Performing Department
(N/A)
Non Technical Summary
Invasive plants pose an unprecedented threat to the ecological, environmental, and economic well-being of natural lands and agricultural systems globally. The management of invasive plants is divided broadly into biological, chemical, and mechanical control techniques and successful strategies typically combine several of these methods. Of particular interest are biological control agents (also known as biocontrols), which are the targeted introduction of natural enemies of invasive species with the intention of reducing or eliminating populations through, for example, herbivory. The establishment of biocontrol agents, however, remains a significant challenge and very little is known about the molecular basis for the interaction between insect herbivores and plants, The application of genomic techniques can be used to improve the suitability of biocontrol agents to their intended targets.The invasive fern Lygodium microphyllum poses significant threats to vulnerable and natural ecosystems and agricultural systems including pine logging activities and ranchlands. Despite the application of chemical, mechanical, and biocontrol methods, current procedures are insufficient for the management of L. microphyllum. Two biocontrol agents are currently employed to manageL. microphyllum, though some populations are resistant to their impacts while others are highly susceptible.We will sequence and compare the genomes of resistant and susceptible varieties of L. microphyllum to determine which parts of the genome may be involved in the herbivore resistance. Through experiments, we will also determine how and when different genes are expressed relative to the presence of the biocontrol and how the biocontrol impacts the health of the plant. Through the development of new genomic resources, we will examine how to improve biocontrol efficacy. The results of this research will provide a valuable foundation for enhancing our understanding of plant-insect interactions and how to improve the suitability of biocontrol agents on invasive plants for the purpose of management.
Animal Health Component
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Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Invasive species pose an unprecedented threat to the ecological, environmental, and economic well-being of natural lands and agricultural systems globally. The establishment of biocontrol agents for the management of invasive taxa, however, remains a significant challenge, especially for plants that have evolved efficient defense responses. This project will use functional genomics to assess the suitability of biocontrol agents to the invasive fern Lygodium microphyllum. We will explore the genetic underpinnings of resistance and susceptibility of L. microphyllum to the biocontrol agents Floracarus perrepae and Neomusotima conspurcatalis and the effect of these biocontrols on the health and performance of the invasive plant. Our ultimate goal is to develop and assess, with the generation of genomic data, whether it will be possible to enhance the efficacy of biocontrol agents on the management of L. microphyllum.Goal 1: Genomics of biocontrol resistanceTo begin to elucidate the genetic contributions to the differential herbivore responses in L. microphyllum, I will first examine the genomes of resistant and susceptible strains of L. microphyllum. Objective 1.1: Phenotype resistant and susceptible strains of Lygodium microphyllum Objective 1.2: Sequence resistant and susceptible strains of Lygodium microphyllum Objective 1.3:Analyze and present resultsGoal 2: Biocontrol effects on gene expressionIdentifying genes involved in establishment and herbivory in the biocontrol agents and the invasive plant will be key for the selection of effective agents and improvement of biocontrol efficacy in management strategies. Objective 2.1: Develop genomic resources for biocontrol agents Objective 2.2: Assess differences in gene expression in the plant and biocontrol agents related to their interaction Objective 2.3: Analyze and present resultsGoal 3: Biocontrol effects on plant health and performanceWe will clarify the impact of biocontrol establishment on the health and performance of the plant to further our understanding of the efficacy of biocontrol as a management tool for L. microphyllum. Objective 3.1: Experimentally test the performance effects of biocontrol agents on L. microphyllum Objective 3.2: Analyze and present resultsGoal 4: MentorshipAn important goal of this project is to interact with and mentor undergraduate students from a wide array of backgrounds and assist in their professional development. Objective 4.1: Involve undergraduate students in both molecular and bioinformatic techniques and analysis throughout the course of this research project. Objective 4.2: Provide opportunities for undergraduate mentees to present research findings at the university, national, and/or international level.Goal 5: Communication of findingsThe dissemination of the findings of the research is a critical goal of this project. I will communicate our findings to a variety of audiences to contribute to increases in knowledge and changes in action with regard to the utility of genomics in the management of weeds through biocontrols agents. Objective 5.1: Publication of results in peer-reviewed journals Objective 5.2: Presentation of results to the scientific community, with particular emphasis on researchers on plant and insect genomics, plant-insect interactions, and invasion ecology andgenomics Objective 5.3: Presentation of results to biocontrol collaborators and experts to improve the suitability of biocontrol agents and invasive plants
Project Methods
Scientific MethodsGenomics of biocontrol resistanceStrains of L. microphyllum from susceptible and resistant genotypes are currently maintained at the USDA ARS IPRL by collaborator Smith. To assess the degree of resistance in these genotypes, biocontrols will be reared and maintained at IPRL and will be introduced and allowed to establish on susceptible and resistant strains. The response to the biocontrol introduction will be scored on a quantitative scale assessing the proportion of above ground tissue impacted. Additional measures of the proportion of pinnae with induced galls and duration to first gall instance will be noted.DNA will be extracted from resistant and susceptible strains of L. microphyllum, extracted DNA will be sequenced with Illumina technology, and raw reads will be trimmed of adaptor and low-quality bases. Filtered reads will be mapped to the reference L. microphyllum genome. Single nucleotide variants will be identified and filtered for quality and depth. Estimates of divergence between the resistant and susceptible strains will be calculated using a sliding window approach and adjusted for false discovery rate to identify highly divergent loci that may be causal to the difference in response to the biocontrol agent. If the resistance phenotype is driven by lineage-specific effects, we would expect to see moderate levels of divergence between susceptible and resistant strains across the genome with no or few highly divergent regions identified as outliers. In contrast, if the phenotype is caused by allelic variants, there should be significant highly divergent loci relative to a less divergent background. Selection on loci conferring the resistance to biocontrol would result in low genetic diversity and differences in allele frequency between the two populations. 2. Biocontrol effects on gene expressionI will use resistant and susceptible strains of L. microphyllum and putative source populations from Australia and China to identify functional genetic pathways responding to biocontrol and their potential sources. Half of the plants will be inoculated with either mites (F. perrepae) or moths (N. conspurcatalis) using standard methods previously reported. The remaining samples will be maintained as a control, maintained in the same conditions as the treatment plants, and not exposed to either biocontrol agent. A bag will be placed over each plant to prevent movement of biocontrols between plants. Once leaflets are removed, plants will not be used for further sampling as mechanical wounding induces defense responses in the fern.For the mite treatment: Leaflets will be excised and immediately flash frozen in liquid nitrogen at two developmental stages: control (no inoculation) and galling/leaf rolling.For the moth treatment: Leaflets will be removed and frozen in liquid nitrogen at three stages: control (no inoculation), oviposition, and feeding by larvae. Samples will be collected at the same time from similar developmental stages of the leaflets to avoid differences in expression due to these extraneous factors.To assess expression changes in the biocontrol agents that are related to the interaction between the biocontrol agent and the plant,For the mites, I will sample from three control colonies with pooled individuals; these data will be used for the development of a de novo transcriptome assembly and as the control treatment gene expression data.The moth developmental stages can be readily separated. To simulate a leaf but not the interaction between the plant and herbivore, I will use wax paper on which adult moths will be allowed to lay eggs and larvae will be reared. Three biological replicates of each stage (eggs, larvae, and adult moths) will be sampled and used for de novo transcriptome assembly and control gene expression data for each developmental stage.Total RNA will be extracted from all samples using an optimized RNA extraction protocol, and sequenced on an Illumina technology. Raw reads will be trimmed of adaptor and low-quality bases. Filtered reads will be mapped simultaneously to the Lygodium reference genome and references for the two biocontrol agents. Unambiguously mapped reads will be segregated and then aligned to their respective reference using the splice-aware aligner. For each species, read counts will be generated and normalized. Significantly differentially expressed transcripts between treatments will be identified. 3. Biocontrol effects on plant health and performanceI will use the same experimental design as in '2) Biocontrol effects on gene expression' to inoculate plants with biocontrol agents. I will track several metrics of plant susceptibility using the developed scaling systems for for mite and moth impacts on Lygodium which include the the proportion of pinnae with mite-induced galls or moth oviposition, mite-induced leaflet tip browning and necrosis, and the proportion of leaflets fed on and windowed by moths larvae. Plant performance will be assessed by measuring rachis growth rate, the proportion of fertile pinnae produced, and the number of rachises with lateral branches. These values will be recorded again after six months. I will compare these values between the resistant and susceptible strains of Lygodium to determine the magnitude of the impact of the biocontrol agents on the success of the plant.EffortsMentoring undergraduate researchers from diverse backgrounds at the University of Arizona, as part of their involvement in these research activities.Presenting results from this project at professional conferences, USDA meetings, and University of Arizona seminars and poster sessions.Presenting results from this project at regional weed management meetings, including the Florida Invasive Plant Council.Development of online repositories for data, code, and walkthroughs of analysesScience communication through the publication of popular press articlesEvaluationMajor milestones that will indicate success in this project include:Obtaining genomic data for the invasive plant Lygodium microphyllumObtaining genomic data for the biocontrol agents Floracarus perrepae and Neomusotima conspurcatalisMentoring of undergraduate students in research techniques and methodsCompleting experimental tests of the effects of biocontrols on the health and performance of resistant and susceptible strains of Lygodium microphyllumPublication of results in peer-reviewed journalsPresentation of results to the scientific community, with particular emphasis on researchers on plant and insect genomics, plant-insect interactions, and invasion ecology and genomicsPresenting of results to biocontrol collaborators and experts to improve the suitability of biocontrol agents and invasive plants