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

Summary

A method of growing germ-free Napa cabbages has been developed which enables researchers to evaluate how single microbial species or multispecies microbial communities interact on cabbage leaf surfaces. A sterile vegetable extract is also presented which can be used to measure shifts in community composition during vegetable fermentation.

Abstract

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial community assembly. This protocol outlines a system for studying microbial community dynamics in the phyllosphere of Napa cabbage plants. It describes how to grow germ-free plants in test tubes with a calcined clay and nutrient broth substrate. Inoculation of germ-free plants with specific microbial cultures provides opportunities to measure microbial growth and community dynamics in the phyllosphere. Through the use of sterile vegetable extract produced from cabbages shifts in microbial communities that occur during fermentation can also be assessed. This system is relatively simple and inexpensive to set up in the lab and can be used to address key ecological questions in microbial community assembly. It also provides opportunities to understand how phyllosphere community composition can impact the microbial diversity and quality of vegetable fermentations. This approach for developing gnotobiotic cabbage phyllosphere communities could be applied to other wild and agricultural plant species.

Introduction

Microbial diversity of the phyllosphere plays an important role in maintaining plant health and can also influence the ability of plants to withstand environmental stress^1,2,3,4,5. In turn, the health of crops directly impacts food safety and quality^6,7. Plants play a role in ecosystem functioning and their associated microbiomes both affect the ability of plants to carry out these activities as well as directly influencing the environment themselves⁸. While scientists have begun to decipher the function and composition of the phyllosphere, the ecological processes that influence phyllosphere microbial community assembly are not fully understood^9,10. The phyllosphere microbiome is an excellent experimental system for studying the ecology of microbiomes¹¹. These communities are relatively simple and many of the community members can be grown on standard lab media^10,12,13.

Fermented vegetables are one system where the community structure of the phyllosphere has important consequences. In both sauerkraut and kimchi, the microbes that naturally occur on vegetable leaves (the phyllosphere of Brassica species) serves as the inoculum for fermentation^14,15. Lactic acid bacteria (LAB) are considered ubiquitous members of vegetable microbiomes, however they can be in low abundance in the phyllosphere¹⁶. Strong abiotic selection during fermentation drives a shift in microbial community composition enabling lactic acid bacteria to increase in abundance. As LAB grow, they produce lactic acid which creates the acidic environment of fermented vegetable products¹⁷. The link between the phyllosphere and the ferment provides an opportunity to use vegetables as a model to understand how microbiomes are structured.

We have developed methods to grow germ-free Napa cabbages and to inoculate them with specific microbial communities using spray bottles. This is an inexpensive and reliable method of evenly inoculating the cabbage with either individual microbes or mixed communities. A sterile vegetable extract (SVE) has also been developed from three different cabbage types/varieties: red and green cabbage (Brassica oleracea) and Napa cabbage (B. rapa). The addition of salt to these SVEs replicates the fermentation environment and allows for small-scale and relatively high-throughput experimental studies of fermentation microbiome assembly. These methods can be used to study microbial community assembly in the phyllosphere and how microbial community dynamics in the phyllosphere can be linked to the success of vegetable fermentation.

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Protocol

1. Growing germ-free cabbages

1. Preparing equipment for growing germ-free cabbages
   1. Cleaning the calcined clay to remove fine dust particles
      1. Rinse calcined clay (Table of Materials) at least 3x with tap water; drain off water.
         CAUTION: Calcined clay produces very fine dust and it is recommended to wear a protective mask (Table of Materials) when washing.
      2. Spread calcined clay out as a thin layer (~4 cm) into an autoclave tray and autoclave on a dry cycle (121 °C heating for 20 min and 20 min drying time) to sterilize.
      3. Allow the calcined clay to fully dry prior to use by spreading out on trays and placing in a warm incubator (30−37 °C) for at least a week. Stir to mix every 3 days to fully dry the calcined clay so that it will absorb an even amount of Murashige and Skoog (MS) nutrient broth (section 1.2).
         NOTE: Drying also helps to keep the volume of calcined clay even when it is weighed into tubes. Drying by other means, such as a drying oven, would also be suitable.
   2. Cleaning the glassware for growing germ-free cabbages
      1. Thoroughly clean and sterilize the glass tubes (Table of Materials) between each use. Soak tubes for 30 min in 30% bleach solution and rinse well with tap water before cleaning in an acid wash on a bacteriology setting. Acid-wash two-way test tube caps (Table of Materials) between uses.
   3. Surface sterilizing cabbage seeds
      1. Place up to 100 Napa cabbage (B. rapa var pekinensis) seeds in a 1.5 mL microcentrifuge tube.
         NOTE: Adding more than 100 seeds to one microcentrifuge tube or changing the size of the tube may affect germination rates of the seeds due to lack of seed coat removal.
      2. Add 1 mL of 70% ethanol to the seeds and vortex for 5 min. Discard the ethanol using a pipette.
      3. Add 1 mL of 50% bleach and vortex for 5 min. Discard the bleach solution using a pipette.
      4. Add 1 mL of autoclaved deionized water and vortex for 5 min. Discard the deionized water using a pipette.
      5. Repeat step 1.1.3.4 3x to rinse off all bleach. Soak the seeds in sterile deionized water for 2−8 h prior to planting to soften the seed coat.
2. Growing germ-free cabbages
   NOTE: Napa cabbages (B. rapa var pekinensis) are grown in glass tubes (15 cm x 2.5 cm) containing calcined clay soaked in Murashige and Skoog (MS) nutrient broth (Figure 1).
   1. Weigh 10 g of clean calcined clay into a clean glass tube (15 cm x 2.5 cm).
   2. Prepare MS nutrient broth by dissolving 4.4 g of MS medium in 1 L of deionized water. Add MS nutrient broth (~9 mL) to each glass tube to cover the calcined clay using a pipette.
      NOTE: Standing liquid in the tube will prevent the seed from germinating so it might benecessary to add slightly less MS broth to some tubes.
   3. Loosely cap glass tubes with 22 mm two-way test tube caps and autoclave (121 °C for 60 min). When removing them from the autoclave, push caps onto the glass tubes to seal them. Cool tubes to room temperature before use.
   4. Gently place one sterile cabbage seed into the center of each tube using sterile, extra-long (25.4 cm) forceps. Place the tubes in a 7-way tray then place under light racks (full-spectrum T5 fluorescent bulbs or other illumination setup for plant growth) with a 16 h light cycle at 24 °C.
      NOTE: Seeds germinate overnight and develop their first true leaf after 5 days. A true leaf is the first vascular leaf after the cotyledons have formed. It has a more wrinkled edge and in Brassica rapa is covered in trichomes.
3. Testing for sterility of germ-free cabbages
   NOTE: To test whether the cabbages are germ-free, select a few (5−10) cabbages from each batch and plate out to determine whether any culturable colonies are present.
   1. Gently remove the cabbage from the glass tubes by gripping the base of the plant with sterilized forceps and pulling it out. Before removing the cabbage fully from the tube, carefully cut off the roots using sterilized dissection scissors. Compact the cabbage leaves into a 1.5 mL microcentrifuge tube.
      NOTE: Larger cabbages might require removing one or two of the larger leaves while the cabbage is still in the tube to make it easier to get the cabbage into the 1.5 mL microcentrifuge tube. These larger leaves can be added to the 1.5 mL tube after the rest of the cabbage has been placed into the tube if the entire cabbage is required.
   2. Add 400 µL of 1x phosphate buffer saline (PBS) to each 1.5 mL microcentrifuge tube. Using a sterile micropestle, homogenize the cabbage by pestling 30x.
   3. Plate 100 µL of the cabbage homogenate onto agar plates to determine whether there are any contaminants present in the sample. Most bacteria found in the phyllosphere will grow on tryptic soy (TS) agar plates. Use wide orifice pipette tips when plating cabbage homogenate as the cabbage homogenate is thick and can clog regular pipette tips.

2. Inoculating the phyllosphere with microbial solutions

1. Making glycerol stocks of inoculation strains
   NOTE: Table 1 lists the microbial isolates that can be used in this step. Other phyllosphere isolates could also be used here.
   1. Densely streak out individual colonies from a fresh streak, onto two/three new plates of the same media to get many colonies.
   2. Let streaks grow for 2−5 days then scrape colonies from all plates into a 15 mL conical tube containing 15 mL of 15% glycerol, and vortex to mix thoroughly.
   3. Transfer an aliquot of 1 mL of the well-mixed glycerol stock into a 1.5 mL microcentrifuge tube and store glycerol stocks at -80 °C until use. Save the remaining 14 mL of glycerol stock at -80 °C as relatively large volumes of inoculation solution are required when inoculating cabbages.
   4. One week before use, thaw the 1.5 mL tube containing 1 mL of glycerol stock (from step 2.1.3) on ice, dilute, and plate at several different dilutions (e.g., 10^-4, 10^-5, and 10^-6) to determine the concentration (colony-forming unit [CFU] per µL) of the 14 mL of inoculation solution.
2. Sterilizing inoculation spray bottles
   1. Disassemble the amber round Boston pump bottles (59 mL) and soak all components (pump, tube, cap and bottle) in 30% bleach solution for 30 min in a large plastic container with a tightly fitting lid.
   2. After soaking, carefully pour out all bleach from the container by lifting just one corner of the lid of the container.
   3. Rinse the bottles by filling the plastic container with autoclaved deionized water (~1 L depending on the container size) and carefully pour out deionized water, again by lifting the lid at one corner.
   4. Sterilize a biosafety cabinet by spraying with 70% ethanol solution and turning on the UV light for 30 min.
      NOTE: Continue this work in the biosafety cabinet so that there is no risk of microbial contamination of the bottles as they air dry.
   5. Remove bottles from the large plastic container and fill each bottle with autoclaved deionized water using a pipette. Reassemble the pumps and place one in each bottle. Pump the deionized water through each bottle (10 sprays per bottle) to remove bleach from the pump component of the bottle.
   6. Repeat step 2.2.5 to ensure that all bleach is removed from the glass bottles.
   7. Test whether bottles are sterile by placing a number on each bottle (sticking lab tape to the side of the bottle when it is fully dry) then add 10 mL of 1x PBS to each of the bottles and pump 3 sprays onto a TS agar plate. After spraying, incubate the plates for one week at room temperature. If any colonies grow on a plate it indicates that the respective bottle was not sterile and should not be used for experiments.
   8. Before storing the sterile bottles, remove all remaining PBS and allow the bottles to dry thoroughly in the biosafety cabinet. Store sterile bottles in a sterile plastic container (typically the container used for bleaching the bottles) until use.
3. Preparing the microbial inoculum and spraying germ-free cabbages
   CAUTION: All steps should be performed in a biosafety cabinet, as spraying aerosolizes the microbial solutions which could contaminate work surfaces or pose a health risk if carried out on a lab bench.
   NOTE: Cabbages will form true leaves after 5 days, so it is advisable to wait one week after planting the cabbage before inoculating with any microbial solutions. As the tubes are sealed, there is no need to water the cabbages. Experiments are best performed within a month of planting, as the small tubes restrict the cabbage's growth.
   1. Thaw glycerol stocks on ice and dilute in 1x PBS to the desired inoculation concentration (concentration determined by thawing and plating a 1 mL aliquot in step 2.1.4).
      NOTE: A variety of different inoculation levels can be used, but phyllosphere isolates can grow from 10⁴ to 10⁸ CFUs/mL of cabbage slurry in 10 days.
   2. Add 10 mL of diluted glycerol stock to the sterile pump bottle and pump 5 sprays into a large waste collection beaker to remove any residual PBS from the bottle pump component.
   3. Remove the lid from the cabbage tube, tilt the cabbage towards the spray bottle, and spray each cabbage with 3 pumps of the inoculation solution, which provides ~600 µL of inoculum.
   4. After inoculating, harvest a subset of the cabbages to assess the actual input inoculation concentration. Remove the cabbage from a tube with sterilized forceps. Cut off the roots with sterile dissection scissors and then carefully place the cabbage in a preweighed sterile 1.5 mL microcentrifuge tube. Record the weight of the cabbage for future calculations if CFUs/g of cabbage is required for calculations.
   5. Add 400 µL of 1x PBS to each 1.5 mL microcentrifuge tube containing cabbage and use a sterile micropestle to homogenize the cabbage into the 1x PBS by grinding it 30x.
   6. Dilute cabbage homogenate (if required) and plate out the pestled cabbage mixture. Use wide orifice tips for pipetting the cabbage slurry because it will be thick and full of plant tissue pieces.

3. Preparing sterile vegetable extract

NOTE: This method is a modified version of cabbage sterile media production^18,19.

1. Purchase a cabbage from a supermarket. In the lab, remove and discard the outermost leaves of the cabbage. Chop all remaining cabbage to fit into a blender and homogenize cabbage to a fine pulp, i.e., the cabbage will not get any finer with further blending.
   NOTE: Any blender which can chop cabbage to a smooth homogeneous pulp should be suitable for this method.
2. Weigh the blended cabbage homogenate and add 2 mL of distilled water per gram of cabbage. Filter the blended cabbage slurry through 2 layers of basket coffee filters (unbleached paper).
3. Dispense the cabbage slurry into centrifuge tubes (size is dependent on the centrifuge). Centrifuge the filtered cabbage slurry at 20,000 x g for 20 min until large particles settle out of solution.
   NOTE: It is essential to centrifuge the cabbage slurry for a long period of time as cabbage particles rapidly clog the filter sterilizer.
4. Using a serological pipette, remove the supernatant from the pelleted cabbage debris taking care not to disturb the pelleted cabbage. If aiming to recreate fermentation conditions where standard salt concentrations are used, add 2% w/v NaCl at this step (i.e., before filter sterilization).
5. Filter sterilize the vegetable extract using a 0.2 µm filter (500 mL or 1 L) attached to a vacuum. Dispense into sterile tubes (either 50 mL centrifuge tubes or 15 mL centrifuge tubes) and freeze at -80 °C until use.

4. Inoculation of sterile vegetable extract

1. Thaw SVE and dispense 490 µL into 1.5 mL microcentrifuge tubes. Use sufficient tubes to have at least five replicates per treatment per timepoint as each timepoint measurement is destructive.
2. Thaw glycerol stocks of microbial isolates on ice and dilute with 1x PBS to the desired concentration. The concentration of lactic acid bacteria can be as low as 5,000 CFU per mL of SVE. To achieve this concentration, dilute stocks to 250 CFUs/µL because 10 µL will be used for inoculation of a total volume of 500 µL.
3. Inoculate SVE with 10 µL of diluted microbial isolate. Pipette up and down a few times to thoroughly mix. Incubate at desired temperature (14 °C for kimchi production temperature or 24 °C for warmest sauerkraut fermentation).
4. Measure rate of growth of microbial isolate in the SVE by harvesting replicate tubes on day 1, day 2, day 4, day 7 and day 14.
   NOTE: Fermentation proceeds rapidly at the outset and slows over time. Therefore, having more initial timepoints gives greater resolution to the dynamics of how fermentation proceeds.
5. At each timepoint, mix the inoculated SVE well by pipetting up and down a few times. Serially dilute the inoculated SVE in 1x PBS and plate onto agar plates. Incubate the agar plates for 4−7 days before counting colonies.
   NOTE: Man, Rogosa, and Sharpe (MRS) agar should be used to enumerate all lactic acid bacteria, yeast peptone dextrose (YPD) should be used for yeast, and TS agar for most other bacteria isolates from the phyllosphere.
6. Record the pH of the samples at each timepoint using a micro pH probe.
   NOTE: This step should be carried out after plating because the pH probe will transfer cells between tubes/treatments.

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Results

Growth rates of Napa cabbages
The seed sterilization method was tested with several different Napa cabbages (B. rapa var pekinese; Supplemental Figure 1) from a number of different suppliers and all grew consistently with similar growth rates. However, testing the methods with different species of Brassica (B. rapa: Turnip Purple Top; B. oleracea: Cairo Hybrid, Tropic Giant Hybrid; B. campestris: Pak Choi...

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Discussion

Germ-free Napa cabbage plants have been used to study dispersal limitation of lactic acid bacteria in the Napa cabbage phyllosphere¹⁷. Germ-free Napa cabbages can also be used to test individual or pair-wise growth in the phyllosphere (Figure 1). Methods for making sterile vegetable extract has been tested for three different varieties of cabbage: red, green and Napa. Each of these SVEs act as a reliable growth media; inoculated microbes grow consistently across the d...

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Materials

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tubes	VWR	20170-650
15 mL conical tubes	Falcon	352096
7-way tray tray	Sigma Magenta	T8654
Amber Round Boston Glass Bottle		GPS 712OZSPPK12BR	Ordered on Amazon.com from various suppliers
Basket coffee filters	If you care		(unbleached paper) Purchased from Wholefoods
Bleach (mercury-free)	Austin's	50-010-45
Borosilicate Glass tubes	VWR	47729-586
Calcined clay	Turface	MVP	Ordered on Amazon.com from Root Naturally 6 Quart Bags. Particle size approximately 3-5 mm
Cuisinart blender	Cuisinart		Cuisinart Mini-Prep Plus Food Processor, 3-Cup
Dissection scissors	7-389-A	American Educational Products	Ordered on Amazon.com
Ethanol	VWR	89125-172
Forceps	Aven	18434	Ordered on Amazon.com
Glycerol	Fisher Scientific	56-81-5
KleenGuard M10	Kimberley-Clark	64240
Large plastic container	Rubbermaid		Ordered on Amazon.com
Light racks	Gardner's Supply	39-357	full-spectrum T5 fluorescent bulbs
Magenta tm 2-way caps	Millipore Sigma	C1934
Man, Rogosa, and Sharpe	Fisher Scientific	DF0881-17-5	This media is for broth and 15 g of agar is added to make plates
Micro pH probe	Thermo Scientific	8220BNWP
Micropestle	Carolina	215828	Also called Pellet Pestle
MS nutrient broth	Millipore Sigma	M5519	Murashige and Skoog Basal Medium
NaCl	Sigma Aldrich	S9888
Napa cabbage seeds	Johnny's Select Seeds	2814G	B. rapa var pekinensis (Bilko)
Petri dish 100 mm x 15 mm	Fisher	FB0875712	Used to make agar plates
Phosphate buffer saline	Fisher Scientific	50-842-941	Teknova
Plant tissue culture box	Sigma	Magenta GA-7
Serologial pipettes	VWR	89130-900
Sterile dowel	Puritan	10805-018	Autoclave before use to sterilize
Sterilizing 0.2 µm filter	Nalgene	974103
Tryptic soy agar	Fisher Scientific	DF0370-17-3	This media is for broth and 15 g of agar is added to make plates
Wide orifice pipette tips	Rainin	17007102
Yeast, peptone and dextrose	Fisher Scientific	DF0428-17-5	This media is suitable but media can also be made using yeast, peptone and dextrose, add 15 g of agar when making plates