Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Summary

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

Abstract

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Introduction

Plant-associated microbiomes consist of dynamic and complex microbial communities comprised of bacteria, archaea, viruses, fungi, and other eukaryotic microorganisms. In addition to their well-studied role in causing plant disease, plant-associated microbes can also positively influence plant health by improving tolerance to biotic and abiotic stresses, promoting nutrient availability, and enhancing plant growth through the production of phytohormones. For this reason, particular interest exists in characterizing the taxa that associate with plant root endospheres, rhizospheres, and the surrounding soil. While some microbes can be cultured in isolation on laboratory g....

Protocol

1. Collection and Separation of Root Endosphere, Rhizosphere, and Soil Samples

1. Prior to entering the field, autoclave ultrapure water (at least 90 mL of water per sample) to sterilize. Prepare epiphyte removal buffer (at least 25 mL per sample) by adding 6.75 g of KH₂PO₄, 8.75 g of K₂HPO₄, and 1 mL of Triton X-100, to 1 L of sterile water. Sterilize the buffer using a vacuum filter with 0.2 µm pore size.
   1. For steps 1.2 to 1.5, wear clean gloves sterilized with ethanol at all times and replace the gloves between each sample to prevent contamination. Sterilize all equipment with 70% ethanol and wipe....

Results

Performing the recommended protocol should result in a dataset of indexed paired-end reads that can be matched back to each sample and assigned to either a bacterial operational taxonomic units (OTU) or exact sequence variant (ESV, also referred to as amplicon sequence variant (ASV) and sub-operational taxonomic unit (sOTU)), depending on downstream analysis. In order to obtain high-quality sequence data, care must be taken at each step to maintain consistency between samples and minimize.......

Discussion

This protocol demonstrates an established pipeline for exploring root endosphere, rhizosphere, and soil microbial community compositions, from field sampling to sample processing and downstream sequencing. Studying root-associated microbiomes presents unique challenges, due in part to the inherent difficulties in sampling from soil. Soils are highly variable in terms of physical and chemical properties, and different soil conditions can be separated by as little as a few millimeters^28,<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.1-10/20 µL filtered micropipette tips	USA Scientific	1120-3810	Can substitute with equivalent from other suppliers.
1.5 mL microcentrifuge tubes	USA Scientific	1615-5510	Can substitute with equivalent from other suppliers.
10 µL multi-channel pipette	Eppendorf	3122000027	Can substitute with equivalent from other suppliers.
10 µL, 100 µL, and 1000 µL micropipettes	Eppendorf	3120000909	Can substitute with equivalent from other suppliers.
100 µL multi-channel pipette	Eppendorf	3122000043	Can substitute with equivalent from other suppliers.
1000 µL filtered micropipette tips	USA Scientific	1122-1830	Can substitute with equivalent from other suppliers.
2 mL microcentrifuge tubes	USA Scientific	1620-2700	Can substitute with equivalent from other suppliers.
2 mm soil sieve	Forestry Suppliers	60141009	Can substitute with equivalent from other suppliers.
200 µL filtered micropipette tips	USA Scientific	1120-8810	Can substitute with equivalent from other suppliers.
25 mL reservoirs	VWR International LLC	89094-664	Can substitute with equivalent from other suppliers.
50 mL conical vials	Thermo Fisher Scientific	352098	Can substitute with equivalent from other suppliers.
500 mL vacuum filters (0.2 µm pore size)	VWR International LLC	156-4020
96-well microplates	USA Scientific	655900
96-well PCR plates	BioRad	HSP9631
Agencourt AMPure XP beads	Thermo Fisher Scientific	NC9933872	Instructions for use: https://www.beckmancoulter.com/wsrportal/ajax/downloadDocument/B37419AA.pdf?autonomyId=TP_DOC_150180 &documentName=B37419AA.pdf
Aluminum foil	Boardwalk	7124	Can substitute with equivalent from other suppliers.
Analytical scale with 0.001 g resolution	Ohaus Pioneer	PA323	Can substitute with equivalent from other suppliers.
Bioruptor Plus ultrasonicator	Diagenode	B01020001
Bovine Serum Albumin (BSA) 20 mg/mL	New England Biolabs	B9000S
Centrifuge	Eppendorf	5811000908	Including 50mL and 96-well plate bucket adapters
Cryogenic gloves	Millipore Sigma	Z183490	Can substitute with equivalent from other suppliers.
DNeasy PowerClean kit (optional)	Qiagen Inc.	12877-50	Previously MoBio
DNeasy PowerSoil kit	Qiagen Inc.	12888-100	Previously MoBio
Dry ice	Any	NA
DynaMag-2 magnet	Thermo Fisher Scientific	12321D	Do not substitute
Ethanol	VWR International LLC	89125-188	Can substitute with equivalent from other suppliers.
Gallon size freezer bags	Ziploc	NA	Can substitute with equivalent from other suppliers.
Gemini EM Microplate Reader	Molecular Devices	EM	Can use another fluorometer that reads 96-well plates from the top.
K2HPO4	Sigma-Aldrich	P3786
KH2PO4	Sigma-Aldrich	P5655
Lab coat	Workrite	J1367	Can substitute with equivalent from other suppliers.
Liquid N2	Any	NA	Can substitute with equivalent from other suppliers.
Liquid N2 dewar	Thermo Fisher Scientific	4150-9000	Can substitute with equivalent from other suppliers.
Milli-Q ultrapure water purification system	Millipore Sigma	SYNS0R0WW
Mini-centrifuge	Eppendorf	5404000014
Molecular grade water	Thermo Fisher Scientific	4387937	Can substitute with equivalent from other suppliers.
Mortars	VWR International LLC	89038-150	Can substitute with equivalent from other suppliers.
Nitrile gloves	Thermo Fisher Scientific	19167032B	Can substitute with equivalent from other suppliers.
Paper towels	VWR International LLC	BWK6212	Can substitute with equivalent from other suppliers.
PCR plate sealing film	Thermo Fisher Scientific	NC9684493
PCR strip tubes	USA Scientific	1402-2700
Pestles	VWR International LLC	89038-166	Can substitute with equivalent from other suppliers.
Plastic spatulas	LevGo, Inc.	17211
Platinum Hot Start PCR Master Mix (2x)	Thermo Fisher Scientific	13000014
PNAs - chloroplast and mitochondrial	PNA Bio	NA	Make sure to verify sequence bioinformatically
Protective eyewear	Millipore Sigma	Z759015	Can substitute with equivalent from other suppliers.
Qubit 3.0 Fluorometer	Thermo Fisher Scientific	Q33216
Qubit dsDNA HS assay kit	Thermo Fisher Scientific	Q32854
Rubber mallet (optional)	Ace Hardware	2258622	Can substitute with equivalent from other suppliers.
Shears or scissors	VWR International LLC	89259-936	Can substitute with equivalent from other suppliers.
Shovel	Home Depot	2597400	Can substitute with equivalent from other suppliers.
Soil core collector (small diameter: <1 inch)	Ben Meadows	221700	Can substitute with equivalent from other suppliers.
Spray bottles	Santa Cruz Biotechnology	sc-395278	Can substitute with equivalent from other suppliers.
Standard desalted barcoded primers (10 µM) (Table 1)	IDT	NA	4 nmole Ultramer DNA Oligo with standard desalting. NGS adapter and sequencing primer (Table 1) are designed for use with Illumina MiSeq using v3 chemistry.
Thermocycler	Thermo Fisher Scientific	E950040015	Can substitute with equivalent from other suppliers.
Triton X-100	Sigma-Aldrich	X100	Can substitute with equivalent from other suppliers.
Weigh boats	Spectrum Chemicals	B6001W	Can substitute with equivalent from other suppliers.