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 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.
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.
1. Growing germ-free cabbages
2. Inoculating the phyllosphere with microbial solutions
3. Preparing sterile vegetable extract
NOTE: This method is a modified version of cabbage sterile media production18,19.
4. Inoculation of sterile vegetable extract
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 Toy Choy Hybrid; B. juncea: Mustard Red Giant) gave limited success (Supplemental Figure 2). Unlike Napa cabbage that forms compact neat rosettes that fit into the glass tubes, these Brassica spp. either had low germination rates after sterilizing or the stem elongated rapidly to make a spindly, unhealthy plant. In addition, sterilizing older seeds (>1 year old) is not recommended as the seed coats dry out making it harder to remove them during the sterilization process. Regularly purchase new seeds and test a subset of cabbages to determine whether they are sterile before carrying out experiments.
Growth of microbial inoculants in the Napa cabbage phyllosphere
Microbial isolates (Table 1) were inoculated either as single strain isolates or in combination with another isolate to look for pairwise interactions in the Napa cabbage phyllosphere. A total of 15 germ-free cabbages were inoculated for each treatment and five cabbages were harvested immediately after inoculation, five were harvested four days after inoculation, and the remaining were harvested 10 days after inoculation. Results show that phyllosphere isolates are capable of rapid growth in the Napa cabbage phyllosphere (Figure 2).
Growth of microbial inoculants in sterile vegetable extract
Two yeasts (Kazachstania barnetti and Pichia membranifaciens) and three bacteria (Lactobacillus koreensis, Pediococcus parvulus, and Leuconostoc mesenteroides) were inoculated into three different types of SVE made from red, green, and Napa cabbage. All samples were incubated at 24 °C and growth of the inoculates over 14 days was recorded by spot plating 5 µL of each treatment onto either MRS or YPD agar plates (n = 5). Results are shown in Figure 3A. The pH of each sample was also recorded throughout the fermentation (Figure 3B) and shows that the lactic acid bacteria were capable of acidifying the SVE to levels below pH 4 (indicating a ferment that is safe for consumption).
Figure 1: Diagram of germ-free cabbage setup. Please click here to view a larger version of this figure.
Figure 2: Growth rates of different bacteria on germ-free Napa cabbage. (A) Growth of single inoculations in the phyllosphere. (B) Growth after inoculating two microbes into the phyllosphere. Growth of microbes was measured as colony forming units counted per g of cabbage homogenate plated onto either TSA or MRS media. n = 5. Error bars = standard deviation. Please click here to view a larger version of this figure.
Figure 3: Growth of lactic acid bacteria and yeasts in sterile vegetable extract (SVE) made with red, green and Napa cabbage. (A) Growth of microbial inoculants was measured by counting colony forming units per mL of SVE plated. Yeasts were plated onto YPD agar plates and bacteria onto MRS agar plates. (B) Acidification of the sterile vegetable extract as microbes grow shown as fall in pH. n = 5. Error bars = standard deviation. Please click here to view a larger version of this figure.
Phyla/Genera of microbial inoculants | Source of microbe |
Firmicutes Bacillus | Phyllosphere |
Firmicutes Lactobacillus | Fermentation |
Proteobacteria Achromobacter | Phyllosphere |
Proteobacteria Rhizobium | Phyllosphere |
Proteobacteria Sphingomonas | Phyllosphere |
Table 1: Microbial isolates inoculated on germ-free Napa cabbage.
Supplemental Figure 1: Growth of Brassica rapa var pekinensis: Bilko in germ-free conditions. Please click here to view a larger version of this figure.
Supplemental Figure 2: Different cabbage varieties growing in germ-free conditions. (A) B. rapa: Turnip Purple Top, (B) B. oleracea: Cairo Hybrid, (C) B. oleracea: Tropic Giant Hybrid, (D) B. campestris: Pak Choi Toy Choy Hybrid, (E) B. juncea: Mustard Red Giant. Please click here to view a larger version of this figure.
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 different media. Single strain growth rates in SVE (Figure 2) show that LAB grow rapidly and acidify the media in the same way that would be anticipated in a ferment17.
Germ-free plants and sterile vegetable extract can be used in combination to address a number of different ecological questions such as priority effects and succession in the phyllosphere or within a ferment. A synthetic community of microbes is simple to construct through plating out homogenized cabbages to obtain phyllosphere isolates, or sauerkraut to obtain lactic acid bacteria16. Pairwise-interactions or leave-one-out experiments with more community members can be carried out in the phyllosphere or in the SVE to assess the importance or function of community members. Environmental selection studies can be carried out in the SVE where the impact of the vegetable fermented can be assessed. There is also potential to use both the germ-free cabbages and SVE to quantify diversification of microbial species and communities using experimental evolution.
A limitation of this germ-free cabbage system is the short timescale of the experiments. Because of the small glass tubes used, the cabbages are not able to grow for periods longer than a month as their leaves are confined by the edge of the tubes. Larger growing containers, such as plant tissue culture boxes (Table of Materials) could be used, but these will still not produce a full-sized cabbage plant. We have also tried growing cabbages in 0.75% agar containing MS broth, but found that this produced inconsistent growth of the cabbage seedlings. Using calcined clay as a growing substrate with enough MS broth to saturate but not flood the clay grains is the optimum method for growing healthy cabbages.
There are a few critical steps to ensure successful growth of germ-free cabbages. Ensuring that the calcined clay is fully dry when adding MS broth allows the clay to fully absorb the MS broth during the autoclave cycle. However, if there is any MS broth over the level of the clay, it must be removed before adding the seeds; seeds will not germinate if they are sitting in MS broth. Another important step to monitor is seed sterilization. Older seeds (>1 year old) will not germinate as quickly or as reliably as young seeds. Changing the size of the tube used for sterilization or overfilling the tubes can also impact sterilization. The sterilization step also helps soften and remove the seed coat so that the seeds rapidly germinate. Note here that reusing the pump spray bottles after using with microbial cultures is not recommended, as it is difficult to remove biofilms from the pump component. Of particular note, caution should be taken with Bacillus species as they are particularly resilient to autoclaving. Any pump bottles that have come into contact with Bacillus spp are not reused.
While sterile vegetable extract does not have the spatial structuring that is present in a fermentation vessel, growth dynamics of LAB suggest that it mimics fermentation progress with a rapid fall in pH and an increase in growth of lactic acid bacteria over the 14 days of fermentation. Leuconostoc mesenteroides is important at the outset of fermentation and it increased in abundance more rapidly than the Lactobacillus and Pediococcus spp, a trend seen in other sauerkraut succession surveys20,21. Work in the lab has also explored using spectrophotometer to obtain optical density (OD) readings for measuring the growth of LAB in SVE dispensed into 96 well plates. Initial results with Napa cabbage extract looked promising, but SVE made with red cabbage extract changed color as the pH dropped resulting in confounded OD readings. Furthermore, using OD readings to enumerate growth limits the use of this system to single strain inoculations. Together, these limitations led us to abandon using OD readings to measure microbial growth.
Testing ecological interactions in the phyllosphere is topical as there is evidence that the phyllosphere affects crop plant health and productivity22. Our model system has only been developed to work with Napa cabbage, but bacteria from the phyla Proteobacteria, Firmicutes, and Actinobacteria are common in the phyllosphere of many plant species13,23. While only three different varieties of cabbage have been tested, SVE can be made with other important agricultural plants. For example, studies investigating microbial community assembly during carrot juice fermentation24 or microbial colonization of maize root25 can be replicated using the protocols outlined in this paper.
Coupling the germ-free cabbage with the SVE to study community assembly in fermentation can show how changes in the phyllosphere microbiome can influence the success of fermentation. Spoilage of ferments or a failure to reach a sufficiently low pH can result if there is not a rapid initial acidification26. These spoiled ferments might be due to manufacturing processes, but variation in phyllosphere microbiomes may also have an important influence on the success of vegetable ferments17. The described system is a useful model for determining what microbiome assembly processes may impact the success of vegetable fermentation.
The authors have nothing to disclose.
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.
1.5 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 |