A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

Using this protocol, we can address key questions regarding how microbes assemble and interact in the phyllosphere. It has the added benefit that by using the sterile vegetable extract, we can link the structure of phyllo sphere communities to their function during vegetable fermentation. These techniques enable scientists to test how microbes interact in the phyllo sphere and then track how these phyllo sphere communities can impact fermentation.

To surface sterilize the cabbage seeds, start by placing 100 seeds in a 1.5 milliliter micro centrifuge tube. Add one milliliter of 70%ethanol to the tube and vortex it for five minutes. Then use a pipette to discard the ethanol.

Add one milliliter of 50%bleach and vortex the tube for five minutes, then discard the bleach. Rinse the seeds four times, by adding one milliliter of autoclaved deionized water vortexing the tube, then discarding the water. After the last rinse, soak the seeds in sterilized deionized water for two to eight hours to soften the seed coat before planting.

Weigh 10 grams of clean calcium clay into a 15 by 2.5 centimeter glass tube. Add approximately nine milliliters of prepared MS Broth to each tube to cover the calciumed clay. Loosely cap the glass tubes with 22 millimeter two way test tube caps.

Autoclave the tubes at 121 degrees Celsius for 60 minutes. When removing the tubes from the autoclave, push the caps onto the glass tubes to seal them, then cool the tubes to room temperature before use. When the tubes have cooled, used extra long forceps to gently place one sterile cabbage seed into the center of each tube.

Lace the tubes in a seven way tray and place the tray under light racks with a 16 hour light cycle at 24 degrees Celsius. The seeds will develop their first true leaf after five days, which is the first vascular leaf after the cotyledons have formed. It has a wrinkled edge and is covered in tricombs.

To test the germ-free cabbage for sterility, gently grip the base of the plant with sterilized forceps and pull it out. Before fully removing the cabbage from the tube, carefully cut off the roots with sterilized dissection scissors. Then, compact the cabbage leaves into a 1.5 milliliter micro centrifuge tube.

Add 400 microliters of PBS to each micro centrifuge tube and use a sterile micro pestle to homogenize the cabbage. Blade 100 microliters of the cabbage homogenate onto agar plates, making sure to use a wide orifice pipette tip. To make glycerol stocks of inoculating strains, gently streak out individual colonies onto two or three new plates of the same media.

Row the colonies for two to five days, then scrape them from the plates into a 15 milliliter conical tube with 15 milliliters of 15%glycerol. Vortex the tube thoroughly to mix. Aliquot one milliliter of the mixed glycerol stock into a micro centrifuge tube and store it 80 degrees Celsius until ready to use along with the remaining 14 milliliters of glycerol stock.

One week prior to use, thaw the one milliliter aliquot on ice and place it at several different dilutions to determine the concentration of the inoculation stock. When ready to inoculate the cabbage, thaw the glycerol stock on ice and dilute it to the desired concentration. Add 10 milliliters of the diluted stock to the sterile pump bottle and pump five sprays into a large waste collection beaker.

Remove the lid from the cabbage tube and tilt the cabbage towards the spray bottle. Spray each cabbage with three pumps of the inoculation solution, then harvest a subset of the cabbages to assess the actual input inoculation concentration. To harvest the cabbage, remove it from the tube with sterilized forceps.

Cut off the roots with sterile dissection scissors and carefully place it in a pre weighed sterile micro centrifuge tube. Record the weight of the cabbage for future calculations. Add 400 microliters of PBS to each tube with the cabbage and grind it 30 times with a micro pestle, then plate the cabbage homogenate using wide orifice pipette tips.

Remove and discard the outermost leaves of the cabbage and chop all remaining cabbage to fit into a blender. Blend it to a fine pulp and weigh the cabbage homogenate. Add two milliliters of distilled water per gram of cabbage.

Then filter the cabbage slurry through two layers of basket coffee filters. Dispense the slurry into centrifuge tubes and centrifuge it at 20, 000 times G for 20 minutes or until large particles settle out of solution. Remove the supernatant, taking care to not disturb the pelleted cabbage debris.

To recreate the fermentation conditions with standard salt concentrations, add 2%weight to volume sodium chloride to the extract. Bilder the vegetable extract with a 0.2 micrometer filter attached to a vacuum, dispense it into sterile tubes and freeze it at minus 80 degrees Celsius. The seed sterilization method was tested with several Napa cabbages and they all demonstrated similar growth rates.

However, other species of brassica gave limited success because the plants either had low germination rates after sterilizing or the stem elongated rapidly to make a spindly unhealthy plant. Microbial isolates were inoculated either as single strain isolates or in combination with another isolate. A total of 15 germ-free cabbages were tested for each treatment and harvested either immediately, at four days or at 10 days after inoculation.

The isolates demonstrated rapid growth in the Napa cabbage Phyllosphere. Two yeasts and three bacteria were inoculated into three different types of sterile vegetable extract or SVE made from red, green and Napa cabbage. Growth of the inoculates was recorded over 14 days by spot plating five microliters of each treatment onto either MRS or YPD agar plates.

Measurements of pH throughout the fermentation showed that the lactic acid bacteria were capable of acidifying the SVE to levels below pH four, indicating a ferment that is safe for consumption. These methods have specifically applied to the cabbage microbiome but could be useful in other plant systems. It can be challenging to keep the system sterile, our outline protocols are detailed, but we found each step to be important.