Back to top

Program Details

In this summer project, we want to test a hypothesis that heritable microbes can be consistently recruited to maize and sorghum roots from various locations across the state of Nebraska, and we want to identify nitrogen fixing microbes in different neighborhood soils.

The science behind the project

The science behind the project

Figure 1: Microbes colonizing plant roots.

  • Plant roots are colonized by millions of bacteria, some of which help the plant by acquiring nutrients (such as nitrogen) or by fighting diseases.

  • It is thought that beneficial bacteria are actively recruited by the plant and picked up from the environment, depending on the plant’s genetics.

  • If this is true, we would expect specific crop species or cultivars to recruit similar microbes across different environments, as long as these microbes are present in the soil where the seeds are planted.

The science behind the project

Figure 1: Microbes colonizing plant roots.

Overall Idea

  • About 10 high school students across Nebraska to be involved in this project.

  • Each participant will plant and manage a 10x10 ft plot (or using plastic pots) with all 4 cultivars in a backyard, field or other suitable location.

  • In the UNL lab, microbiome DNA will be processed for pooled DNA sequencing.

  • After the data collection, a workshop will be held through online tutorials to develop a data analysis pipeline.

Expected Participants

Figure 2: Expected geographic distribution of the potential participants.

We will recruit about 10 high school students across Nebraska to be involved in this project. Under remote supervision by UNL professors, graduate students, and/or undergraduate students, each participant will receive a field lab kit by mail with seeds for 2 maize and 2 sorghum cultivars, sampling equipment, and detailed instructions for each step.

Figure 2: Expected geographic distribution of the potential participants.

Materials and methods

Figure 3: Field layout of the experiment in each location.

Each participant will plant and manage a 10x10 ft plot (or using plastic pots) with all 4 cultivars in a backyard, field or other suitable location. Participants are expected to measure the morphological traits as the plants develop. About five weeks after planting, rhizosphere soil (soil attached to plant roots, which contains many bacteria) will be collected and shipped back to the UNL lab.

Figure 3: Field layout of the experiment in each location.

Microbiome sequencing

Figure 4: DNA sequences present in the soil samples will identify root-colonizing microbes, selective culture media will reveal nitrogen fixing microbes

In the UNL lab, microbiome DNA will be extracted from the collected soil samples to prepare the 16S rRNA libraries for pooled DNA sequencing. Meanwhile, participants will also use selective media to isolate a subset of the microbes that are capable of fixing nitrogen (this means they can take nitrogen out of the air rather than from foodstuff). Online group meetings will be scheduled as needed to discuss the project progress and to solve potential problems.

Figure 4: DNA sequences present in the soil samples will identify root-colonizing microbes, selective culture media will reveal nitrogen fixing microbes

Timeline

Timeline

Figure 5: Timeline from seed planting to harvesting.

Timeline

Figure 5: Timeline from seed planting to harvesting.

Summary of Experimental Procedure

Field prep & planting

In each location, participants will designate and measure a 10x10 foot plot in a backyard, field or other suitable location. Vegetation and debris will be removed from each plot, the plot will be tilled and one row of 6 plants for each cultivar will be sowed (see Fig. 3). A ~1 lb bulk soil sample is collected from the center of the 10x10 ft plot and stored in a household freezer. The bulk soil sample will serve to assess the chemical composition of the soil as well as the background microbiome from which the plants in each location are expected to recruit root microbes.

Measure time to germination

During the first week, the plot is checked daily for emerging seedlings. The date of emergence will be recorded for each plant.

Record plant traits

Starting upon emergence, morphological traits of each plant are recorded 2x per week. Traits to measure include plant height, number of leaves and base stem circumference. For each time point a photo of the plot will be taken and submitted to the university mentors.

Root soil sampling

3 plants per cultivar will be cut and roots dug up with a shovel. Loose soil is shaken off to expose roots with tightly adherent soil (called “rhizosphere”). We are interested in this soil as it is home to a great number of microbes. Root pieces with adherent soil are collected in a 50 ml sample tube, sample buffer is added and rhizosphere soil is shaken off the roots. The rhizosphere suspension is filtered and stored in a fresh freezer tube. Before freezing, a small sample of the soil suspension pipetted off to be analyzed for nitrogen fixing bacteria.

Isolation of nitrogen fixing microbes

For each of the 4 plant cultivars, the 3 replicates will be mixed and processed in 2 ways: 1 ml rhizosphere soil suspension will be transferred into a screw cap tube, mixed with glycerol solution, and frozen for later analysis at UNL (glycerol keeps microbes alive during freezing). A second 1 ml rhizosphere soil suspension will be diluted and serial dilutions will be plated out on nitrogen deficient media and growth of microorganisms will be monitored (guess which kind of microbes will survive on that media).

Sample pickup and data analysis at UNL

From each of 10 locations, 1 bulk soil sample, 12 rhizosphere soil samples and 4 glycerol stocks will be collected by UNL teams. This will also be a chance for a meet & greet with the scientists! Once all samples are collected, DNA will be isolated from 120 rhizosphere and 10 bulk soil samples for Illumina 16S rDNA sequencing. This is expected to take several weeks. Sequencing data will be analyzed by UNL researchers. 16S reads will be quality filtered, reads will be annotated to microbial taxa and microbiomes will be summarized for all samples at the level of microbial genera. A final table of sequence counts for all detected microbial genera (rows) in all 130 samples (columns) will be able to answer the question stated in the hypothesis.

Kits for the Experiments

Two kits for conducting the experiments will be shipped to the participant’s home address.

Field Lab Kit

expected delivery date: June 16th

  • 6 seeds each for 2 maize and 2 sorghum cultivars
  • Plant and row labels, stakes, stapler, sharpie
  • Plastic box to collect roots
  • Nylon gloves
  • Ziploc bags (bulk soil sample and freezer bag for all samples)
  • Falcon tubes (50 ml) + rack
  • Sample labels (pre-printed)
  • 2x 500 ml bottle with PBS buffer (autoclaved)
  • Labels (pre printed)
  • Pots (for participants who need these)
  • Manual with detailed instructions

Optional field tools

  • Hoe for tilling/weeding, shovel for root harvesting,
  • Planting Pots
  • Field gloves, pruner, 10 ft tape measure

Microbiome Isolation Kit

expected delivery date: July 21st

  • Tubes pre-filled with PBS or MgCl2 (for serial dilutions)
  • Sterilized glass beads in eppendorf tubes (for plate spreading)
  • Tube rack (for “mixing to mimic vortexing” and to collect dilutions)
  • Nitrogen-deficient media (with preprinted labels)
  • Sharpie (for labeling tubes)
  • Plastic container
  • Tubes to use as a way to measure roots for similar volume (instead of weight)
  • Prepaid Shipping Box with packaging material
  • Sterile transfer pipettes
  • Nylon gloves