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 themselve....

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 ....

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 T.......

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.......

Materials

List of materials used in this article

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