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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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.......

Representative 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 fungi5,7. 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.......

Acknowledgements

This work was supported by research funds provided by the Department of Energy Biological and Environmental Research (DOE-BER 0000217519 to E.A.S.), the National Science Foundation (INSPIRE IOS-1343020 to E.A.S). SLH was also supported by the National Science Foundation Graduate Research Fellowship Program. We thank Dr. Jeffery Dangl for providing bacterial strains and invaluable insight. We thank Dr. Andrew Klein and Matthew J. Powers for experimental suggestions. Finally, SLH would like to thank connections on social media for reminding us that disseminating science is a privilege and a responsibility, especially through creative and accessible means.

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Materials

NameCompanyCatalog NumberComments
Required Materials
1.5 mL eppendorf tubesanyN/A
24-well platesBD Falcon1801343
AerasealExcel ScientificBE255A2
AutoclaveanyN/A
Bacteria of InterestanyN/AStored at -80ËšC in 40% glycerol preferred
BactoAgarBD2306428; REF 214010
bleachanyN/A
ConvironanyN/AShort 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 plasticanyN/A
EthanolanyN/A
FlameanyN/A
ForcepsanyN/A
IncubatoranyN/AAt optimal temperature for growth of specified bacteria
Hydrochloric AcidanyN/A
Lennox LB BrothRPIL24066-1000.0
MicrocentrifugeanyN/A
MicropipettersanyN/AVolumes 5 µL to 1000 µL
Microscope (preferably fluorescence)anyN/ACould be light if best definition not important
MS Salts + MESRPIM70300-50.0
Orbital Plate ShakeranyN/ACapable of running at 220 rpm for at least 96 hours
Petri DishesanyN/A50 mL total volume
ReservoirsanyN/A
SpectrophotometeranyN/A
Standard Hole PunchanyN/AApproximately 7mm punch diameter
Sterile wateranyN/A
Surgical Tape3MMMM1538-1
Teflon MeshMcMaster-Carr1100t41
UltrasonicatoranyN/A
Vortex MixeranyN/A
X-galGoldBiox4281cother vendors available
Suggested Materials
24 Prong Ultrasonicator attachmentanyN/AFor sonicating multiple samples at once. Can be done individually
Alumaseal IIExcel ScientificFE124F
Glass beadsanyN/A
Multipetter/RepetteranyN/A
Sterile 96-well platesanyN/AFor serial dilutions. Can be replaced by eppendorf tubes
Biological Materials Used
Arabidopsis thaliana seedsanyN/AWe recommend Arabidopsis Biological Resource Center for seed stocks
Arthrobacter nicotinovoransLevy, et al. 2018
Curtobacterium oceanosedimentumLevy, et al. 2018
Microbacterium oleivoransLevy, et al. 2018
Pseudomonas simiae WCS417rPublished in a similar system in Haney, et al. 2015. Strain used developed in Cole, et al. 2017

References

  1. Strange, R. N., Scott, P. R. Plant disease: a threat to global food security. Annual Review of Phytopathology. 43, 83-116 (2005).
  2. Cook, A. M., Grossenbacher, H., Hütter, R. Isolation and cultivation of microbes with biodegradative p....

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