This work was supported by the USDA-NIFA grant: 2017-67013-26520. Tracy Debenport and Claire Fogan provided technical support and Ruby Ye and Casey Cosetta provide helpful comments on early versions of this manuscript.

1. Growing germ-free cabbages
<ol>
	<li>Preparing equipment for growing germ-free cabbages
	<ol>
		<li>Cleaning the calcined clay to remove fine dust particles
		<ol>
			<li>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.</li>
			<li>Spread calcined clay out as a thin layer (~4 cm) into an autoclave tray and autoclave on a dry cycle (121 &#176;C heating for 20 min and 20 min drying time) to sterilize.</li>
			<li>Allow the calcined clay to fully dry prior to use by spreading out on trays and placing in a warm incubator (30&#8722;37 &#176;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.</li>
		</ol>
		</li>
		<li>Cleaning the glassware for growing germ-free cabbages
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Surface sterilizing cabbage seeds
		<ol>
			<li>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.</li>
			<li>Add 1 mL of 70% ethanol to the seeds and vortex for 5 min. Discard the ethanol using a pipette.</li>
			<li>Add 1 mL of 50% bleach and vortex for 5 min. Discard the bleach solution using a pipette.</li>
			<li>Add 1 mL of autoclaved deionized water and vortex for 5 min. Discard the deionized water using a pipette.</li>
			<li>Repeat step 1.1.3.4 3x to rinse off all bleach. Soak the seeds in sterile deionized water for 2&#8722;8 h prior to planting to soften the seed coat.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>Weigh 10 g of clean calcined clay into a clean glass tube (15 cm x 2.5 cm).</li>
		<li>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.</li>
		<li>Loosely cap glass tubes with 22 mm two-way test tube caps and autoclave (121 &#176;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.</li>
		<li>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 &#176;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.</li>
	</ol>
	</li>
	<li>Testing for sterility of germ-free cabbages 
	NOTE: To test whether the cabbages are germ-free, select a few (5&#8722;10) cabbages from each batch and plate out to determine whether any culturable colonies are present.
	<ol>
		<li>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.</li>
		<li>Add 400 &#181;L of 1x phosphate buffer saline (PBS) to each 1.5 mL microcentrifuge tube. Using a sterile micropestle, homogenize the cabbage by pestling 30x.</li>
		<li>Plate 100 &#181;L 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.</li>
	</ol>
	</li>
</ol>
2. Inoculating the phyllosphere with microbial solutions
<ol>
	<li>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.
	<ol>
		<li>Densely streak out individual colonies from a fresh streak, onto two/three new plates of the same media to get many colonies.</li>
		<li>Let streaks grow for 2&#8722;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.</li>
		<li>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 &#176;C until use. Save the remaining 14 mL of glycerol stock at -80 &#176;C as relatively large volumes of inoculation solution are required when inoculating cabbages.</li>
		<li>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 &#181;L) of the 14 mL of inoculation solution.</li>
	</ol>
	</li>
	<li>Sterilizing inoculation spray bottles
	<ol>
		<li>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.</li>
		<li>After soaking, carefully pour out all bleach from the container by lifting just one corner of the lid of the container.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Repeat step 2.2.5 to ensure that all bleach is removed from the glass bottles.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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&#39;s growth.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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 &#181;L of inoculum.</li>
		<li>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.</li>
		<li>Add 400 &#181;L 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Preparing sterile vegetable extract
NOTE: This method is a modified version of cabbage sterile media production18,19.
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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).</li>
	<li>Filter sterilize the vegetable extract using a 0.2 &#181;m 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 &#176;C until use.</li>
</ol>
4. Inoculation of sterile vegetable extract
<ol>
	<li>Thaw SVE and dispense 490 &#181;L 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.</li>
	<li>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/&#181;L because 10 &#181;L will be used for inoculation of a total volume of 500 &#181;L.</li>
	<li>Inoculate SVE with 10 &#181;L of diluted microbial isolate. Pipette up and down a few times to thoroughly mix. Incubate at desired temperature (14 &#176;C for kimchi production temperature or 24 &#176;C for warmest sauerkraut fermentation).</li>
	<li>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.</li>
	<li>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&#8722;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.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

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...

Microbial diversity of the phyllosphere plays an important role in maintaining plant health and can also influence the ability of plants to withstand environmental stress1,2,3,4,5. In turn, the health of crops directly impacts food safety and quality6,7. 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 understood9,10. 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 media10,12,13.
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 fermentation14,15. 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.

<table><tbody><tr><td>1.5 mL microcentrifuge tubes</td><td>VWR</td><td>20170-650</td><td></td></tr><tr><td>15 mL conical tubes</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>7-way tray tray</td><td>Sigma Magenta</td><td>T8654</td><td></td></tr><tr><td>Amber Round Boston Glass Bottle</td><td></td><td>GPS 712OZSPPK12BR</td><td>Ordered on Amazon.com from various suppliers</td></tr><tr><td>Basket coffee filters</td><td>If you care</td><td></td><td>(unbleached paper) Purchased from Wholefoods</td></tr><tr><td>Bleach (mercury-free)</td><td>Austin&#039;s</td><td>50-010-45</td><td></td></tr><tr><td>Borosilicate Glass tubes</td><td>VWR</td><td>47729-586</td><td></td></tr><tr><td>Calcined clay</td><td>Turface</td><td>MVP</td><td>Ordered on Amazon.com from Root Naturally 6 Quart Bags. Particle size approximately 3-5 mm</td></tr><tr><td>Cuisinart blender</td><td>Cuisinart</td><td></td><td>Cuisinart Mini-Prep Plus Food Processor, 3-Cup</td></tr><tr><td>Dissection scissors</td><td>7-389-A</td><td>American Educational Products</td><td>Ordered on Amazon.com</td></tr><tr><td>Ethanol</td><td>VWR</td><td>89125-172</td><td></td></tr><tr><td>Forceps</td><td>Aven</td><td>18434</td><td>Ordered on Amazon.com</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>KleenGuard M10</td><td>Kimberley-Clark</td><td>64240</td><td></td></tr><tr><td>Large plastic container</td><td>Rubbermaid</td><td></td><td>Ordered on Amazon.com</td></tr><tr><td>Light racks</td><td>Gardner&#039;s Supply</td><td>39-357</td><td>full-spectrum T5 fluorescent bulbs</td></tr><tr><td>Magenta tm 2-way caps</td><td>Millipore Sigma</td><td>C1934</td><td></td></tr><tr><td>Man, Rogosa, and Sharpe</td><td>Fisher Scientific</td><td>DF0881-17-5</td><td>This media is for broth and 15 g of agar is added to make plates</td></tr><tr><td>Micro pH probe</td><td>Thermo Scientific</td><td>8220BNWP</td><td></td></tr><tr><td>Micropestle</td><td>Carolina</td><td>215828</td><td>Also called Pellet Pestle</td></tr><tr><td>MS nutrient broth</td><td>Millipore Sigma</td><td>M5519</td><td>Murashige and Skoog Basal Medium</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S9888</td><td></td></tr><tr><td>Napa cabbage seeds</td><td>Johnny&#039;s Select Seeds</td><td>2814G</td><td>&lt;em&gt;B. rapa var pekinensis (Bilko)&lt;/em&gt;</td></tr><tr><td>Petri dish 100 mm x 15 mm</td><td>Fisher</td><td>FB0875712</td><td>Used to make agar plates</td></tr><tr><td>Phosphate buffer saline</td><td>Fisher Scientific</td><td>50-842-941</td><td>Teknova</td></tr><tr><td>Plant tissue culture box</td><td>Sigma</td><td>Magenta GA-7</td><td></td></tr><tr><td>Serologial pipettes</td><td>VWR</td><td>89130-900</td><td></td></tr><tr><td>Sterile dowel</td><td>Puritan</td><td>10805-018</td><td>Autoclave before use to sterilize</td></tr><tr><td>Sterilizing 0.2 &amp;micro;m filter</td><td>Nalgene</td><td>974103</td><td></td></tr><tr><td>Tryptic soy agar</td><td>Fisher Scientific</td><td>DF0370-17-3</td><td>This media is for broth and 15 g of agar is added to make plates</td></tr><tr><td>Wide orifice pipette tips</td><td>Rainin</td><td>17007102</td><td></td></tr><tr><td>Yeast, peptone and dextrose</td><td>Fisher Scientific</td><td>DF0428-17-5</td><td>This media is suitable but media can also be made using yeast, peptone and dextrose, add 15 g of agar when making plates</td></tr></tbody></table>

a gnotobiotic system for studying microbiome assembly in the phyllosphere and in vegetable fermentation

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.

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...

Watch this Scientific Journal Video about A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation at JoVE.com

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

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.

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 ...

a-gnotobiotic-system-for-studying-microbiome-assembly-phyllosphere

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

7.1K Views.  Tufts University. 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.

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

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Genetics

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Immunology and Infection

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and ...

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various &#34;omics&#34; analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e.&#160;microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. ...

Biology

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

 Microscale SEM Analysis of Microbial Communities in Fungus-Growing Ant Cultures

Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy

In macroscale ecosystems, such as rainforests or coral reefs, the spatial localization of organisms is the basis of our understanding of community ecology. In the microbial world, likewise, microscale ecosystems are far from a random and homogeneous mixture of organisms and habitats. Accessing the spatial distribution of microbes is fundamental for understanding the functioning and ecology of the microbiota, as cohabiting species are more likely to interact and influence each other&#39;s physiology.
An interkingdom microbial ecosystem is at the core of fungus-growing ant colonies, which cultivate basidiomycete fungi as a nutritional resource. Attine ants forage for diverse substrates (mostly plant-based), metabolized by the cultivated fungus while forming a spongy structure, a &#34;microbial garden&#34; that acts as an external gut. The garden is an intertwined mesh of fungal hyphae growing by metabolizing the substrate, opening niches for a characteristic and adapted microbiota to establish. The microbiota is thought to be a contributor to substrate degradation and fungal growth, though its spatial organization is yet to be determined.
Here, we describe how we employ Scanning Electron Microscopy (SEM) to investigate, with unprecedented detail, the microbiota and biofilm spatial organization across different fungiculture systems of fungus-growing ants. SEM imaging has provided a description of the microbiota spatial structure and organization. SEM revealed that microbiota commonly assemble in biofilms, a widespread structure of the microbial landscapes in fungiculture. We present the protocols employed to fix, dehydrate, dry, sputter coating, and image such a complex community. These protocols were optimized to deal with delicate and heterogeneous samples, comprising plant and fungal biomass, as well as the microbiota and the biofilm.

We propose an optimized Scanning Electron Microscopy protocol for visualizing highly heterogeneous and delicate samples containing plant and fungal biomass, together with microbiota and biofilm. This protocol allows describing the spatial dimensions of the microbiota organization.

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

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Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy