Recipient Organization
University of Maryland Eastern Shore
11868 College Backborne Road
Princess Anne,MD 21853
Performing Department
(N/A)
Non Technical Summary
Globally escalating demands for both food and energy have raised concerns about the potential for food-based biofuels to be sustainable, abundant, and environmentally beneficial energy sources. Current biofuel production competes for fertile land with food production, increases pollution from fertilizers and pesticides, and threatens biodiversity when natural lands are converted to biofuel production. To meet the rising food and energy demands in a sustainable manner, i.e., to produce more output from existing farmland in ways that minimize environmental tradeoffs, modifying our agricultural system at different levels, ranging from soil and crop husbandry to plant genetic and microbiome management, will need to be implemented. Given the importance of soil microbial communities and their biological processes in maintaining soil health and crop productivity, one approach to the productivity-sustainability challenge is to harness soil microbial resources for enhanced food- and energy crops performance. Our specific objectives are: (i) Characterize the microbial community at the root-soil interface of different switchgrass genotypes, and Miscanthus, (ii) Screen the plant growth promoting microorganisms (PGPMs) among switchgrass core microbiome, (iii) Explore the possibility that different switchgrass genotypes recruit genotype-specific set of microbes under specific climatic conditions, (iv) Microbiota reconstitution in switchgrass plants to understand the role of interactions on plant fitness, and (v) Develop a strong community engagement and education program.
Animal Health Component
60%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Goals / Objectives
The overall goal of the proposed project is to exploit the most effective PGP microbes isolated from resilient root microbiome of bioenergy crops for improved biomass yield of bioenergy crops. The project has five main objectives:I. Characterize the microbial community at the root-soil interface of different switchgrass genotypes,and MiscanthusII. Screen the plant growth promoting microorganisms (PGPMs) among switchgrass resilient/core microbiomeIII. Explore the possibility that different switchgrass genotypes recruit genotype-specific set of microbes under specific climatic conditionsIV. Design microbial consortia containing bacteria and fungi displaying PGP traitsV. Develop a strong community engagement and education program
Project Methods
Characterize the microbial community at the root-soil interface of different switchgrass genotypes, and Miscanthus:Switchgrass plants will be harvested at the same developmental stage.Microbial community profiles of the harvested switchgrass roots will be compared to those of neighboring miscanthus grass. Roots of plants will be separated from the main soil particles, and themicrobial communities in rhizosphere, and root will be profiled.DNA isolation will be performed from rhizosphere and rhizoplane root samples andsubjected to microbial profiling, using specific genomic regions for each microbial group of study:16s rRNA gene for bacterial profiling (V2-V4 region and V4-V7) & ITS1 and ITS2 for fungal profiling. PCR amplicon libraries will be generated using primers 799F-1192Rand 341F-806R for bacterial communities profiling, ITSand ITS2 for fungal community profiling. Sequencing data analysis will be conducted using the Mi-SeqTM platform.The Sebacinales identity of the fungal isolates will be verified by the specific PCR amplification of a DNA fragmentcomprising SSU; 18S, ITS1, the 5.8S subunit, ITS2, and the D1/D2 region of the large subunitof the ribosomal DNA.For sequencing purposes, this PCR will be followed by a second PCR with the universal fungal primers ITS1F and NL4. RNA extraction for quantification of fungal colonization and RNA-seq, cDNA generation and RT-PCR will be performed.- Screen the plant growth promoting microorganismsamong switchgrass core microbiomeThe root samples will be collected from previous steps without surface sterilization will be crushed, and diluted gradually. 100 μL of bacterial suspensions will be spread on TSA plates. The plates will be incubated at 30 C, and colonies of different morphologies will bepicked and streaked on new plates. ForIsolation root bacterial/fungal endophytes, the roots will be thoroughly washed with tap water to remove any adhering soil. Then, they will be rinsed in water and scrubbed lightly to remove any soil. The roots will be subjected to surface sterilization to fully remove any microbe from the root surface. To isolate bacterial endophytes, plant tissues will be homogenized andincubated at room temperaturein an orbital shaker. Aliquots of 100 μl of each dilution will be plated on TSA, LA, and KBA plates in duplicate, and incubated. To separate the fungal endophytes, the surface sterilized roots will be cut in 1cm pieces and the root fragments will be incubatedon CM, MEA, MYP, PDA.- Biochemical assays for PGP traits:All bacterial isolates will be first grown in TSB, washed twice, and resuspended in PBSto obtain OD600 0.5. Then, they will be screened to assess their ability to produce IAA, Growth on N-free medium (N2 fixation), P solubilization, siderophore production, and sequencing the 16s rRNA region.Explore the possibility that different switchgrass genotypes recruit genotype-specific set of microbes under specific climatic conditions:a. Field transplantation experiment: AtUMES Agricultural Experiment Station, a transplantation experiment will be set up where the adapted and non-adaptedswitchgrass genotypes will be planted. In this way, we can assess climate and genotype effect on soil and root-associated microbial communities, as well as the impact of climate and soil on plant fitness. Also, to learn more about microbial resilience over time, plant and soil samples will be harvested 3 consecutive years.Harvesting will be done by taking each plant and its surrounding soil, removing as much soil attached to the root as possible and keeping this root as a technical replicate (this will be made between 4 and 6 technical replicates).b. Greenhouse transplantation experiment: In order to reduce the environmental background inherent to the previous experiment, the same experimental set-up will be reconstituted in greenhouse. The experiment will be performed using a trapping system consisting of the rhizosphere soil as inoculum and switchgrass genotypes as host plants. Soil samples collected from the rhizosphere ofswitchgrass genotypes will be bulked into one composite sample and used as source of inoculum and planted with 2 adapted (Kanlowa, and Alamo) and 2 non-adapted switchgrass genotypes. Seeds of each genotype will be surface sterilized, germinated, transplanted into the soil, and underwent a full life cycle in greenhouse. Plants and their surrounding soil will be harvested trying not to disturb the root system, and transported to the laboratory.c. Microbial community profiling: DNA extraction, library preparation, and Sequencing data analysis of both greenhouse and field transplantation experiments will be performed.Microbiota reconstitution in switchgrass plants to understand the role of interactions on plant fitness:- Design microbial consortia containing bacteria and fungi displaying PGP traits: Based on the culturable microbial isolates, PGPM (bacteria and fungi) strains representing the prevalent taxa will be selected. Clearly, the number of bacterial and fungal isolates we will select, is by no means the only combination possible to create an inoculum with a minimal number of microbial isolates.Thus, through a series of colonization assays with all community combinations lacking one species, we will exclude, add, or replace some of the isolates to develop a more simplified one.a. Bacterial strains growth and inoculation: The selected bacterial strains (5-10) will be grown separately in liquid TSB medium at 28º andshaken at 120 rpm for 1 to 3 days. The bacterial cells will be centrifuged at 5000 rpm for 15 min and the pellets will be washed 3 times with sterile water. Prior to plant inoculation, an optical density of 0.2 will be adjusted to maintain uniform bacterial cell density. The bacterial consortium will be obtained by mixing the 20 bacterial solutions and diluted the final inoculum to an OD600 of 0.02.b. Fungal strains growth and inoculation: Fungal strains (2-5) isolated from switchgrass and miscanthus as well as the reference fungi strains from the UMES microbial collection, will be grown on PGAat 25ºC for 2 weeks. On the inoculation day, 500 mg of mycelium of each fungus will be harvested from the agar, placed in a 5-mL screw-lid tubecontaining 4mL of sterile 10 mM MgCl2), and grinded in paint shaker to disrupt the mycelium. Then, 2 mL of fungal homogenates will be pooled together in 2final fungal stocks of 50 mg/mL concentration. 1mL of these stocks will be utilized to inoculate seedlings. The remaining of fungal homogenates will be used to control fungal survival, by pipetting part of this (100 μL) on PGA plates and incubated for a week at 25ºC.- Gnotobiotic experiment: Sterile switchgrass seedlings will be inoculated with different microbial combinations:Bacteria only (B),fungi only (F), and two groups together (BF), also, an un-inoculated control will be included (microbefree, MF). Fungal strains will be prepared (Week 1) as mentioned above and incubated for 2 weeks before the inoculation day. The bacterial strains will be grown in liquid media as previously described, and the switchgrass seeds will be sterilized and entrained for four days. On the inoculation day, the seedlings will be transferred into big pots filled with a sterilized soil (collected from the same field site as our microbiome study), aseptically inoculated with desired input microbiota, and kept in greenhouse. After 3-4 months, plant shoots were harvested, along with root and matrix samples. Then, roots will be washed thoroughly with clean sterile water, dried,placed in falcon tubes and snap-frozen in liquid nitrogen for storage at -80ºC.We will compare plant height, root length, fresh and dry weights of shoot and root, biomass composition, and theoretical ethanol yield of the inoculated plants with those parameters in control plants.