Name: a gnotobiotic system for studying microbiome assembly in the phyllosphere and in vegetable fermentation
Uploaded: 2020-06-03T14:00:00
Description: Watch this Scientific Journal Video about A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation at JoVE.com

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 aut...

<ol>
	<li>Grady, K. L., Sorensen, J. W., Stopnisek, N., Guittar, J., Shade, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+seasonality+of+core+phyllosphere+microbiota+on+perennial+biofuel+crops.">Assembly and seasonality of core phyllosphere microbiota on perennial biofuel crops.</a> Nature Communications. 10 (1), 4135 (2019).</li><li>Pii, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+interactions+in+the+rhizosphere:+beneficial+influences+of+plant+growth-promoting+rhizobacteria+on+nutrient+acquisition+process.+A+review.">Microbial interactions in the rhizosphere: beneficial influences of plant growth-promoting rhizobacteria on nutrient acquisition process. A review.</a> Biology and Fertility of Soils. 51 (4), 403-415 (2015).</li><li>Berendsen, R. L., Pieterse, C. M. J., Bakker, P. A. H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rhizosphere+microbiome+and+plant+health.">The rhizosphere microbiome and plant health.</a> Trends in Plant Science. 17 (8), 478-486 (2012).</li><li>Bai, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+overlap+of+the+Arabidopsis+leaf+and+root+microbiota.">Functional overlap of the Arabidopsis leaf and root microbiota.</a> Nature. 528 (7582), 364-369 (2015).</li><li>Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van Themaat, E., Schulze-Lefert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functions+of+the+bacterial+microbiota+of+plants.">Structure and functions of the bacterial microbiota of plants.</a> Annual Review of Plant Biology. 64, 807-838 (2013).</li><li>Dinu, L. D., Bach, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+viable+but+nonculturable+Escherichia+coli+O157:H7+in+the+phyllosphere+of+lettuce:+a+food+safety+risk+factor.">Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor.</a> Applied and Environmental Microbiology. 77 (23), 8295-8302 (2011).</li><li>Heaton, J. C., Jones, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+contamination+of+fruit+and+vegetables+and+the+behaviour+of+enteropathogens+in+the+phyllosphere:+a+review.">Microbial contamination of fruit and vegetables and the behaviour of enteropathogens in the phyllosphere: a review.</a> Journal of Applied Microbiology. 104 (3), 613-626 (2008).</li><li>Bringel, F., Cou&#233;e, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+roles+of+phyllosphere+microorganisms+at+the+interface+between+plant+functioning+and+atmospheric+trace+gas+dynamics.">Pivotal roles of phyllosphere microorganisms at the interface between plant functioning and atmospheric trace gas dynamics.</a> Frontiers in Microbiology. 6, 486 (2015).</li><li>Maignien, L., DeForce, E. A., Chafee, M. E., Eren, A. M., Simmons, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+succession+and+stochastic+variation+in+the+assembly+of+Arabidopsis+thaliana+phyllosphere+communities.">Ecological succession and stochastic variation in the assembly of Arabidopsis thaliana phyllosphere communities.</a> mBio. 5 (1), e00682 (2014).</li><li>Carlstr&#246;m, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+microbiota+reveal+priority+effects+and+keystone+strains+in+the+Arabidopsis+phyllosphere.">Synthetic microbiota reveal priority effects and keystone strains in the Arabidopsis phyllosphere.</a> Nature Ecology &amp; Evolution. 3 (10), 1445-1454 (2019).</li><li>Meyer, K. M., Leveau, J. H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiology+of+the+phyllosphere:+a+playground+for+testing+ecological+concepts.">Microbiology of the phyllosphere: a playground for testing ecological concepts.</a> Oecologia. 168 (3), 621-629 (2012).</li><li>Humphrey, P. T., Nguyen, T. T., Villalobos, M. M., Whiteman, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+abundance+of+phyllosphere+bacteria+are+linked+to+insect+herbivory.">Diversity and abundance of phyllosphere bacteria are linked to insect herbivory.</a> Molecular Ecology. 23 (6), 1497-1515 (2014).</li><li>Williams, T. R., Marco, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phyllosphere+microbiota+composition+and+microbial+community+transplantation+on+lettuce+plants+grown+indoors.">Phyllosphere microbiota composition and microbial community transplantation on lettuce plants grown indoors.</a> mBio. 5 (4), e01564 (2014).</li><li>Di Cagno, R., Coda, R., De Angelis, M., Gobbetti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploitation+of+vegetables+and+fruits+through+lactic+acid+fermentation.">Exploitation of vegetables and fruits through lactic acid fermentation.</a> Food Microbiology. 33 (1), 1-10 (2013).</li><li>K&#246;berl, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+microbiome+shift+during+fermentation+of+medicinal+plants.">Deciphering the microbiome shift during fermentation of medicinal plants.</a> Scientific Reports. 9 (1), 13461 (2019).</li><li>Yu, A. O., Leveau, J. H. J., Marco, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance,+diversity+and+plant-specific+adaptations+of+plant-associated+lactic+acid+bacteria.">Abundance, diversity and plant-specific adaptations of plant-associated lactic acid bacteria.</a> Environmental Microbiology Reports. 12 (1), 16-29 (2020).</li><li>Miller, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+limitation+constrains+the+abundance+of+lactic+acid+bacteria+in+the+Napa+cabbage+phyllosphere.">Establishment limitation constrains the abundance of lactic acid bacteria in the Napa cabbage phyllosphere.</a> Applied and Environmental Microbiology. 85 (13), e00269 (2019).</li><li>Stamer, J. R., Stoyla, B. O., Dunckel, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+rates+and+fermentation+patterns+of+lactic+acid+bacteria+associated+with+sauerkraut+fermentation.">Growth rates and fermentation patterns of lactic acid bacteria associated with sauerkraut fermentation.</a> Journal of Milk and Food Technology. 34 (11), 521-525 (1971).</li><li>Yildiz, F., Westhoff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+growth+of+lactic+acid+bacteria+in+cabbage+juice.">Associative growth of lactic acid bacteria in cabbage juice.</a> Journal of Food Science. 46 (3), 962-963 (1981).</li><li>Zabat, M. A., Sano, W. H., Wurster, J. I., Cabral, D. J., Belenky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+community+analysis+of+sauerkraut+fermentation+reveals+a+stable+and+rapidly+established+community.">Microbial community analysis of sauerkraut fermentation reveals a stable and rapidly established community.</a> Foods. 7 (5), 77 (2018).</li><li>Lee, S. H., Jung, J. Y., Jeon, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Source+tracking+and+succession+of+kimchi+lactic+acid+bacteria+during+fermentation.">Source tracking and succession of kimchi lactic acid bacteria during fermentation.</a> Journal of Food Science. 80 (8), M1871 (2015).</li><li>Trivedi, P., Schenk, P. M., Wallenstein, M. D., Singh, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiny+Microbes,+Big+Yields:+enhancing+food+crop+production+with+biological+solutions.">Tiny Microbes, Big Yields: enhancing food crop production with biological solutions.</a> Microbial Biotechnology. 10 (5), 999-1003 (2017).</li><li>Knief, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaproteogenomic+analysis+of+microbial+communities+in+the+phyllosphere+and+rhizosphere+of+rice.">Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice.</a> The ISME Journal. 6 (7), 1378-1390 (2012).</li><li>Wuyts, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carrot+Juice+Fermentations+as+Man-Made+Microbial+Ecosystems+Dominated+by+Lactic+Acid+Bacteria.">Carrot Juice Fermentations as Man-Made Microbial Ecosystems Dominated by Lactic Acid Bacteria.</a> Applied and Environmental Microbiology. 84 (12), AEM.00134 (2018).</li><li>Niu, B., Paulson, J. N., Zheng, X., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+and+representative+bacterial+community+of+maize+roots.">Simplified and representative bacterial community of maize roots.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (12), E2450-E2459 (2017).</li><li>Steinkraus, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactic+acid+fermentation+in+the+production+of+foods+from+vegetables,+cereals+and+legumes.">Lactic acid fermentation in the production of foods from vegetables, cereals and legumes.</a> Antonie van Leeuwenhoek. 49 (3), 337-348 (1983).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39;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&#39;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&#39;s Select Seeds</td>
			<td>2814G</td>
			<td>B. rapa var pekinensis (Bilko)</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 &#181;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 ...

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