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

Summary

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.

Abstract

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’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.

Introduction

Crop destruction by bacterial and fungal diseases results in lowered food production and can severely disrupt global stability¹. Based on the discovery that microbes in suppressive soils are responsible for increasing plant health², scientists have asked whether the plant microbiome can be leveraged to support plant growth by modifying the presence and abundance of particular bacterial species³. Bacteria found to aid in plant growth or development are collectively termed plant growth-promoting bacteria (PGPB). More recently, studies have shifted from simply identifying potential PGPB to understand....

Protocol

NOTE: The experimental setup is described for clarity and used to generate the representative results included in this report, but conditions can be modified as desired. All steps should be performed using PPE and following institutional and federal reccomendations for safety, according to the BSL status of the bacteria used.

1. Characterization of bacteria

1. Determine the morphology of bacteria on the growth medium agar plate. Resuspend cells at an approximate OD₆₀₀ = 0.5 and plate a 1 µL volume onto agar medium of choice. Add X-gal to agar plates to a final concentration of 20 mg/mL to better differentiate indivi....

Results

The well-characterized PGPB P. simiae WCS417r is known to colonize the roots of A. thaliana in hydroponic culture. This naturally fluorescent bacterium can easily be visualized using microscopy on the roots of seedlings following colonization (Figure 2). Although it is possible to image the full length of these A. thaliana seedlings’ (4–6 mm length) roots, doing so for many plants would take a prohibitive amount of time........

Discussion

Plants in all environments interact with thousands to millions of different bacteria and fungi^5,7. These interactions can either negatively and positively impact plant health, with potential effects on crop yield and food production. Recent work also suggests that variable colonization of crops by PGPBs may account for unpredictable plant size and crop yield in field trials²². Understanding the mechanisms behind these interactions might al.......

Materials

Name	Company	Catalog Number	Comments
Required Materials
1.5 mL eppendorf tubes	any	N/A
24-well plates	BD Falcon	1801343
Aeraseal	Excel Scientific	BE255A2
Autoclave	any	N/A
Bacteria of Interest	any	N/A	Stored at -80˚C in 40% glycerol preferred
BactoAgar	BD	2306428; REF 214010
bleach	any	N/A
Conviron	any	N/A	Short Day Light-Dark Cycles: 460-600 µmoles/m²/s set at 9/15 hours light/dark at 18/21˚C, with inner power outlet
Dessicator Jar: glass or heavy plastic	any	N/A
Ethanol	any	N/A
Flame	any	N/A
Forceps	any	N/A
Incubator	any	N/A	At optimal temperature for growth of specified bacteria
Hydrochloric Acid	any	N/A
Lennox LB Broth	RPI	L24066-1000.0
Microcentrifuge	any	N/A
Micropipetters	any	N/A	Volumes 5 µL to 1000 µL
Microscope (preferably fluorescence)	any	N/A	Could be light if best definition not important
MS Salts + MES	RPI	M70300-50.0
Orbital Plate Shaker	any	N/A	Capable of running at 220 rpm for at least 96 hours
Petri Dishes	any	N/A	50 mL total volume
Reservoirs	any	N/A
Spectrophotometer	any	N/A
Standard Hole Punch	any	N/A	Approximately 7mm punch diameter
Sterile water	any	N/A
Surgical Tape	3M	MMM1538-1
Teflon Mesh	McMaster-Carr	1100t41
Ultrasonicator	any	N/A
Vortex Mixer	any	N/A
X-gal	GoldBio	x4281c	other vendors available
Suggested Materials
24 Prong Ultrasonicator attachment	any	N/A	For sonicating multiple samples at once. Can be done individually
Alumaseal II	Excel Scientific	FE124F
Glass beads	any	N/A
Multipetter/Repetter	any	N/A
Sterile 96-well plates	any	N/A	For serial dilutions. Can be replaced by eppendorf tubes
Biological Materials Used
Arabidopsis thaliana seeds	any	N/A	We recommend Arabidopsis Biological Resource Center for seed stocks
Arthrobacter nicotinovorans			Levy, et al. 2018
Curtobacterium oceanosedimentum			Levy, et al. 2018
Microbacterium oleivorans			Levy, et al. 2018
Pseudomonas simiae WCS417r			Published in a similar system in Haney, et al. 2015. Strain used developed in Cole, et al. 2017