Performing Department
Davis College of Agriculture
Non Technical Summary
Growing evidence shows that the microbiome of plants influences their productivity, health, drought tolerance, and reproductive fitness. The proposed research will use a function focused approach leveraging novel techniques and field experiments to understand plant-microbe interactions in response to fertilization and drought using Miscanthus × giganteus as a model bioenergy crop. Miscanthus x giganteus is a promising feedstock for sustainable bioenergy due to its high productivity and robust growth on sub optimal soil. In comparison to other candidate bioenergy crops such as corn and switchgrass, Miscanthus has higher biomass and energy yields and lower cultivation greenhouse gas emissions. Indeed, Miscanthus can enhance soil carbon (C) stocks making the crop "C-negative", that is, a system that cumulatively stores more C than it respires to the atmosphere. Appalachia, is an ideal region to perform research on microbiomes of bioenergy crops on marginal lands due to the regional legacy of coal energy production. The environmentally disruptive nature of coal mining has resulted in an abundance (~4 million acres) of land that is unsuitable for the growth of most economically valuable crops (i.e. marginal land). However, recent evidence suggests that marginal land is suitable for bioenergy crop production, particularly crops like Miscanthus that grow robustly even under harsh conditions. Estimates suggest that ~3.5 million hectares of land marginalized by surface mining could be utilized for growing bioenergy crops, most of which is in Appalachia. As the energy industry shifts away from coal and toward renewable sources, Appalachia possesses the unique opportunity to utilize the agricultural potential of our mined lands to produce biofuel crops like Miscanthus. Efforts to utilize marginal lands for biofuel production would diversify the economy of Appalachia, a region economically depressed with the declining availability of jobs in the fossil fuel sector. The first aim to identify and characterize the function of the Miscanthus core microbiome (i.e., organisms that reliably associate with the host plant) on marginal Appalachian soils. The core microbiome of Miscanthus provides the plant with water and nutrients in exchange for carbon rich root exudates. If fertilization reduces root colonization by beneficial members of the microbiome and changes microbiome function this may reduce the crops ability to withstand drought stress. To test this hypothesis, we will use a field experiment and cutting-edge molecular tools to characterize plant microbiome interactions. Lastly, we will assess if microbiome manipulation, through the addition of beneficial microorganisms can enhance yield and drought tolerance in a field trial. In summary, the proposed will advance our theoretical understanding of plant-microbiomes and provide practical guidance for bioenergy crop production. The results of this work will help build a predictive understanding of the Miscanthus microbiome function as well as plant-microbiome responses to fertilization and drought stress.
Animal Health Component
0%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
Project Objectives:The overarching aim of the proposed work is to build a predictive understanding of key microbiome functions that influence Miscanthus performance and modulate plant-microbiome responses to fertilization and drought stress. This work will leverage both taxon-specific and community level measurements of microbiome function. Specific objectives and hypotheses (H1-H4) are as follows:Objective 1) Identify and functionally characterize the core microbiome of Miscanthus on Appalachian marginal soils.H1A) Miscanthus has a core microbiome consisting of taxa that are taxonomically or phylogenetically clustered.H1B) The Miscanthus rhizosphere microbiome has a distinct functional profile relative to adjacent soil not planted in Miscanthus.Objective 2) Determine how fertilization alters plant-microbe interactions, plant performance, and microbiome function in response to drought stress. H2) Fertilization weakens Miscanthus-microbiome associations and alters plant and microbial resistance and resilience to drought stress. For instance, we expect fertilization to reduce root colonization with AM fungi and beneficial bacteria ultimately making plant performance and microbiome function (e.g. rates of soil respiration, N fixation, and N mineralization) less resistant and resilient to drought.Objective 3) Design a novel, targeted biofertilizer based on the core microbiome of Miscanthus and test the efficacy of this biofertilizer to improve microbiome function, plant performance, and drought tolerance in a field experiment. H3) Additions of targeted biofertilizer will enhance rhizosphere and endosphere colonization by beneficial members of the core microbiome, stimulate microbiome function, and improve Miscanthus establishment, performance, and drought tolerance.Objective 4) Connect the functions of individual microbial taxa with community-level microbiome function and plant performance in Miscanthus systems. H4) Functional attributes of the Miscanthus microbiome covary with microbiome function, plant performance, and drought tolerance. For example, we expect the growth rates of diazotrophs to predict rates of N2 fixation in the rhizosphere, and the extent of root colonization with AM fungi to predict plant performance during drought stress.
Project Methods
Core Microbiome Study -Addresses Objective 1 and supports Objective 2-4 It is beyond the scope of this work to sample exhaustively enough to characterize the Miscanthus core microbiome in soils across the nation. Consequently, we will focus the characterization of the Miscanthus core microbiome to the Appalachian region. In year 1 we will compare replicate Miscanthus rhizosphere and endosphere microbial communities to adjacent soil (not planted in Miscanthus) at five distinct sites across Appalachia and the fertilization experiment on the WVU Animal Sciences Farm discussed above. These sites vary in soil type, chemistry, mineralogy, pH, and bulk density.To understand how management practices influence the core microbiome this study will also leverage the fertilization experiment initiated in 2019 on the WVU Animal Sciences farm. The experiment has a set of randomly established plots (5m2) experiencing one of four treatments: no fertilization, organic fertilizer, high input and low input inorganic NPK fertilizer with 8 replicates per treatment (32 plots in total). The high and low N-P-K fertilizer treatments receive applications of 19-19-19 N-P-K fertilizer yearly at a rate of 300 and 100 kg ha-1, respectively, rates commonly applied to bioenergy cropping systems on marginal lands. The organic amendment consists of composted dairy manure (25:1 C:N) that is applied at a rate of 300 kg kg ha-1 N yearly. By comparing the rhizosphere and endosphere communities to those of adjacent soil not planted in Miscanthus we will be able to identify the core microbiome of Miscanthus. This study will produce 213 samples for microbiome analysis (including rhizosphere and endosphere) wherein we will perform amplicon sequencing of the 16S rRNA and ITS2 genes, and assess root colonization by AM fungi as described below. To connect variation in the Miscanthus microbiome with plant performance, plant physiological and phenotypic traits will be measured including yield, height, stem number, fine root biomass, root respiration, root mucilage production, and leaf gas exchange as described below. Microbiome functional measurements will include soil respiration (measured in the field and in the lab), net N mineralization, and N fixation rates. Soil properties, such as soil C and N contents, pH, bulk density, moisture and water holding capacity, will also be assessed.Nitrogen Fixation Study - Addresses the Objective 1 and 4We propose to use qSIP with 15N2 to confirm and quantify N fixation activity in putative diazotrophs within the Miscanthus rhizosphere. This will help characterize the core microbiome of Miscanthus . We propose to a pioneering application of qSIP with 15N2 which will provide more sensitive detection of DNA enrichment with 15N than traditional DNA SIP and enable a quantitative measure of N assimilation from N2.During mid growing season of year 2 we will collect samples from 3 of the established Miscanthus sites described above as well as the control and high inorganic fertilization plots from the above descried fertilization experiment on the Animal Sciences Farm described above. The sites will be selected using the results of the Core Microbiome Study to have distinct putative diazotroph community composition and rates of N fixation. From each of the sites and experimental treatments we will collect rhizosphere soil samples from 4 replicate plots.A qSIP experiment will be conducted within 72 hours of collection, subsamples of soil (~4g) will be adjusted to 60% water holding capacity using a solution contain sterile filtered, leached DOC from Miscanthus roots applied at a rate of 400ugC per g soil. Subsamples will be incubated in 12ml Exetainer vials in an artificial atmosphere of 10% O2 and either 20% ~97 atom % 15N2 or natural abundance N2 and 70% He. Soils will be incubated for 21 days and the atmosphere will be refreshed, and additional root exudate solution will be added on day 10. The remaining soil will be analyzed for 15N to determine rates of N fixation.Fertilization and Drought Experiment - Addresses Objectives 2 and 4 In years 3 and 4 we will perform a field experiment to investigate how fertilization alters plant-microbiome interactions and microbiome function in responses to drought stress. This project will leverage the control, organic fertilization, and high inorganic fertilization treatments of the field experiment on the WVU Animal Sciences established in 2019. High replication in this experiment (n=8 per treatment) will allow us to overlay a drought treatment by applying rain-out shelters to half of the plots during two growing seasons. This will produce an experimental design with (6 treatments: Control, Control + Drought, Organic Fertilization, Organic Fertilization + Drought, Inorganic Fertilization, Inorganic Fertilization + Drought) with four plots per treatment for a total of 24 plots. Rainout shelters will be to intercept and divert approximately 75% of rainfall. Shelters will be in place between May and October for two growing seasons. Miscanthus traits (height, leaf fluorescence, leaf gas exchange, fine root biomass, etc.), root colonization with endophytic AM fungi and diazotrophs, as well as community level microbiome function (soil respiration, net N mineralization, and rates of N fixation) will be measured twice per year during the peak growing season (July) and before senescence (October). During the July sampling rhizosphere soil samples will be used for a qSIP experiment within 72 hours of collection to measure the taxon-specific relative growth rates microbes in the rhizosphere of Miscanthus following fertilization and drought stress. Soil subsamples from each plot (3g) will be adjusted to 60% water holding capacity with either natural abundance water or ~97 atom % 18O labeled water and incubated for 5 days (similar to Morrissey et al., 2019). Soil respiration and net N mineralization rates will be measured in parallel incubations with 40 grams of soil.Biofertilizer and Drought Experiment - Addresses Objectives 3 and 4 We will conduct a field trail of Miscanthus biofertilization with 1) a targeted inocula designed to contain beneficial members of the core microbiome and 2) a microbiome transplant made using soil from a well-established and highly productive Miscanthus stand. The targeted inocula will be produced by isolating and purchasing microbial taxa identified using the results from the Core Microbiome studyFor this field experiment we will establish 10m2 plots at least 3m apart that will receive biofertilization and drought treatments in a full factorial design. This will produce an experimental design with (6 treatments: Control, Drought, Biofertilizer, Biofertilizer + Drought, Microbiome Transplant, Microbiome Transplant + Drought) with four plots per treatment for a total of 24 plots. The plots will be established in year 2 and maintained for 4 years. Each year biofertilization treatments will be applied 3 weeks after Miscanthus emergence. Drought treatments will experience a 75% reduction in rainfall (as described for the previous experiment). To facilitate establishment, the drought treatments will not be implemented until after the first growing season. Miscanthus traits (height, leaf gas exchange, fine root biomass, etc.) as well as microbiome function (soil respiration, net N mineralization, and rates of N fixation) will be measured twice per year during the peak growing season (July) and before senescence (October). Samples for microbiome analysis will be collected each July.