Source: THE UNIVERSITY OF TEXAS AT AUSTIN submitted to
DEVELOPING THE PHYTOBIOME TO BENEFIT PLANT DROUGHT RESPONSES: FOLIAR FUNGAL ENDOPHYTES IN AGRICULTURAL AND BIOFUELS GRASSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1011976
Grant No.
2017-67013-26196
Project No.
TEXW-2016-10506
Proposal No.
2016-10506
Multistate No.
(N/A)
Program Code
A1152
Project Start Date
Feb 15, 2017
Project End Date
Feb 14, 2020
Grant Year
2017
Project Director
Hawkes, C. V.
Recipient Organization
THE UNIVERSITY OF TEXAS AT AUSTIN
101 EAST 27TH STREET STE 4308
AUSTIN,TX 78712-1500
Performing Department
Dept. of Integrative Biology
Non Technical Summary
Drought is an important source of stress for plants that causes billions of dollars in agronomic losses worldwide. With the prevalence of extreme drought increasing, there is a critical need for the development of new tools to improve crop drought tolerance. We propose to leverage the microbial symbionts that already live inside plants. In general, plant responses to drought and other abiotic stresses have been studied outside the context of the plant microbiome. Yet symbiotic fungi living inside plant leaves can directly alter plant physiology and improve plant drought tolerance. Understanding the mechanisms by which fungi improve plant drought tolerance will increase our basic understanding of what controls plant physiology and will help us to develop these fungi as agronomic drought management tools. Specifically, we propose to address how symbiotic fungi affect plant drought physiology and to test how we can use those fungi in field conditions to improve crop production and survival under drought.To accomplish these objectives, we will examine how plants and fungi interact across multiple levels, from genes to physiology. We will initially use controlled approaches in the greenhouse to isolate and characterize these mechanisms in a model warm-season grass, Panicum hallii. We will then move to slightly more realistic conditions important for treatment success, including how target fungi fare in the face of other fungi that arrive later and attempt to invade the same plant and whether the outcomes we see for individual fungi scale up to a diverse fungal community. Finally, we will test how fungal effects on plant drought responses translate to switchgrass, maize, and grain sorghum in a field setting with different levels of irrigation.Ultimately, we hope to identify the ways in which fungi and plants interact to produce drought benefits and determine how to exploit those interactions to increase the resilience of our agricultural systems to drought.
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20316291070100%
Goals / Objectives
Our ultimate goal is to develop fungal endophytes (or their genetic or chemical products) as a novel tool for plant drought management. We propose three specific objectives that will help move us toward that goal: (1) identification of mechanisms and development of a predictive framework for the outcome of plant-endophyte symbiosis, (2) scaling from single fungi to communities of fungi for real-world application, and (3) testing generality across plant hosts and feasibility for field application. Moreover, measuring the same set of responses and mechanisms across a set of greenhouse to field experiments supports interpretation of complex field results. These objectives are described in more detail below.Objective 1: Develop a mechanistic framework for predicting fungal effects on plantsUnderstanding how fungal endophytes ranging from mutualistic to antagonistic mechanistically affect plant drought responses will allow us to develop a predictive framework for fungal mediation of plant drought responses. We will address ecological, physiological, metabolomic, and gene expression mechanisms by which fungi affect plants. By combining empirical tests of endophytes on plant drought response with "multi-omics" measurements, we can use a comparative approach to identify mechanisms at multiple levels. Results will form the baseline for design and interpretation of Objectives 2 and 3.We will develop genomic resources, through whole genome assembly of target fungi, to link the ecological, physiological, metabolomic, and transcriptomic mechanisms in Objs. 1a, 2a, and 3a to the fungal genome. In addition, we will use a comparative genomics approach with target fungi and congeners with existing genomes to help identify genes and pathways characteristic of drought-beneficial symbionts.Objective 2: Scale predictions from individual to multiple fungi interacting in a single host in the context of priority fungal applicationsFungi occur as multi-species communities in plants and therefore scaling up to include realistic diversity and interactions will be critical for maintaining targeted functions. Based on niche theory, we expect that functionally similar mixtures of fungi will have antagonistic effects on plants due to competition, whereas functionally diverse mixtures of fungi will have synergistic effects on plants due to complementarity. We will test a range of numbers of fungal species (0, 1, 2, 4, 8) in plant hosts. We will also quantify niche overlap and predict additive vs. non-additive outcomes. Because fungal interactions in the plant may lead to unique mechanisms affecting the outcome of symbiosis, we will quantify how interactions alter patterns of gene expression and chemical production relative to single fungal inocula.In field-settings, applications of endophytes would likely take advantage of putative priority effects where the first fungus to arrive can exclude or minimize later arrivals and thus have the greatest impact on the plant host. To examine whether arrival order can be used as a tool to influence the outcome of symbiosis, we will give priority to the most drought-beneficial endophyte taxa (Obj. 1) and challenge them with later arriving individuals and communities that are less beneficial. We will determine which individuals or communities are best able to resist invasion by later arrivals and therefore maintain function (which will be used in Obj. 3).Objective 3: Determine host generality and field feasibility of fungal endophytes in plant drought managementTransfer of endophyte effects to other host species in a field setting is key to their development as a useful plant drought management tool. We propose to examine how the effects of fungal endophytes translate from our target hosts (Panicum virgatum and P. hallii) to two other agriculturally relevant plants, maize (Zea mays) and grain sorghum (Sorghum biocolor), under well-watered and drought conditions in the field. We will plant outdoors at a site with controlled irrigation, and will monitor inoculated fungi for persistence and effects on plant physiology growth, survival, and gene expression.
Project Methods
Fungal isolates and library maintenance. Existing fungal isolates are preserved in two ways: in sterile water at room temperature for short-term use and cryopreserved at -80°C for long-term storage to prevent loss of traits due to serial transfer. Fungi required for the proposed work will be cultured from frozen stocks on solid potato dextrose (PD) agar and then grown in large volumes in PD broth.Growing and harvesting greenhouse plants. P. hallii will be grown in transparent microcosms with 20-um nylon mesh windows to allow water to escape. Fungi will be inoculated as described below. Soil moisture will be maintained under either well-watered (15%, ca. -15 KPa) or drought (5%, ca. -275 KPa) conditions. Weekly measurement of soil moisture by whole-pot weight will be used to adjust water levels and maintain treatments. For all plant measurements, entire boxes will be placed inside a sterile benchtop enclosure. Between boxes, the enclosure will be UV-sterilized and all instruments wiped with ethanol to avoid cross-contamination. Plants will be destructively harvested at peak growth for biomass of leaves and roots. Leaf subsamples will be flash-frozen in liquid N and stored at -80°C for RNA-seq, community sequence analysis, and metabolomics.Growing and harvesting field plants. We will grow P. virgatum, S. bicolor, and Z. mays outdoors in a randomized block design with two irrigation treatments (drought = 400 mm/yr and well-watered = 850 mm/yr). The design includes 10 plants per plot to support 9 fungal treatments and 1 fungus-free control, with each species separated into 2 plots per block over 4 blocks. To create realistic growing conditions, we will sow plants in 1-m2 patches with seeds of each species sown at recommended densities for this region. Patches will be arranged in a checkerboard in the 25-m2 plots. We will only measure a single central plant in the patch to avoid edge effects. Survival, flowering phenology, and indicators of stress such as leaf rolling will be recorded every two weeks; growth rates will be measured monthly; leaf-level physiology will be measured on a subset of plants just prior to harvest. Full aboveground leaf and reproductive biomass will be cut down, dried, and weighed. Leaf subsamples will be flash-frozen in liquid N and stored at -80°C for RNA-seq, community sequence analysis, and metabolomics.Plant inoculations with fungi. To create fungal treatments, plants will be inoculated with fungi grown in liquid culture. Fungi will be grown in 1-L culture flasks of PD broth, hyphal filaments will be disaggregated and chopped in a blender, aliquots will be created with equal hyphal fragment densities, and these will be pipetted (greenhouse) or sprayed (field) directly onto new host plant leaves. To confirm inoculation success in single-fungus experiments, plant leaves will be subcultured on potato dextrose agar plates and stained for microscopy to quantify colonization rates. For multi-fungi and field experiments, fungi in leaves will be quantified by sequencing.Fungal genomics. For the 20 target fungi, we will grow cultures on PDA, collect mycelia after 2-3 days, and DNA will be extracted using Zymo Genomic DNA kits; extracts will be purified and submitted to the UT Genome Sequence and Analysis Facility (GSAF) for library preparation and sequencing using Illumina HiSeq 4000 at 100 x coverage (2x150 paired-end reads). The fungal genome sizes are estimated at 25-40Mb. Genomes will be assembled against a reference in the same genus (available for 87% of fungi in our collection) using Soap3-dp. Assembled genomes will be checked for quality and completeness with BUSCO. Structural and functional gene annotation will be performed following the JGI Annotation Pipeline on repeat-masked assemblies. In addition, we will use the SMURF and antiSMASH to predict secondary metabolite gene clusters. Genome assembly and analyses will be carried out on the Stampede supercomputer at the Texas Advanced Computing Center (TACC). For comparative genomics, we will focus on genes that prove to be differentially expressed compared to sterile controls and significantly different between water levels and fungal treatments from RNA-seq data. Comparisons will be among the fungal genomes generated here and available congener genomes with known lifestyles (e.g., pathogen, saprotroph, endophyte) available in public databases. We will quantify variation across genomes in gene presence and abundance profiles, clustering, and synteny.Plant-fungal RNA-seq. For plants and fungi in leaves, total RNA will be extracted from leaf tissue by grinding in liquid N with RNAse-Zap-treated beads and extracting using a TRIzol/chloroform/isoamyl alcohol extraction. RNA-seq library samples will be prepared using RNAtag-seq to quantify gene expression responses of both plants and fungi. Briefly, from each sample 10 µg total RNA will be fragmented and reverse transcribed. The cDNA constructs are prepared from fragmented RNA, labeled with sample-specific oligonucleotide barcodes, then pooled and sequenced using 1x150 bp reads on Illumina Hiseq 4000. Tag reads will be mapped against the P. hallii genome and fungal genomes generated as described above. Reads will be parsed into those mapped to a plant, fungal, or ambiguous biological source and independent analyses will be completed on each set.qPCR. We will evaluate the accuracy of RNA-seq expression profiles by using qPCR on the same RNA samples used for RNA-seq. We will select 8 genes that are differentially expressed among the treatments, and 4 genes that show constant expression to act as references. qPCR will be carried out with Sybr Master Mix on ViiA7 instruments (Life Technologies) available through the UT DNA Sequencing Core Facility. Reference genes will be chosen and expression profiles will be normalized by the geometric mean of the reference genes.Quantifying fungal composition and abundance in multi-species and field experimentsFungal community composition and abundance will be characterized for all samples in the diversity, priority, and field experiments based on ITS rRNA with the fungal primers ITS-1F and ITS2. To standardize fungal abundance, we will sequence a control sample on each sequence run containing 100 spores isolated from cultures of the 20 fungi for which we will obtain genomic sequences. Given a known number of spores/species, we can adjust for bias to obtain accurate abundances. Samples will be sequenced on Illumina MiSeq v3 with 33,500 2x250 reads per sample.Chemical extraction, purification, and identification. Fungal cultures as well as plant samples in the P. hallii endophyte pair trials (Obj. 1a), diversity scaling experiment (Obj. 2a), and priority experiment (Obj. 2b) will be screened for bioactive compounds that improve drought tolerance in plants. Chloroform-methanol will be used for plant extractions in a 5:1 ratio of solvent to plant material. The plant-organic solvent mixtures will be homogenized with a mortar and pestle and incubated at 4°C for 3-24 hrs. Following incubation, the solid plant material will be removed from the liquid phase (i.e., supernatant) via filtration or centrifugation. For both fungal and plant extractions, organic and aqueous phases will be separated and concentrated to dryness by vacuum or lyophilization. A comparative metabolomics approach for identifying known and unknown compounds will be performed on the extracts. Dried fungal and plant extracts will be re-dissolved in methanol, analyzed by LCMS, and mass data will be searched against the METLIN metabolite database to identify any known bioactive metabolites. Principal components analysis along with orthogonal least squares discriminant analysis will be used to distinguish metabolic profiles for all fungal and plant samples.

Progress 02/15/17 to 02/14/20

Outputs
Target Audience:Students, researchers, biotechnology firms, agricultural managers. Changes/Problems:We have one major change to the project.Objective 3 was originally intended to simultaneously test both how greenhouse results in switchgrass would translate to field conditions and how observed effects in switchgrass would translate to other C4 crops (maize, sorghum). Unfortunately, in mid-June all the maize and sorghum plants were destroyed by herbivores. As a result, we continued the field experiment with only switchgrass. After discussions with Dr. Liang-Shiou Lin, we will add a second experiment to this objective to examine how the fungi behave across crop species. Specifically, we will carry out greenhouse experiments in 2020-21 to test a subset of fungi (or consortia depending on the outcome of Objective 2 experiments) with different effects on switchgrass drought physiology. We will minimally test maize and sorghum in these experiments, and will attempt to expand to several other crops. What opportunities for training and professional development has the project provided?We mentored three undergraduate students in independent research projects that leverage this research. These are detailed below. (1) Tina Bui, a microbiology major, completed her senior honors thesis on how fungal diversity affects growth and production of secondary metabolites. She applied for and received funding from the University of Texas Undergraduate Research Fellowship program for this work. Tina is currently enrolled in the PhD program in microbiology at the University of Rochester. (2) Kenia Segura Alba, a biochemistry major, was awarded a summer research fellowship by the University of Texas Freshman Research Initiative. She was trained in all lab methods related to this project. In addition, Kenia developed her own independent research question addressing how the leaf metabolome shifts between old and new switchgrass leaves inoculated with different fungi in our Objective 3 field experiment. She collected those leaves as part of the summer harvest and is currently processing samples. Kenia is in her junior year and expects to complete this project by spring 2019. (3) Nicholas Birk, a joint math-biology major, continues to pursue his senior project to understand source-sink dynamics in leaf endophytic fungi that are key to understanding how communities assemble in the field. He completed an initial analysis of an existing data set using the QIIME pipeline and SourceTracker in spring, then spent the summer comparing approaches for network analyses. He is currently finalizing the network analysis and plans to write up his thesis this year. Along with collaborator Dr. Moriah Sandy, we continued to leverage a portion of the research in our undergraduate course, BioProspecting, which is part of the UT Freshman Research Initiative (FRI) Program. The primary objective of FRI is early involvement of students in scientific research while obtaining lower-division course credit. In summer 2018, three freshman undergraduate researchers, Amy Marshall, Tara Piyapanee and Alex Staten, followed up on extraction-method development carried out by Fall 2017 FRI BioProspecting students. The Summer 2018 students developed a Python program for rapidly compiling HPLC UV data into a chemical matrix for downstream statistical analysis. The students also compared different statistical packages in R for analyzing UV and mass metabolomic data and are currently optimizing their graphics scripts to visualize results. Students enrolled in the Fall 2018 FRI BioProspecting course (~25 students) will use the Python program and statistical methods developed by the summer 2018 research students for analyzing UV and mass metabolomic data. How have the results been disseminated to communities of interest?In the past 6 months, we finalized the agreement with IndigoAg, Inc. in Boston, MA to test development of fungi in our collection as products to minimize drought stress in agricultural crops. The licensing agreement includes access to the fungi and associated fungal data. Funding from this agreement will provide support for our educational efforts through the Freshman Research Initiative (FRI) BioProspecting course.In spring 2018, we also hosted an interactive table at the Explore UT outreach event. In late summer 2018, we presented a research poster at the FRI Meet and Greet. Finally, throughout spring and summer 2018, we hosted lab tours and research Q&A sessions for various outreach events. What do you plan to do during the next reporting period to accomplish the goals?During the remainder of 2018-2019, we will hire a new postdoc at North Carolina State University (interviews in progress), who will process frozen samples from Objectives 1a and 3 for RNA-seq and carry out comparative genomics in Objective 1b. We will also analyze the data from these experiments to select subsets of fungal taxa for use inObjective2in 2019-20.

Impacts
What was accomplished under these goals? Objective 1a -Understand how a range of fungi affect plant drought responses to develop a predictive framework of fungal benefits based on multi-omics measurements. We completed the greenhouse experiment. Plants were grown in microcosms with a single fungus or with no fungi, under well-watered or drought conditions. Water treatments were maintained by weight twice weekly and plants were measured for growth (height, number of leaves) and signs of stress (wilt, browning) once per week. After 10 weeks, plants were measured for leaf physiology (photosynthesis, conductance) then harvested, weighed for fresh biomass, flash-frozen in liquid nitrogen, and stored at -80C. A separate set of plants were used to develop a relationship between fresh and dry biomass. Frozen leaf samples will be ground in liquid nitrogen for characterization of transcriptomic and metabolomic profiles. Frozen root and soil samples were sent to collaborators at Lawrence Livermore National Lab, who will similarly characterize the root transcriptome metabolome, as well as impacts on soil carbon pools. Objective 1b -Develop genomic resources for 20 fungi All 20 fungi were re-isolated from archival stocks, tested for contamination by both bacteria and non-target fungi, and verified against former identifications via ITS and 28S sequences. These are now ready for genomic DNA extraction, sequencing, and assembly via the optimized protocol we developed previously. Objective 2: Scale predictions from individual to multiple fungi in a single host (a) Examine how fungal diversity affects plant drought responses (b) Determine how priority of arrival affects the outcome of symbiosis. Both of these objectives will be carried out in 2019-20. Objective 3: Determine host generality and feasibility in field conditions. The field experiment was completed. Any switchgrass plants in the subplots that died over winter were replaced with new transplants in February 2018 to maintain the appropriate densities. The annuals, sorghum and maize, were planted from seed in early April 2018. Fungal treatments were inoculated twice in May 2018 by spraying all leaves with a known quantity of fungal hyphae. Drought treatments were imposed beginning in June 2018. All subplots were measured bi-weekly for leaf area index and maximum height. In mid-June 2018, the corn and sorghum were destroyed by herbivores (see section on "Changes" below), so we proceeded with only switchgrass. In August 2018, we measured leaf physiology (photosynthesis, conductance). Immediately afterwards, the same leaves were harvested for transcriptomic and metabolomic analysis by flash-freezing in liquid nitrogen and storing on dry ice for transport to the lab and storage at -80C. These samples have been ground in liquid nitrogen and the ground tissue was split into two portions: one for RNA extraction and one for metabolite extraction. Additional leaves were collected for analysis of fungal community composition (flash-frozen) and for colonization rates by fungal hyphae (stored in ethanol). The remaining aboveground plant tissue was cut and dried for biomass measurement. Roots and soils were collected for parallel analyses by collaborators at Lawrence Livermore National Lab, with additional measurements of soil carbon and nutrients.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Bui TI (2018) Effects of diversity and environmental stress on growth of fungal symbionts. Undergraduate Senior Honors Thesis. University of Texas at Austin.


Progress 02/15/17 to 02/14/18

Outputs
Target Audience:Our target audience includes undergraduate and graduate students, other researchers, biotechnology firms, and agricultural managers. Changes/Problems:We have had two changes to the project schedule. Our postdoctoral researcher was unable to start work on the project until June 2017, and then left the project 3 months later for family reasons. In addition, Hawkes is moving from UT Austin to North Carolina State University (NCSU) in summer 2018. This resulted in two scheduling changes. First, we prioritized the Objective 3 field experiment to complete the outdoor fungal work in Texas prior to moving. Second, we were unable to find a new postdoc to work at UT for less than one year, so a postdoc with molecular skills will be hired at NCSU to carry out the RNA-Seq and comparative genomics analysis in Objective 1 on frozen samples. Instead of a postdoc, an existing technician supported by in-house funds at UT has been assisting Hawkes with the day-to-day experimental work associated with Objectives 1 and 3 and all harvested tissue samples will be stored at -80C for the future postdoc. Objective 2 will be carried out at NCSU. We also discovered a bacterial contaminant in some of our culture library stocks, which has led to additional QC work on the strains prior to their use in the next round of experiments. The QC work will be completed by March 2018. What opportunities for training and professional development has the project provided?We mentored three undergraduate students in independent research projects that leverage this research. These are detailed below. (1) Brianna Barry, a biochemistry major, focused her senior thesis on fungal genome assembly methods and graduated in May 2018; she is currently working on a manuscript for submission and is enrolled in the PhD program in molecular biology at Johns Hopkins University. (2) Tina Bui, a microbiology major, is pursuing her senior thesis on how fungal interactions affect the production of secondary metabolites. She had previously received extensive training in working with endophytic fungi through our Freshman Research Initiative course on BioProspecting. For Tina's thesis, we further mentored her in grant proposal writing and she submitted two proposals to fund her work (still under consideration). Tina presented a poster of her work at the2017 SACNAS National Diversity in STEM conference in Salt Lake City, UT. Tina is currently interviewing for PhD programs in microbiology. (3) Nicholas Birk, a joint math-biology major still in his junior year, is pursuing an independent project to understand source-sink dynamics in endophytic fungi that are key to understanding how communities assemble in the field. He is using one of our existing data sets. He was trained in bioinformatic methods to analyze microbial communities, including the use of the QIIME pipeline to define OTUs, dada2 to identify unique sequence variants, and SourceTracker to estimate contributions between fungal pools. In addition, one undergraduate student, Amelia Ellis, volunteered in the lab and was trained in fungal culturing methods prior to graduating in May 2018 with a BS degree in Biochemistry. She is currently working as a Quality Assurance Specialist in Microbiology at 3M in New Haven, CT and applying to graduate programs in microbial ecology. Hawkes and Sandy incorporated portions of the research into their undergraduate course, BioProspecting, which is part of the UT Freshman Research Initiative (FRI) Program. The primary objective of FRI is to involve undergraduates in conducting scientific research from the outset of their college careers. Students are able to gain lower-division course credit for FRI, while conducting research projects. Specifically, our FRI BioProspecting course focuses on isolating and structurally characterizing novel molecules with agricultural and medicinal potential from fungi. First-year students join the course in their second semester, where they learn basic skills in the ecology, molecular biology, and chemistry of endophytic fungi. The majority of students continue through the following fall semester where they undertake semi-independent research projects. Nearly 57% of students enrolled are from underrepresented ethnicities, first generation college students, low socioeconomic backgrounds (<$40K annual household income), with low SAT scores (<1100), or women in targeted STEM majors. Significantly more FRI students in at-risk groups go on to graduate from UT (81% vs. 68%) and 15-30% more obtain higher degrees or STEM jobs. Initially, two undergraduate students in Fall 2017 were involved in optimization of plant tissue extraction methods as part of their class projects. Specifically, students surveyed literature for experimental methods, compared and presented different methods to their peers in a group meeting setting, and began preliminary methods tests and analysis on different solvent extraction methods for plant tissues. They compared initial results to published results in the literature and discovered inconsistencies - highlighting the importance of clear communication in publication, the value of reproducibility of reported findings. From their earlier literature survey, the students were able to propose testing of additional methods that might improve diversity in metabolites isolated from plant tissue. In Spring 2018, 35 students in the class will use the data generated in these studies to develop skills in analyzing and interpreting liquid chromatography and mass spectrometry data. The Spring 2018 students will also be testing different solvent systems on their fungal samples with the aim of diversifying our fungal metabolite profiles and making them more comparable to plant metabolite profiles. Finally, through our BioProspecting course, two freshman undergraduate researchers, Amanda Pittman and Kenia Segura Aba, contributed to testing and optimization of metabolomics methods as described in the Activities section. Specifically, they were trained in harvesting plant tissue and preparing plant material for extraction, plant tissue extraction methods, and methods to analyze HRLCMS data, including use of the metabolomics software XCMS. How have the results been disseminated to communities of interest?We have worked to disseminate our findings in several ways. First, we published a perspectives/review paper on the ideas behind the project in open-access Phytobiomes Journal, which targets researchers addressing the full range of fundamental to translational work. That paper has been downloaded 1955 times as of February 5, 2018, ranking it fourth at the journal. Second, we have been working with IndigoAg, Inc. in Boston, MA to test development of fungi in our collection as products to minimize drought stress in agricultural crops. As part of the licensing agreement, we have shared our fungi and associated fungal data (traits, plant effects, genomics) with Indigo. The company has already launched microbial inoculants for five crops (cotton, maize, rice, soybeans, wheat) and we hope to contribute to that effort. Third, by giving the keynote address at the International Turfgrass Conference, we introduced a new audience to the idea of microbiome manipulation to improve crops. Finally, we hosted interactive tables at UT outreach events including Explore UT (Spring 2017 and 2018), the Welch Groundbreaking ceremony (Spring 2017) and has hosted lab tours and research Q&A sessions for outreach events periodically throughout the year. These events were carried out with students in our BioProspecting course, which is part of the UT Freshman Research Initiative described in the Activities section. What do you plan to do during the next reporting period to accomplish the goals?During the 2018-2019 period, we will complete the Objective 1a greenhouse test and the Objective 3 field experiment. Samples for metabolomics will be processed in summer 2018. Samples for gene expression will be frozen at -80C and stored. A new postdoctoral researcher will be hired for a target start date of September 2018. The postdoc will be responsible for carrying out the RNA-Seq analysis on frozen leaf tissues in Objective 1a and 3, and for comparative genomics in Objective 1b. We will also analyze the data from these experiments to select subsets of fungal taxa for use in testing Objectives 2a and 2b in 2019-2020.

Impacts
What was accomplished under these goals? Objective 1a We completed two major activities: (1) we tested the pot system for our controlled greenhouse experiments based on reviewer comments, and (2) we revised and optimized our metabolite extraction protocol based on initial results and new advances in the literature. Details are provided below. Experimental pot system Based on reviewer comments from the proposal, we tested the size of the pot used in our controlled greenhouse experiments with Panicum hallii. The reviewers felt that the magenta boxes we routinely use were too small and wanted us to use larger and deeper pots. The magenta boxes are square, 3-inches in diameter, and 3.8 inches deep. We tested larger square pots that were 8-inches deep, but the same diameter to allow the magenta lid to fit over the top and maintain sterile conditions (MT38 mini-treepots, Steuwe and Sons, Inc.). Pot drainage holes were taped shut in order to prevent loss of water and infiltration by contaminants. We grew P. hallii in the pots for 12 weeks, a period in which the plants can go from seedling to flowering. Root sizes achieved during that time were only 0.01-0.37 g dry weight and occupied a small fraction of the pot volume. In addition, plant size and physiology data from this work demonstrated that the larger pots prevented plants from experiencing water stress as rapidly as needed for the drought treatment to be successfully applied. The larger pots were also subject to contamination from the bottom. Thus, we determined that the magenta boxes were the most appropriate for our target plant. We are preparing to carry out the full objective 1 experiment in spring 2018: magenta boxes are sterilized, soil is autoclaved, and fungi are being cultured in preparation for a mid-March start date. Metabolomics methods In addition, in our original proposal we proposed to use the classic chloroform:methanol:water (CH3Cl:MeOH:H2O) solvent system for plant tissue extraction to characterize secondary metabolites. However, we discovered problems with reproducibility, phase separation, and limitations on types of molecules extracted. Therefore, we tested three solvent extraction methods with the goal of maximizing access to diverse metabolites: (1) methanol:water, (2) ethyl acetate: methanol: water, and (3) methyl tertiary butyl ether (MTBE):methanol:water. Our results from testing methanol:water contradicted with the published results: we were unable to detect non-polar secondary metabolites (e.g., terpenes) and we were unable to detect primary metabolites (e.g., amino acids and sugars). For ethyl acetate: methanol: water, we ran into difficulties with consistent phase separation, similar to the CH3Cl:MeOH:H2O system. Often it was difficult to recover the aqueous phase, or the aqueous phase would vary in volume, which ultimately interfered with accurate measurements of metabolite identity and abundance. Using MTBE:methanol:water, we were able to reliably reproduce results reported in the literature. The extractions were clean, consistent and afforded accessibility to the broadest range of metabolites including pigments (chlorophylls and carotenoids), lipids, secondary metabolites and primary metabolites. There is also potential to expand the extraction phase analysis to include protein, cell wall and starch quantification. Objective 1b The major activity of Objective 1b was to optimize the fungal genome sequencing and assembly process. We used six fungal species to test two DNA extraction methods and three analytical approaches to genome assembly. We compared DNA extracted using the 1000 Fungal Genomes Project protocol and the Omega Bio-Tek EZNA High Performance Fungal Genomic DNA kit. Paired-end sequencing (2x150) with 100-fold coverage was conducted using the Illumina HiSeq 4000. Comparative genome assembly was completed using three approaches: MaSuRCA, Velvet, and SOAPdenovo2. Assemblies were improved when DNA extractions were performed with the Omega kit instead of the 1000 Fungal Genomes Project protocol; in some cases, DNA extracted using the latter method produced data that could not be successfully assembled because gaps could not be closed. MaSuRCA consistently outperformed both Velvet and SOAPdenovo2 in genome assembly based on N50 and completeness metrics. The N50 values were 38-300% larger with MaSuRCA. Contigs and scaffold assemblies also performed equally well with MaSuRCA, but other assemblers were improved through the use of scaffolds. Based on BUSCO analysis, genome assembly completeness ranged from 98.3-99.7%, with 0.3% fragments and 0-1.4% missing with MaSuRCA. In contrast, the other assemblers were at best 43.5-79.3% complete using the same data. The MaSuRCA assemblies are on par with published high quality genomes of congeneric taxa, which ranged from 98.3-99.1% complete. Based on these assessments, we will use MaSuRCA for assembling the remaining fungal genomes. Objective 3 The field experiment is designed to test how fungi perform in more realistic conditions on multiple host plant species and will be carried out during the 2018 growing season. There are three host plants, all warm-season grass crops raised for fuel or food: Panicum virgatum (switchgrass), Sorghum bicolor (sorghum), and Zea mays (maize). The major activity to date has been preparation of the field plots for the Objective 3 field experiment. In summer 2017, we did the following to prepare the field plots: (1) applied herbicide twice to kill previous plants (various P. virgatum genotypes), (2) tilled to break up previous root systems and homogenize soils, (3) solarized to reduce weed seeds, and (4) removed old roofing material. We grew the perennial Panicum virgatum plants in the greenhouse and these were transplanted to the field in early October 2017. The annuals, sorghum and maize, will be planted from seed in late March or early April 2018 depending on weather. In addition, the poly roofing material for the rain-out shelter will be ordered in February 2018 for installation in late April 2018 to impose drought treatments in late May 2018. We tested the field irrigation system and replaced solenoids, valves, and sprinkler heads as needed after below-freezing temperatures in January 2018.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Hawkes CV, Connor EW (2017) Translating phytobiomes from theory to practice: ecological and evolutionary considerations. Phytobiomes Journal 1: 57-69
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Hawkes CV, Connor EW, Giauque H, Sandy M (2017) Microbial tools in agriculture require an ecological context: stress-dependent non-additive symbiont interactions. Keynote presentation at the 13th International Turfgrass Research Conference, New Brunswick, NJ
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bui TI, Hawkes CV (2017) Diversity and environmental effects on secondary metabolite production of fungal symbionts. Poster Presentation at SACNAS: The National Diversity in STEM Conference. Salt Lake City, UT.
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Connor EW (2017) Fungal endophyte interactions and mechanisms of fungal-mediated plant drought tolerance. PhD Dissertation, University of Texas at Austin.