Online, Aug 2-6. Aimone, C. D., Lavington, E., Hoyer, J. S., Deppong, D., Mickelson-Young, L., Jacobson, A., Kennedy, G. G, Carbone, I. Hanley-Bowdoin, L., and S. Duffy. 2021. Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation. American Society for Virology Annual Meeting Online. Jul 19-23, 2001. Additionally, the Carbone Lab contributed a genome analysis of 221 loci examining the evolutionary placement of anastomosis groups (AG) in Rhizoctonia solani on the fungal tree of life that was included in Dr Marc Cubeta's MSA presidential address. The analysis showed that anastomosis group AG1 evolved before the other AG groups and has a smaller genome size and number of proteins. This analysis highlighted the importance of placing Rhizoctonia and its nearest relatives within the context of a larger reference tree. (Cubeta, Marc A. 2021. Rhizoctonia fungi: subterranean and airborne pandemics of imperfect nuclear splendor. MSA Presidential Address. Botany Virtual 2021. July 18-23.) ADDITIONAL PROFESSIONAL DEVELOPMENT ACTIVITIES: Green participated in a virtual workshop on Diversity, Equity, and Inclusion in Socio-Environmental Synthesis Research. Interactive activities and skills-based trainings spanning team science, interdisciplinary proposal writing and shared research design, science communication and delivering actionable outcomes. Hosted by the National Socio-Environmental Synthesis Center. Two hours per week for seven weeks ending September 15, 2020. Geyer participated in the 2021 Evolutionary Medicine Summer Institute run by the Triangle Center for Evolutionary Medicine. Geyer and Grunberg participated in the Virtual Participant in the (virtual) 2020 AAAS Science and Technology Policy Forum. Grunberg attended the (virtual) 2020 annual meeting of the Helminthological Society of Washington. Grunberg attended the (virtual) 2020 annual meeting of the American Society of Naturalist. Grunberg attended the (virtual) 2021 Evolution and Ecology of Infectious Disease Conference. How have the results been disseminated to communities of interest?In November 2020, UNC's Morehead Planetarium and Science Center opened the exhibit that was proposed as this project's major outreach activity. It is anticipated to remain open for at least 3-5 years. Joyner made a six-minute video of a visit to the exhibit: https://youtu.be/BN2vK7WC-38 In February 2021, Green and Joyner worked with the Museum of Life and Science in Durham, for their Field Trip Friday series, to create a video tour (16 minutes plus live Q&A) of the lab and field experiments that was available to Durham Public Schools classrooms with over 600 students: https://youtu.be/Dltzxt_o79A In June 2021,Green, Joyner, and Bergquist worked with UNC's WinSPIRE (Women in Science Promoting Inclusion in Research Experiences) to lead a virtual lab tour (10 minutes) and ran an activity on dichotomous keys (50 minutes) for 12 high school students. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test our overarching hypothesis that epidemics are commonly driven (and controlled) by microbial interactions, specifically testing the roles of population and community genetics, as well as community structure. More specifically, we will continue nearly all the activities we planned to do in this reporting period, as most were delayed or slowed by COVID-19. With our mathematical model, we will conduct simulations to quantify how within-leaf parasite competition, combined with the degree of within-plant vs. between-plant transmission, influences the epidemics of two parasite species, particularly when one parasite species has a head-start in abundance each year owing to earlier phenology. Ultimately, this will allow us to make predictions of when and how competition within leaves can allow a parasite that establishes earlier in the host population can determine whether another parasite can establish later. We will analyze genetic variation in Rhizoctonia at spatial scales from centimeters to the state of North Carolina. To test effects of soil fertility on microbial interactions and epidemics, we will continue our field plot experiment and conduct a growth-chamber experiment in which a fertilizer treatment is factorially crossed with inoculations of Colletotrichum and Rhizoctonia. To test the effects of one key component of the seasonal environment on microbial interactions, we will conduct a growth-room experiment manipulating temperature. Together, these activities will provide further insight into how microbial communities may be manipulated to slow or prevent parasite epidemics.
Impacts
What was accomplished under these goals?
This project investigated how transmission of microbial parasites across a population of hosts is influenced by the host individuals' microbiomes. To do this, we conducted surveys in the field, and experiments in the field and lab, with the grass tall fescue, and extended our empirical results using a mathematical model. Sequencing revealed that the Rhizoctonia population varies genetically among locations within a single field. This suggests that brown patch epidemics in our experimental plots will be the product of multiple genetically distinct lineages of Rhizoctonia, which could respond independently to microbial interactions. In a field experiment, initial inoculation with different symbionts altered parasite community assembly within individual plants, which could impact transmission to other plants. Ultimately, this project contributed fundamental knowledge that can be used to develop targeted approaches that suppress parasite epidemics by leveraging the plant microbiome. Question 1 1-ACTIVITIES A-To test effects of soil fertility on microbial interactions and parasite epidemics, Green established a field experiment adding NPK fertilizer at three levels to replicated plots of intact vegetation dominated by tall fescue, then surveyed all diseases every two weeks. To quantify long-term effects, e.g. mediated by shifts in plant community composition, the experiment is planned to run for three years. B-As the leaf microbiome composition typically shifts as the leaf ages, understanding such shifts in the leaf microbiome may be necessary to understand impacts of the leaf microbiome on parasite infection and disease. To quantify such shifts, Geyer conducted a greenhouse experiment to characterize the fungal and bacterial leaf microbiome at different leaf ages. Library prep for amplicon sequencing on Illumina is in progress. This experiment was planned for spring 2020, but COVID-19 delayed completion until early 2021, then supply-chain limitations in 2021 further delayed library prep. C-To test how effects of soil nutrients on susceptibility to infection by multiple parasites depends on seasonal environment, Green conducted an outplant experiment. Individual potted plants of tall fescue received nitrogen fertilizer at one of three rates, then were deployed into the field at three times of the season to serve as sentinel hosts for disease transmission. This experiment was also delayed by COVID-19. D-To test how infection with foliar microbes alters the potential for Rhizoctonia to have an epidemic in different seasonal environments, Joyner and Bergquist conducted a field mesocosm experiment in which tall fescue plants were started in the greenhouse, inoculated with Rhizoctonia, then transplanted into field mesocosms. To manipulate exposure to field microbes, the mesocosms were embedded, or not, for one week in fescue-dominated vegetation. E-To test effects of Epichloë, Colletotrichum and Rhizoctonia on parasite community assembly, Joyner (mock) inoculated individual potted tall fescue in the lab, then deployed them in the field as sentinel outplants to expose them to ambient transmission. In this reporting period, Grunberg completed data analysis and prepared a manuscript for submission to a journal. 2-DATA A-Plot experiment: disease prevalence and severity every two weeks. Plant community composition will be quantified later in the season, and leaf nitrogen concentration will also be quantified after samples are prepped in the lab. B-Greenhouse experiment: leaf age and leaf size. After sequencing, data will include fungal composition based on ITS sequence and bacterial composition based on 16s sequence. C-Fertilized outplant experiment: disease prevalence and severity twice a week. Leaf nitrogen concentration will also be quantified after samples are prepped in the lab. D-Mesocosm experiment: time series of prevalences of all diseases. E-Inoculated outplant experiment: longitudinal biweekly surveys (13 total) of severity of all diseases. Epichloë infection at end of experiment. 3-RESULTS A-Plot experiment: No results yet, as data are being analyzed. B-Greenhouse experiment: No results yet, as library prep is in progress. C-Fertilized outplant experiment: Many plants were destroyed by rats. No quantitative results yet, as data are being analyzed. D-Mesocosm experiment: Experiment was completed as planned. Data are being processed. E-Inoculated outplant experiment: Parasite community trajectories generally diverged over time between treatment groups and the magnitude of divergence depended on the symbiont species inoculated. For example, co-inoculation with both Colletotrichum and Rhizoctonia resulted in faster rates of divergence and greater temporal change in parasite communities relative to hosts inoculated with only the parasite Colletotrichum. 4-OUTCOMES A-Plot experiment: No outcomes yet, as data are being analyzed. B-Greenhouse experiment: No outcomes yet, as library prep is in progress. C-Fertilized outplant experiment: No outcomes yet, as data are being analyzed. D-Mesocosm experiment: No outcomes yet, as data are being processed. E-The inoculated outplant experiment indicated that initial infection by different symbionts can alter parasite community assembly within an individual plant, which could impact transmission to other plants. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetic structure, Joyner and Stiver continued to culture and isolate fungi from brown patch lesions on tall fescue leaves at Widener Farm, our long-standing field site. Additionally, to investigate variation at larger spatial scales in contrasting agricultural environments, we expanded our survey to two Field Stations of the North Carolina Department of Agriculture and Consumer Services: Tidewater and Upper Mountain. Isolates that, based on both morphology and ITS, fell in the Rhizoctonia species complex were sequenced on Illumina MiSeq at five loci by Jones and by the Carbone lab using ddRADseq. Cornish, White and Carbone isolated DNA, inferred trees in T-BAS based on ITS and multi-locus haplotypes, clustered sequences based on SNPs, and performed in vitro compatibility assays. COVID-19 prevented sampling in 2020, and delayed lab prep and sequencing in 2021. 2-DATA For each isolate of Rhizoctonia, we obtained data on morphotype and ITS sequence. For a subset of isolates from Widener Farm, we obtained multi-locus haplotype, and 7,585 SNPs from ddRADseq. Similar sequencing of all isolates, including from the two other sites, is in progress. 3-RESULTS At Widener Farm, ordination of isolates based on SNPs revealed four genetic clusters of isolates. Pairwise in vitro vegetative compatibility assays indicated that each cluster was incompatible with the others. In the field, two of the four clusters were each restricted to single, different sampling location. 4-OUTCOMES The genetic composition of the Rhizoctonia population varied among sampling locations within what appears to be a relatively homogenous host population in a single field. Earlier in the season, different lineages of Rhizoctonia may have colonized leaves in different parts of the field by chance, then maintained local transmission as the season progressed. Alternatively, the different lineages could occur in different locations because they are sensitive to fine-scale variation in the environment. Either way, the observed genetic variation suggests that brown patch epidemics are the product of multiple genetically distinct lineages of Rhizoctonia. Question 3 1-ACTIVITIES, 2-DATA, 3-RESULTS, 4-OUTCOMES None new. We completed work on Question 3 with a journal publication in a previous reporting period.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
O⿿Keeffe, K.R., F.W. Halliday, C.D. Jones, I. Carbone, C.E. Mitchell, 2021. Parasites, niche modification, and the host microbiome: A field survey of multiple parasites. Molecular Ecology 30:2404-2416. (https://doi.org/10.1111/mec.15892).
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
O⿿Keeffe, K.R., A. Simha, C.E. Mitchell, 2021. Indirect interactions among co-infecting parasites and a microbial mutualist impact disease progression. Proceedings of the Royal Society B. 288(1956):20211313 (https://doi.org/10.1098/rspb.2021.1313).
|
Progress 09/01/19 to 08/31/20
Outputs
Target Audience:During this reporting period, we reached several target audiences. We reached fellow scientists by releasing software online, by delivering presentations at symposia, conferences and universities, and by publishing papers in peer-reviewed journals. We reached undergraduate and graduate students through classroom teaching, including guest lectures and panels at other universities. Additionally, via outreach, we reached several other target audiences. We reached high school students and their teachers via several virtual classroom visits. We also reached K-12 teachers through a workshop to develop classroom activities based on our research. We reached people in the social media network of the UNC Microbiome Club. Finally, we reached recipients of the Duke Forest newsletter, a general audience of about 1600 people, including recreational users of the Forest, neighbors, and local government officials. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, this project trained 1 postdoc, 2 graduate students, 2 undergraduate students, 1 technical IT staff, 2 lab managers, and 2 research technicians who were recent college graduates planning to pursue graduate work in ecology or related fields. Each undergraduate student, graduate student, and postdoc received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students and postdoc additionally received training in publishing research in scientific journals. Additionally, all students, postdocs and research technicians received mentoring in career development. GUEST LECTURES: Carbone presented a guest lecture on "Species delimitation methods accounting for gene flow" in the Systematic Biology course (BIOLOGY 556) at Duke University (Enrollment: 8 students, 3 credit hours) in Spring 2020 (on Mar 31). In that class' laboratory component Carbone also taught a full day workshop (on Apr 2) on using T-BAS and other tools in the DeCIFR toolkit. INVITED PRESENTATIONS: Mitchell, C.E. 2019. Parasite phenological responses to climate change: impacts on multi-species interactions and epidemics. Symposium on Disease Ecology and Evolution in a Changing World (the Triangle Center for Evolutionary Medicine, Duke University; September 26, 2019). O'Keeffe, K.R. 2019. Bryn Mawr College (September 16, 2019). O'Keeffe, K.R. 2020. Academy of Natural Sciences at Drexel University (Jul 24, 2020). Two additional invited presentations were impacted by COVID-19 by being postponed until after the end of this reporting period. CONTRIBUTED PRESENTATIONS: Harrington, A. H., Lutzoni, F., Carbone, I., Miadlikowska, J., Tanney, J. B., U'Ren, J. M. and A. E. Arnold. 2020. Unidentified barcode sequences: novel fungal diversity or artifacts of incomplete databases? MSA Virtual Meeting, "Mycology from the Cloud!" July 21-22. ADDITIONAL PROFESSIONAL DEVELOPMENT ACTIVITIES: Geyer completed a graduate course in professional development, UNC (Biweekly, January - April 2020). Geyer participated in a workshop for building CVs and Resumes, UNC Graduate School (1 day, February 2020). Geyer participated in a writing workshop, UNC Biology Graduate Student Association (1 day, January 2020). Geyer participated in the Beyond the Bench Seminar Series, the Graduate Business and Consulting Club, UNC (recurring monthly during the semester). Geyer participated in the First Friday Microbiome Seminar, UNC, (recurring monthly during the semester). Geyer served as the Social Media and Outreach Coordinator for the UNC Microbiome Club. This included helping coordinate and advertise a joint microbiome seminar series with UNC and MIT. Green participated in a virtual workshop on Diversity, Equity, and Inclusion in Socio-Environmental Synthesis Research. It included interactive activities and skills-based trainings spanning team science, interdisciplinary proposal writing, shared research design, science communication and delivering actionable outcomes. Hosted by the National Socio-Environmental Synthesis Center. Two hours per week for seven weeks beginning July 28, 2020. Grunberg attended the American Society of Naturalists Meeting (Asilomar, CA, January 2020). Grunberg served on the education committee for the American Society of Parasitologists. Mitchell, Geyer, Green, and Grunberg attended the Triangle Center for Evolutionary Medicine's Symposium on Disease Ecology and Evolution in a Changing World at Duke University 26 Sept 2019. How have the results been disseminated to communities of interest?We have eight outreach activities to report. Green participated in the SciREN (Scientific Research and Education Network) Lesson Plan Workshop, bringing together educators and researchers to turn research into lesson plans that fit within the North Carolina science education curriculum (Jan 25, 2020). Grunberg and Newberry participated in Parasite Week 2020 (in March) organized by the American Society of Parasitologists. They gave a two-day online presentation to a group of high school students, introducing them to the world of parasites in general, and to our study system and research more specifically. Mitchell participated in Duke Forest's online Ask A Scientist. Duke Forest solicited questions about our research project via their website and newsletter, which goes out to a general audience of about 1600 people, including recreational users of the Forest, neighbors, and local government officials. Then, Mitchell answered these questions in a half-hour discussion with Duke Forest staff. This discussion was posted in August 2020 and is available: https://www.youtube.com/watch?v=esP-DCkoXno. Newberry served as a guest panelist for Virtual Backyard Botany in April 2020 with a classroom of biology students from the University of Mount Olive. She shared her experiences in plant biology and how she arrived at her current position as a researcher in a plant disease ecology lab. She also helped students identify plants from photos and their surroundings. Newberry worked with UNC's Morehead Planetarium and Science Center to develop content for the exhibit that was proposed as this project's major outreach activity. It is scheduled to open in October 2020 (pending COVID-19 response) and anticipated to remain open for at least 3-5 years. O'Keeffe, as part of Skype a Scientist, conducted a video chat about ecology of infectious disease in spring 2020 with a 5th grade class in Los Angeles. O'Keeffe, as part of Skype a Scientist, conducted regular video chats in summer 2020 with two students in first grade, speaking with them about infectious diseases, climate change, and backyard ecology. O'Keeffe, as part of Skype a Scientist Live, spoke about her research on within-host microbial interactions and infectious disease on 7 October 2019. Skype A Scientist Live hosts live-streamed and recorded talks by scientists where students can submit questions beforehand. Its goal is to make connecting with scientists accessible to K12 classrooms, regardless of the quality of a class's internet connection. Her talk is available: https://www.youtube.com/watch?v=YkRToeBkBrw&feature=youtu.be. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test our overarching hypothesis that epidemics are commonly driven (and controlled) by microbial interactions, specifically testing the roles of population and community genetics, as well as community structure. With our mathematical model, we will conduct simulations to quantify how within-leaf parasite competition, combined with the degree of within-plant vs. between-plant transmission, influences the epidemics of two parasite species, particularly when one parasite species has a head-start in abundance each year owing to earlier phenology. Ultimately, this will allow us to make predictions of when and how competition within leaves can allow a parasite that establishes earlier in the host population to reduce the epidemic of a parasite that establishes later. To experimentally test whether within-host interactions among parasite species scale up to determine the size of epidemics, we will analyze our data from the tall fescue mesocosm experiment. We will test whether nitrogen enrichment alters the timing of infections by multiple parasite species, or interactions among parasite species within individual plants, by conducting an experiment in which individual potted tall fescue plants receive a range of nitrogen fertilizer rates and are deployed in the field as sentinel outplants to expose them to ambient transmission of parasites and other microbes. We will extend our work characterizing tall fescue's fungal leaf microbiome and its relationship with parasite infection to include the bacterial leaf microbiome, and the successional dynamics of the microbiome over time. We will expand our study of genetic variation in Rhizoctonia from high-throughput sequencing of multiple marker loci to a genome-wide scan using ddRADseq, then characterize genetic mechanisms of seasonal succession, including population genetic and community genetic responses to microbial interactions. Together, these activities will provide further insight into how microbial communities may be manipulated to slow or prevent parasite epidemics.
Impacts
What was accomplished under these goals?
This project investigated how transmission of microbial parasites across a population of hosts is influenced by the host individuals' microbiomes. To do this, we conducted surveys in the field, and experiments in the field and lab, with the grass tall fescue, and extended our empirical results using a mathematical model. In a field experiment, early infection of a plant by a parasite altered the subsequent progression over time of infections by multiple parasites. Our mathematical model predicted that, when a parasite that establishes in the host population early in the season competes within leaves with a parasite that establishes later in the season, it can reduce the epidemic of the later arriving parasite. Ultimately, this project contributed fundamental knowledge that can be used to develop targeted approaches that suppress parasite epidemics by leveraging the plant microbiome. Question 1 1-ACTIVITIES A-To test effects of parasite phenology on parasite epidemics, Newberry, Grunberg, Halliday, and O'Keeffe quantified plant community structure as well as Epichloë and parasite infections of tall fescue in the seasonal spray experiment. We ceased spray (fungicide) treatments and surveys after three growing seasons, in February 2020. In August 2020, we quantified parasite recolonization. COVID-19 prevented us from collecting parasite infection data between February and August 2020, and from hiring additional non-students and undergraduates to expand our activities. B-To test effects of Epichloë on parasite epidemics, our field experiment manipulating Epichloë infection of tall fescue populations needed liming, fertilization, and reseeding, but COVID-19 prevented this. C-To test effects of Colletotrichum and Rhizoctonia on each other's epidemics, Newberry, Knorr and Cook grew tall fescue populations in field mesocosms (kiddie pools), then factorially inoculated with Colletotrichum and Rhizoctonia. D-To explore shifts in the tall fescue leaf microbiome associated with fungal parasites, O'Keeffe Illumina-sequenced fungal ITS1 of naturally (un)infected leaf segments, then used DADA2 to analyze data. In this reporting period, O'Keeffe et al. published this study as a preprint, and submitted it to a journal, which reviewed it and invited us to revise and resubmit it. E-To determine the conditions under which parasites can suppress each other's epidemics, Umbanhowar refined the mathematical model to fully separate parasite transmission into two levels (within-plant and between-plant), then conducted numerical simulations. He also developed a deterministic approximation of the full stochastic model. F-To test effects of Epichloë, Colletotrichum and Rhizoctonia on parasite community assembly, Grunberg, Newberry, Knorr and Cook (mock) inoculated individual potted tall fescue in the lab, then deployed them in the field as sentinel outplants to expose them to ambient transmission. 2-DATA A-Seasonal spray experiment: infections of tall fescue by Epichloë and all parasites. Percent cover by plant species, plant community aboveground biomass at the end of the fall growing season. B-Epichloë field experiment: infections by Epichloë. C-Mesocosm experiment: infections by Epichloë and all parasites. D-Microbiome survey: abundances of 2961 unique amplicon sequencing variants in 252 leaf segments. E-Mathematical model: predicted population growth rates of each parasite. F- Outplant experiment: Longitudinal biweekly surveys (13 total) of parasite infections, including disease severity and numbers of lesions/pustules. Epichloë infection and aboveground biomass at end of experiment. 3-RESULTS A-Seasonal spray experiment: Excluding foliar fungal parasites did not affect percent cover of tall fescue, but increased plant community biomass and decreased plant species richness. B-Epichloë field experiment: Prevalence of Epichloë was high (68% even in the low-Epichloë treatment). C-Mesocosm experiment: Prevalences of the inoculated parasites were low in uninoculated controls. D-Microbiome survey: Leaf segments symptomatic of Rhizoctonia solani had lower fungal diversity and different fungal composition compared to segments that were asymptomatic or symptomatic of other parasites. E-Mathematical model: Parasite population growth rates were sensitive to the degree to which parasite phenology gives one species a head-start in population growth relative to its competitor. F-Outplant experiment: Parasite communities in plants inoculated with Colletotrichum, Rhizoctonia, or both parasites diverged over time from parasite communities in plants mock-inoculated with both parasites. 4-OUTCOMES A-The seasonal spray experiment indicated that fungal parasites decrease plant community biomass and help maintain plant species diversity. B-The Epichloë field experiment suggested that establishment of tall fescue from Epichloë-free seed requires greater soil fertility than from Epichloë-inoculated seed. C-The mesocosm experiment confirmed the utility of this approach to study experimental epidemics in the field. D-The microbiome survey supported the hypothesis that parasites that kill host tissue act as niche modifiers for the host's microbiome. E-The mathematical model predicted that competition within leaves can allow a parasite that establishes in the host population earlier to reduce the epidemic of a parasite that establishes later. F-The outplant experiment indicated that initial parasite infection can alter parasite community assembly within an individual plant. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetics and community genetic structure, O'Keeffe, Newberry, Knorr and Cook cultured fungi from Rhizoctonia lesions on tall fescue leaves in the seasonal spray experiment and the surrounding hayfield. Cultures that, based on both morphology and ITS, fell in the Rhizoctonia species complex were sequenced by Jones on Illumina MiSeq at five loci. Cornish, White and Carbone isolated DNA and built trees in T-BAS based on ITS and multi-locus haplotypes. 2-DATA For each culture of Rhizoctonia, we obtained data on morphotype, ITS sequence, and multi-locus haplotype. 3-RESULTS In nonexperimental areas of the Widener Farm hayfield, the Rhizoctonia population was spatially structured at a scale between about 10 and 100 meters. In the seasonal spray experiment (also located within the Widener Farm hayfield), the Rhizoctonia population was not spatially structured, and had reduced genetic variation. Intriguingly, some Rhizoctonia lineages may have incorporated genetic material from other fungi, including an endophyte of tall fescue and a hyperparasite of Rhizoctonia. 4-OUTCOMES Our results are consistent with genetic hitchhiking and the hypothesis that seasonal exclusion of foliar fungal parasites induced a selective sweep in Rhizoctonia. More generally, our results are helping define the spatial and temporal scales of genetic change in Rhizoctonia, suggest that its population is more genetically dynamic than expected. Question 3 1-ACTIVITIES, 2-DATA, 3-RESULTS, 4-OUTCOMES None new. We completed work on Question 3 in the previous reporting period with this publication: O'Keeffe, K.R., and C.D. Jones, 2019. Challenges and solutions for analysing dual RNA?seq data for non?model host-pathogen systems. Methods in Ecology and Evolution 10(3):401-414. (https://doi.org/10.1111/2041-210X.13135).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
F.W. Halliday, R.W. Heckman, P.A. Wilfahrt, C.E. Mitchell, 2020. Eutrophication, biodiversity loss, and species invasions modify the relationship between host and parasite richness during host community assembly. Global Change Biology 26(9):4854-4867. (https://doi.org/10.1111/gcb.15165).
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
OKeeffe, K.R., F.W. Halliday, C.D. Jones, Ignazio Carbone, C.E. Mitchell, 2020. Parasites as niche modifiers for the microbiome: A field test with multiple parasites. bioRxiv 2020.03.31.018713. (https://doi.org/10.1101/2020.03.31.018713).
|
Progress 09/01/18 to 08/31/19
Outputs
Target Audience:During this reporting period, we reached several target audiences. We reached fellow scientists by conducting a workshop at the annual meeting of the Mycological Society of America, releasing software online, delivering presentations at conferences and universities, and publishing papers in peer-reviewed journals. Additionally, via outreach, we reached several other target audiences. We reached high school students and their teachers via a classroom visit. We reached people in the social media network of the UNC Microbiome Club. Finally, we reached recipients of the Duke Forest newsletter, a general audience of about 1600 people, including recreational users of the Forest, neighbors, and local government officials. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, this project trained 1 postdoc, 2 graduate students, 4 undergraduate students, 2 technical IT staff, 2 lab managers, and 5 research technicians who were recent college graduates planning to pursue graduate work in ecology or related fields. Each undergraduate student, graduate student, and postdoc received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students and postdoc additionally received training in publishing research in scientific journals. Additionally, all students, postdocs and research technicians received mentoring in career development. Carbone organized a one-day workshop at the 2019 Mycological Society of America meeting (August 10-14, University of Minnesota, Minneapolis, MN) to highlight the tools available in DeCIFR, including T-BAS and others; introduce users to example analyses; and provide an opportunity for users to upload their own phylogenetic trees and analyze their own data sets. Graduate students, postdocs, faculty, and other mycologists/fungal biologists participated (14 participants). In terms of professional development, during this reporting period, this project supported the following oral and poster presentations at conferences and universities. Invited presentations: Halliday, F.W. 2019. Biotic and abiotic drivers of disease risk across space and time. University of Zurich: Behaviour, Ecology, Environment, and Evolution Seminar. Halliday, F.W. 2019. Biotic and abiotic drivers of disease risk across space and time. University of Bern. Contributed presentations: Cullen, M., Jacob, M. E., Cornish, V., VanderSchel, I. Q., Cotter, H. V. T., Cubeta, M. A., Carbone, I. and B. C. Gilger. 2019. Association of fungal species, in vitro susceptibility, and in vivo antifungal agent efficacy for isolates sampled from equine fungal keratitis. 2019 ARVO Annual Meeting, Vancouver, Canada, April 28 - May 2, 2019 Halliday, FW. 2019. Biotic and abiotic drivers of disease risk in wild plant communities. Wild Plant Pathosystems Conference, Schmitten, Germany (Oral Presentation). Luis, J. M. S., Carbone, I., Heiniger, R., Weaver, M. A., Abbas, H. K., Allen, T., Isakeit, T. S., Bowen, K. L. and P. S. Ojiambo. 2019. Genetic factors influencing the efficacy and persistence of biocontrol of aflatoxin contamination in maize. The APS Annual Meeting. Cleveland, Ohio. Aug 3 - 7. O'Keeffe, K., F.W. Halliday, C.D. Jones, I. Carbone, and C.E. Mitchell, 2019. The fungal microbiome of a grass host under natural infections by diverse parasites. Ecology & Evolution of Infectious Diseases Conference (17th Annual), Princeton University (Poster presentation). This project provided other professional development activities for several participants: Mitchell, Halliday, and O'Keeffe attended the 17th Ecology and Evolution of Infectious Disease (EEID) Conference at Princeton University 10-13 June 2019. O'Keeffe had a UNC ImPACT internship from TIBBS, which provided support to teach an introductory biology course for non-majors at Meredith College (Fall 2018). Newberry completed a one-day UNC training course in Mental Health First Aid. Geyer completed a one-day UNC workshop on next-generation sequencing (Fall 2018). Geyer attended UNC's First Friday Microbiome Seminar (recurring monthly seminar). Geyer attended UNC's Global Health Forum (recurring monthly forum). How have the results been disseminated to communities of interest?We have four outreach activities to report. First, Duke Forest Teaching and Research Laboratory (site of our research) published a Research Update on this project in their newsletter on 1 August 2019. Their newsletter goes out to a general audience of about 1600 people, including recreational users of the Forest, neighbors, and local government officials. Second, the Carbone Lab participated in the NC State CAALS 3D (Creating Awareness of Agriculture and Life Sciences Disciplines, Degree Programs and Discoveries) outreach program (July 17-18, 2019). Students received wet lab and computational training on methods for next generation sequencing, data analysis and visualization using T-BAS and DeCIFR tools (2 participants). Third, O'Keeffe spoke to a club for females interested in STEM (FemStem) at Chapel Hill High School about her research on this project, her career path, and what it's like to be a scientist (March 19, 2019). Finally, Geyer served as the Social Media and Outreach Coordinator for the UNC Microbiome Club. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test our overarching hypothesis that epidemics are commonly driven (and controlled) by microbial interactions, and specifically testing the roles of population and community genetics, as well as community structure. Now that the seasonal spray experiment has run for three growing seasons, we will critically assess it and decide whether it needs to be continued in its current form. We will reseed the Epichloë field experiment to increase the number of Epichloë-free plants. We will further analyze and perhaps elaborate our mathematical model to dissect the roles of leaf-level, plant-level, and host population-level parasite interactions in epidemics. We will extend our work characterizing the fungal leaf microbiome and its relationship with parasite infection to include the bacterial leaf microbiome, and the successional dynamics of the microbiome over time. Based on our recent work showing that nutrient addition can drive epidemics in host communities dominated by tall fescue (Halliday et al. 2019), we will conduct experiments to dissect the underlying processes at the levels of the tall fescue individual and population. We will conduct a simplified version of the proposed parasite density - population growth experiment to quantify the joint effects of Epichloë, Colletotrichum, and Rhizoctonia infection on susceptibility of individual plants to natural transmission of parasites under field conditions. We will continue to characterize genetic mechanisms of seasonal succession, including population genetic and community genetic responses to microbial interactions. To more directly test the hypothesis of plot-scale clonal reproduction by Rhizoctonia, we will analyze mating types. Together, these activities will provide further insight into how microbial communities may be manipulated to slow or prevent parasite epidemics.
Impacts
What was accomplished under these goals?
This project investigated how transmission of microbial parasites across a population of hosts is influenced by the hosts' microbiomes. To do this, we conducted field experiments, field surveys, and a lab experiment with the grass tall fescue, and extended our empirical results using a mathematical model. A field survey of the leaf microbiome indicated that parasite species that kill host tissue modify the microbiome more than other parasites. A ubiquitous component of the tall fescue microbiome is a non-parasitic fungus transmitted in the plant's seed. A lab experiment demonstrated that excluding this fungus from the host microbiome ameliorated the combined impact of two parasite species on host growth and survival. Ultimately, this project contributed fundamental knowledge that can be used to develop targeted approaches that suppress parasite epidemics by leveraging the plant microbiome. Question 1 1-ACTIVITIES A-To test effects of parasite phenology on parasite epidemics, Halliday and O'Keeffe (with Newberry, Knorr and Cook) continued to survey parasite and Epichloë infections in the seasonal spray experiment. To quantify associations between parasite infection and the root microbiome, Rúa microscopically examined roots of outplants deployed in that experiment in 2017. B-To test effects of Epichloë on parasite epidemics, Newberry, Knorr and Cook surveyed our field experiment manipulating Epichloë infection of host populations, using immunoblot to quantify prevalence of Epichloë infection. C-To test effects of Colletotrichum and Rhizoctonia on each other's' epidemics, Newberry, Knorr and Cook grew tall fescue in field mesocosms (kiddie pools with 13 multi-tillered plants per pool, and 12 replicates per treatment), then factorially inoculated with Colletotrichum and Rhizoctonia. The purpose of the pools was to reduce transmission from outside each pool; to check this, additional pools were left uninoculated. D-To explore shifts in the leaf microbiome associated with fungal parasites, O'Keeffe Illumina-sequenced fungal ITS1 of naturally (un)infected leaf segments, then used DADA2 to analyze data. E-To determine the conditions under which parasites can suppress each other's epidemics, Umbanhowar elaborated the mathematical model to include two levels of parasite transmission, across the host population and among leaves on the same host plant, then conducted preliminary simulations. F-To test whether an endophyte can modify interactions between two parasites, O'Keeffe conducted a growth chamber experiment that factorially inoculated plants with Epichloë, Colletotrichum and Rhizoctonia. 2-DATA A-Seasonal spray experiment: (i) monthly and mid-monthly prevalence of parasite infections, stratified by tiller and leaf relative age, (ii) infection by Epichloë (lab tests ongoing), (iii) infection of roots by arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE). B-Epichloë field experiment: Epichloë infection status for 5 tillers per plot (600 tillers total). C-Pool experiment: infection of each plant by Epichloë, parasite infection prevalence each week, and parasite infection histories of individual leaves (including infection severity). D-Microbiome survey: abundances of 2961 unique amplicon sequencing variants in 252 leaf segments. E-Mathematical model: predicted population growth rates of each parasite. F-Growth chamber experiment: plant survival, biomass, and infection with Epichloë, Colletotrichum and Rhizoctonia. 3-RESULTS A-Seasonal spray experiment: Tall fescue roots were lightly (30% colonization) infected by AMF, and more heavily infected by DSE. B- Epichloë field experiment: Prevalence of Epichloë infection was >95%. C-Pool experiment: Since the experiment was only harvested 3-4 Sept 2019, we do not have results yet. D-Microbiome survey: Leaf segments symptomatic of Rhizoctonia solani had lower fungal diversity and different fungal composition compared to segments that were asymptomatic or symptomatic of other parasites. E-Mathematical model: Parasite population growth rates were sensitive to the relative amount of parasite transmission among leaves on the same plant. F-Growth chamber experiment: Epichloë -infected plants that were co-inoculated with both parasites had lower survival and biomass. 4-OUTCOMES A-The seasonal spray experiment indicated that dark septate endophytes may be an important component of the tall fescue microbiome. B-We determined that we need to reseed the Epichloë field experiment to increase the number of Epichloë-free plants. C-The pool experiment confirmed the utility of this mesocosm approach. D-The microbiome survey supported the hypothesis that parasites that kill host tissue act as niche modifiers for the host's microbiome. E-The mathematical model suggested that whether a parasite epidemic is suppressed by another parasite may depend on the relative amount of parasite transmission among leaves on the same plant. F-The growth chamber experiment demonstrated that an endophyte can modify interactions between two parasites, to the detriment of the host. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetics and community genetic structure, O'Keeffe, Newberry, Knorr and Cook cultured fungi from leaves symptomatic of Rhizoctonia in the seasonal spray experiment and the surrounding hayfield. Cultures that, based on both morphology and ITS, fell in the Rhizoctonia species complex were sequenced by Carbone's group at five loci. Carbone built trees in T-BAS based on ITS and multi-locus haplotypes. 2-DATA For each culture of Rhizoctonia, we obtained data on morphotype, ITS sequence, and multi-locus haplotype. 3-RESULTS The Rhizoctonia population was somewhat genetically differentiated among plots. Within plots, genetic variation was generally consistent with clonal reproduction. On the other hand, we also detected signs of genetic exchange and recombination. Over time, we detected some shifts in population genetic structure on a relatively short timescale of a few weeks. 4-OUTCOMES Results to date indicate that our approach will allow us to characterize genetic mechanisms of seasonal succession, including population genetic and community genetic responses of the Rhizoctonia species complex to other parasite species. Question 3 1-ACTIVITIES To test whether RNA-seq can be used for symbiotic species without a reference genome, O'Keeffe and Jones computationally simulated RNA-seq of a fungus-infected plant, and mapped the reads to reference genomes of four species varying in evolutionary distance, using four different aligners. New in this reporting period: the manuscript was revised, resubmitted, accepted and published (O'Keeffe and Jones, 2018). 2-DATA Data was collected on the proportion of simulated RNA-seq reads and contigs that mapped to each reference genome. 3-RESULTS Mapping RNA-seq reads to a congeneric reference genome instead of a conspecific reference genome resulted in either a failure to map the reads (with two aligners) or (with the other two aligners) mismapping of reads from plant RNA to the fungal reference genome. Mismapping was prevented by first performing a de novo assembly of the reads into contigs. 4-OUTCOMES The RNA-seq simulations revealed a previously unappreciated problem: mapped RNA-seq reads from fungal infections of plants can be from plant RNA rather than fungal RNA, compromising the integrity of the data. Further simulations and analysis demonstrated a solution to that problem.
Publications
|
Progress 09/01/17 to 08/31/18
Outputs
Target Audience:During this reporting period, we reached at least three target audiences: fellow scientists, K-12 teachers, and middle school students. Specifically, we reached fellow scientists by publishing papers in peer-reviewed journals. Additionally, via outreach, we reached other target audiences, including both K-12 teachers and middle school students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During this reporting period, this project trained 3 graduate students, 4 undergraduate students, 3 technical IT staff, and 7 research technicians who are recent college graduates planning to pursue graduate work in ecology or related fields. Each undergraduate and graduate student received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students additionally received training in publishing research in scientific journals. Additionally, all students and research technicians received mentoring in career development. In terms of professional development, during this reporting period, this project supported the following oral and poster presentations at conferences and universities. Invited presentations: O'Keeffe, K.R. 2018. The effect of within-host microbial interactions on disease: from parasite individuals to populations. Invited oral presentation at the UNC Department of Biology Annual Symposium (one of two invited graduate student speakers out of 100+ graduate students). Mitchell, C.E. 2018. Pathogen species interactions across scales. North Carolina State University (Department of Entomology and Plant Pathology). Mitchell, C.E. 2018. Symbiont species interactions across scales. Rice University (Department of BioSciences series in Ecology and Evolutionary Biology). O'Keeffe, K.R. 2018. The effect of within-host microbial interactions on disease: from parasite individuals to populations. Stanford University (Department of Biology, ecology group). Halliday, FW. 2017. Diversity and interactions of foliar fungal parasites, from host organs to communities. University of Virginia (Department of Biology EBio Seminar Series). Contributed presentations: O'Keeffe, K., B.T. Wheeler, and C.E. Mitchell, 2018. A defensive symbiont may decrease the spread of a fungal plant pathogen through a host population but not within hosts. Ecological Society of America, New Orleans LA (Oral presentation). Halliday, F.W., J. Umbanhowar, C.E. Mitchell, 2018. A defense hormone and viral infection modify parasite epidemics and within-host priority effects in a grass host. Ecology & Evolution of Infectious Diseases Conference (16th Annual), University of Glasgow (Poster presentation). O'Keeffe, K., B.T. Wheeler, and C.E. Mitchell, 2018. A defensive symbiont decreases the spread of a fungal plant pathogen through a host population but not within hosts. Ecology & Evolution of Infectious Diseases Conference (16th Annual), University of Glasgow (Poster presentation). This project provided professional development activities for several participants: Mitchell, Halliday, and O'Keeffe attended the 16th Ecology and Evolution of Infectious Disease (EEID) Conference in Glasgow Scotland 29 May - 1 June 2018. Mitchell, Halliday, and O'Keeffe attended Duke University Symposium on Disease and Health: ecological perspectives from individuals to ecosystems 12 April 2018. Halliday attended one-day workshop on Presenting Data and Information by Edward Tufte, Raleigh, NC. O'Keeffe participated in the 2017 UNC 3 Minute Thesis Competition, in which she had to describe her dissertation research in under 3 minutes for a broad audience. O'Keeffe took a class in Fall 2017 through UNC's SPIRE postdoc program on college teaching practices. Geyer completed a one-day UNC workshop on using QIIME2 to process and analyze prokaryotic 16S microbiome sequencing data. Geyer completed a one-day UNC workshop on using the UNC computing cluster, Longleaf. How have the results been disseminated to communities of interest?Through the SciMatch program run through the NC Science Festival, O'Keeffe was connected to a middle school teacher at York Chester Middle School in Gastonia, North Carolina. In May 2018, she visited (in person, a 2.5 hour trip each way) three of the teacher's classes and taught students about epidemics with a lesson plan she had previously designed, and also discussed her research. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test our overarching hypothesis that epidemics are commonly driven (and controlled) by microbial interactions, and specifically testing the roles of population and community genetics, individual plasticity, and community structure. We will continue the seasonal spray experiment and the Epichloë field experiment. To test the role of microbial interactions in parasite epidemics at a more tractable scale under field conditions, we will conduct further kiddie pool experiments that build on O'Keeffe's pool experiments with Epichloë and Rhizoctonia. To improve on the less-than-expected percent of Epichloë-inoculated plants that tested positive for Epichloë infection, we will consult with experts on Epichloë, including our seed source at the University of Kentucky. To test whether Epichloë can modulate interactions between Rhizoctonia and Colletotrichum within a leaf, including responses of Rhizoctonia individuals, we will conduct a growth chamber experiment. We will further analyze and perhaps elaborate our mathematical model to dissect the roles of leaf-level and host population-level parasite interactions in epidemics. We will extend our work characterizing the fungal leaf microbiome and its relationship with parasite infection to include the prokaryotic leaf microbiome. We will continue to characterize genetic mechanisms of seasonal succession, including population genetic and community genetic responses to microbial interactions.
Impacts
What was accomplished under these goals?
This project investigated how transmission of microbial parasites across a population of hosts is influenced by differences between host individuals in their microbiomes. To do this, we conducted several field experiments and field surveys with the grass tall fescue, and extended our field results using a mathematical model. To understand the genetic basis of effects on parasite transmission, we developed new ways to analyze genetic shifts across known microbial populations and species, and new ways to analyze the genetic diversity of unknown microbial populations and species. To test the effects of shifts in the level of expression of microbial genes on parasite transmission, we identified a previously overlooked problem that may have undermined previous studies, and found a way for future studies to avoid that problem. Ultimately, this project contributed fundamental knowledge that can be used to develop targeted approaches that suppress parasite epidemics by leveraging the plant microbiome. Question 1 1-ACTIVITIES A-To test effects of parasite phenology on parasite epidemics, Halliday and O'Keeffe (with Newberry, Crews, Long, and Muirhead) continued the seasonal spray experiment, surveying leaves within tillers for parasite infection and leaf relative age each month year-round. We longitudinally tracked parasite infections of each leaf on 1152 tillers weekly June-October; each tiller was harvested and is being tested for infection by the non-parasite Epichloë. B-To test effects of Epichloë on parasite epidemics, we planted our proposed field experiment manipulating Epichloë infection of host populations, at three levels: 99%, 55%, and 10% of seed from plants putatively infected by Epichloë. Forty replicates per treatment yielded 120 fully randomized plots (each 2x6m). C-To test effects of Epichloë on Rhizoctonia epidemics, O'Keeffe and Wheeler grew either Epichloë-inoculated or Epichloë-free tall fescue in kiddie pools (13 multi-tillered plants per pool, and 13 replicates per treatment), then inoculated with Rhizoctonia. The purpose of the pools was to reduce transmission from outside each pool; to check this, additional pools were left uninoculated with Rhizoctonia. Twice weekly, we surveyed seven leaves per plant for infection by each parasite. Weekly, we also surveyed one tagged tiller per plant for percent leaf area infected. D-To explore shifts in the leaf microbiome associated with fungal parasites, we Illumina-sequenced fungal ITS1 of naturally (un)infected leaf sections. Now O'Keeffe is applying a pipeline developed by Carbone's group in T-BAS (see Other Products): First BLAST to place each sequence in a fungal family, then place it within that family's phylogeny, then use DADA2 (see Other Products) to test hypotheses using all unique sequence variants (not OTUs). E-To determine the conditions under which parasites can suppress each other's epidemics, Umbanhowar revised the structure of the mathematical model to better examine seasonal dynamics. He then performed a nullcline analysis. 2-DATA A-In the seasonal spray experiment, we collected data on: (i) monthly prevalence of parasite infections, stratified by tiller and leaf relative age, (ii) weekly parasite infection histories of individual leaves, and (iii) infection of each tagged tiller by Epichloë (lab tests ongoing). B-In the Epichloë field experiment, we observed the density and growth of fescue. C-In the pool experiment, we collected data on infection prevalence, and parasite infection histories of individual leaves (including infection severity). Lab tests are ongoing for infection of each plant by Epichloë. D-In the microbiome survey, we obtained data on genetic sequences of foliar fungi at ITS1. E-In the mathematical model, we observed predicted population growth rates of each parasite. 3-RESULTS A-In the seasonal spray experiment, the monthly prevalence survey yielded 768 plot-level observations based on about 61,000 leaf-level observations (stratified by tiller and leaf relative age). We obtained about 10,000 infection histories of individual leaves. We are still testing tillers for Epichloë. B-In the Epichloë field experiment, a tall fescue population established in each plot. Populations varied in density; most appeared modest but adequate. Individual plants produced up to 12 tillers. C-In the pool experiment, the prevalence survey yielded 208 pool-level observations based on 2704 plant-level observations. We obtained 944 infection histories of individual leaves. Of Epichloë-inoculated plants tested for Epichloë so far, 44% tested positive; this is less than expected, but seems adequate. The pools largely prevented colonization by parasites except Puccinia and Magnaporthe grisea (cause of gray leaf spot on tall fescue). D-In the microbiome survey, we obtained 6,650,600 paired-end reads. DADA2 (see Other Products) detected 3659 unique sequencing variants. Data analysis is ongoing. E-In the mathematical model, the nullcline analysis indicated that the parasite species can compete both at the level of a leaf and at the level of the host population. 4-OUTCOMES We made substantial progress by establishing our proposed field experiment, conducting the second year of the seasonal spray experiment, and moving three other activities toward completion. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetics and community genetic structure, O'Keeffe cultured fungi from symptomatic leaves of tagged tillers in the seasonal spray experiment. Cultures that, based on both morphology ITS, fell in the Rhizoctonia species complex are being sequenced by Carbone's group at multiple loci. 2-DATA For each culture, morphospecies was recorded. ITS sequences were obtained, and ongoing sequencing is yielding multi-locus haplotypes, including multiple alleles, if present, in a single individual. 3-RESULTS The seasonal spray experiment yielded 78 cultures of the target parasite taxon, the Rhizoctonia species complex. However, very few of the tagged tillers tested negative for Epichloë. 4-OUTCOMES This year's approach of using tagged tillers yielded a much larger sample of the target parasite taxon, but a surprising lack of hosts uninfected by Epichloë. These samples may not allow us to test effects of Epichloë, but should allow us to characterize genetic mechanisms of seasonal succession, including population genetic and community genetic responses of the Rhizoctonia species complex to other parasite taxa. Question 3 1-ACTIVITIES To test whether RNA-seq can be used for symbiotic species without a reference genome, O'Keeffe and Jones computationally simulated RNA-seq of a fungus-infected plant, and mapped the reads to reference genomes of four species varying in evolutionary distance, using four different aligners. New in this reporting period: the fourth aligner (MapSplice2) was analyzed, and the manuscript was submitted, revised, then accepted. 2-DATA Data was collected on the proportion of simulated RNA-seq reads and contigs that mapped to each reference genome. 3-RESULTS Mapping RNA-seq reads to a congeneric reference genome instead of a conspecific reference genome - as is common practice with non-model organisms - resulted in either a failure to map the reads (with two aligners) or (with the other two aligners) mismapping of reads from plant RNA to the fungal reference genome. Mismapping was prevented by first performing a de novo assembly of the reads into contigs. 4-OUTCOMES The RNA-seq simulations revealed a previously unappreciated problem: mapped RNA-seq reads from fungal infections of plants can be from plant RNA rather than fungal RNA, compromising the integrity of the data. Further simulations and analysis then demonstrated a solution that problem (O'Keeffe and Jones, in press).
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
OKeeffe, K.R., and C.D. Jones, in press. Challenges and Solutions for Analyzing Dual RNA-seq Data for Non-Model Host/Pathogen Systems. Methods in Ecology and Evolution.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
F.W. Halliday, J. Umbanhowar, C.E. Mitchell, in press. A host immune hormone modifies parasite species interactions and epidemics: insights from a field manipulation. Proceedings of the Royal Society B. (https://doi.org/10.1098/rspb.2018.2075).
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
F.W. Halliday, R.W. Heckman, P.A. Wilfahrt, C.E. Mitchell, in press. Past is prologue: Host community assembly and the risk of infectious disease over time. Ecology Letters. (https://doi.org/10.1111/ele.13176).
|
Progress 09/01/16 to 08/31/17
Outputs
Target Audience:During this reporting period, we reached at least three target audiences: fellow scientists, K-12 teachers, and middle school students. Specifically, among scientists, we reached plant biologists by publishing O'Keeffe et al. 2017 in Current Opinion in Plant Biology, and we reached ecologists by publishing Halliday et al. 2017 in Ecology Letters. Additionally, via outreach, we reached other target audiences, including both K-12 teachers and middle school students. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In terms of training, during this reporting period, this project trained 3 graduate students, 2 undergraduate students, 2 technical IT staff, and 3 research technicians who are recent college graduates planning to pursue graduate work in ecology. Each undergraduate and graduate student received training, to varying degrees, in field experiments, laboratory procedures, and computational approaches. The graduate students additionally received training in publishing research in scientific journals. Additionally, all students and research technicians received mentoring on career development. Finally, UNC PhD student Kayleigh O'Keeffe completed training on science communication (IMPACTS, based on the NSF-funded program, Portal to the Public) provided by UNC's Morehead Planetarium and Science Center, the project's partner in outreach. In terms of professional development, during this reporting period, this project supported the following oral and poster presentations at conferences and universities. Invited presentations: Mitchell, C.E., 2017: Parasite species interactions across scales. Fudan University, Shanghai (School of Life Sciences). Mitchell, C.E., J. Umbanhowar, 2017. Parasite species interactions and epidemics. SIAM (Society for Industrial and Applied Mathematics) Central States Section Conference. Colorado State University. Mitchell, C.E., F.W. Halliday, K.R. O'Keeffe, A. Simha, J. Umbanhowar, 2017. Parasite species interactions across spatial and temporal scales. Ecology & Evolution of Infectious Diseases Conference. University of California - Santa Barbara. Contributed presentations: O'Keeffe, K. and C.E. Mitchell, 2017. Effects of endophyte symbiosis and pathogen intraspecific variation on the growth of a fungal plant pathogen. Ecological Society of America, Portland OR (Poster presentation). O'Keeffe, K., C.D. Jones, C.E. Mitchell, 2017. Challenges for analyzing dual RNA-seq data for non-model host/pathogen systems. Ecology & Evolution of Infectious Diseases Conference (15th Annual), University of California - Santa Barbara (Poster presentation). How have the results been disseminated to communities of interest?During the reporting period, the project's three outreach activities were all conducted by UNC PhD student Kayleigh O'Keeffe. SciREN (The Scientific Research and Education Network): Kayleigh designed a lesson plan for educators based on our research project. She then took the lesson plan to the 2016 SciREN Triangle event. At the event, approximately 30-35 NC teachers (chiefly middle school) approached her to discuss the lesson plan, expressing interest in adopting it for their classrooms. Skype-a-scientist: Kayleigh participated in Skype-a-scientist throughout the 2016-2017 school year. She Skyped with 3 middle school classes, each with 15-20 students, at a charter school in Kentucky, and discussed what it's like to be a scientist, how she got to be a PhD student at UNC working on plant disease, and what her research is on. Scientists of NC: Kayleigh was featured on this online project (modeled on the popular Humans of New York), which has hundreds of followers and has the goal of humanizing scientists. The feature discussed topics including how she chose her career path, some of the challenges of science, and how she spends her free time. What do you plan to do during the next reporting period to accomplish the goals?We will continue to test our overarching hypothesis that epidemics are commonly driven (and controlled) by microbial interactions, and specifically testing the roles of individual plasticity, population genetics, and community structure. To do this, our main approach will be to conduct further experiments, both in growth chambers and in the field. We will extend the results of our experiments using observational data and mathematical models.
Impacts
What was accomplished under these goals?
For the grass tall fescue, grown for hay, a field experiment demonstrated that a parasite that establishes first in a host population can obtain a head-start advantage over other parasites. Specifically, establishing first can allow that parasite to (1) infect leaves prior to other parasites, (2) thereby alter the composition of the leaf microbiome, and (3) ultimately suppress epidemics by other parasites. Which parasites had epidemics was determined by the order in which the parasites established. Therefore, which parasites have epidemics may be altered by factors that change the order in which parasites establish. Such factors could include weather, no-till farming, planting date, and timing of pesticide applications. This project provides a potentially general principle that may be applied to suppress parasite epidemics by leveraging the plant microbiome. Question 1 1-ACTIVITIES This grant supported publication of Halliday et al. 2017 in Ecology Letters (above impact statement). To extend Halliday et al. 2017, UNC PhD students Fletcher Halliday and Kayleigh O'Keeffe attempted to manipulate the order in which parasites infected host populations (the "seasonal spray experiment"). For each of three focal parasites, a corresponding treatment sprayed replicate host populations (plots) with fungicide until that parasite's epidemic started. In the 64 plots, we deployed 1712 Epichloë-inoculated and Epichloë-free sentinel hosts in 3 cohorts. To execute our proposed field experiment manipulating Epichloë infection of replicate host populations, we moved forward on two fronts. (1) We planted a pilot experiment. (2) UNC PhD student Bradley Saul used a power analysis to estimate our ability to test the hypothesis that a defensive symbiont can indirectly protect symbiont-uninfected hosts from parasite transmission. To test the effects of Epichloë on Rhizoctonia epidemics, UNC undergraduate Brandon Wheeler, mentored by Kayleigh O'Keeffe, grew Epichloë-inoculated and Epichloë-free tall fescue in replicate kiddie pools, then inoculated them with Rhizoctonia. The purpose of the pools was to reduce transmission from outside each pool; to check this, additional pools were left uninoculated with Rhizoctonia. To investigate shifts in the leaf microbiome associated with infection by fungal parasites, we are using a metabarcoding approach. O'Keeffe and Halliday collected 420 leaf sections infected and uninfected by each of our three focal parasites from 60 gridded locations in the field. We are now extracting DNA; we will characterize the fungal microbiome on Illumina. To extend Halliday et al. 2017 by determining the conditions under which Colletotrichum and Rhizoctonia can suppress each other's epidemics, co-PD James Umbanhowar developed a mathematical model. 2-DATA In the seasonal spray experiment, we collected data on: (A) leaf-level parasite infection of each sentinel plant (weekly longitudinal survey), (B) infection of each sentinel plant by Epichloë (end-of-season harvest; preserved for testing), and (C) leaf-level parasite infection of resident plants (monthly), (D) sentinel plant root biomass and colonization by arbuscular mycorrhizal fungi (end-of-season harvest; shipped to collaborator Megan Rúa). In the pilot field experiment, we observed the density of fescue and other plant species. In the pool experiment, we collected data on the proportion of leaves infected by each parasite (weekly). At the end of the season, we also surveyed 5 leaves per plant for percent leaf area infected by each parasite. 3-RESULTS In the seasonal spray experiment, the surveys of infection of resident plants yielded 23,000 observations. The surveys of sentinel plants yielded 100,000 observations. In the pilot field experiment, the density of fescue was low relative other plant species. Therefore, we are conducting a pilot 2.0, and seeding earlier. Based on the power analysis, we will increase replication to at least 100 plots. The pools were highly effective at preventing colonization by all parasites except Puccinia. The weekly survey yielded 3900 observations. The severity survey yielded 1300 observations. This experiment also serves as a pilot for the parasite density - population growth experiments. In the metabarcoding survey, we collected data on cover of tall fescue at each of the 60 locations. In the mathematical model, if there is no soil transmission of Rhizoctonia, alternative stable states are possible. If there is soil transmission, its rate determines whether there is stable coexistence or whether Rhizoctonia excludes Colletotrichum. This non-spatial model can be expanded into a spatially explicit dynamical model. 4-OUTCOMES Halliday et al. 2017 described a major change in knowledge regarding the role of the microbiome in determining which parasites can have epidemics. Question 2 1-ACTIVITIES To quantify effects of microbial interactions on population genetics, O'Keeffe cultured fungi from symptomatic leaves of sentinel plants in the seasonal spray experiment. O'Keeffe et al. 2017 synthesizes scientific understanding on population genetics and on phenotypic plasticity, arguing that studies directly integrating both areas are required. 2-DATA For each culture from the seasonal spray experiment, morphospecies was recorded. Amplicon sequencing will be used to distinguish fungal taxa. 3-RESULTS The seasonal spray experiment yielded 250 cultures. Cultures will be sequenced at multiple loci, and we will use coalescent models to characterize population genetic responses to microbial interactions. 4-OUTCOMES O'Keeffe et al. 2017 describes a change in knowledge regarding the scientific approaches required to understand the evolutionary consequences of interactions in the plant microbiome. Question 3 1-ACTIVITIES To test whether RNA-seq can be used for symbiotic species without a reference genome, O'Keeffe (with co-PD Jones) computationally simulated RNA-seq of a fungus infecting a plant, and mapped the reads to reference genomes of four species varying in evolutionary distance, using three different aligners. To test the importance of the order in which parasites infect a leaf, UNC undergraduate Anita Simha (mentored by O'Keeffe) conducted a growth chamber experiment (GCE1) manipulating the order of infection by Rhizoctonia and Colletotrichum. To test for genetic variation in microbial interactions, O'Keeffe conducted a growth chamber experiment (GCE2) in which plants were grown from endophyte-inoculated or endophyte-free seed and exposed to one of three isolates of Rhizoctonia. 2-DATA In the computational study of RNA-seq, data was collected on the proportion of simulated RNA-seq reads and contigs that mapped to each reference genome. In both growth chamber experiments, data was collected on the length of each Rhizoctonia lesion (daily). 3-RESULTS Mapping RNA-seq reads to a congeneric reference genome instead of a conspecific reference genome - as is common practice with non-model organisms - resulted in either a failure to map the reads (with one aligner) or (with two aligners) mismapping of reads from plant RNA to the fungal reference genome. Mismapping was prevented by first performing a de novo assembly of the reads into contigs. In GCE1, Colletotrichum facilitated Rhizoctonia. The strength of facilitation depended on order of arrival. In GCE2, the Rhizoctonia isolates differed in lesion growth rate, but this was not influenced by Epichloë. 4-OUTCOMES The RNA-seq simulations revealed a previously unappreciated problem: mapped RNA-seq reads from fungal infections of plants can be from plant RNA rather than fungal RNA, compromising the integrity of the data. Further simulations and analysis then demonstrated a solution that problem. Together, these results comprise a substantial change in knowledge (O'Keeffe and Jones, manuscript in preparation).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
F.W. Halliday, J. Umbanhowar, C.E. Mitchell, 2017. Interactions among symbionts operate across scales to influence parasite epidemics. Ecology Letters 20(10):12851294. (http://dx.doi.org/10.1111/ele.12825).
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
OKeeffe, K.R., Ignazio Carbone, C.D. Jones, C.E. Mitchell, 2017. Plastic potential: how the phenotypes and adaptations of pathogens are influenced by microbial interactions within plants. Current Opinion in Plant Biology 38:7883. (https://doi.org/10.1016/j.pbi.2017.04.014). (Invited).
- Type:
Websites
Status:
Published
Year Published:
2016
Citation:
http://fescuefungi.org/
|
|