Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Summary

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

Abstract

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD₆₀₀ of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 °C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

Introduction

There are quantitative and qualitative methods for detecting rhizosphere colonization by a single bacterial strain. For the qualitative method, a strain that constitutively expresses fluorescence should be used, and the fluorescence distribution and intensity should be examined under fluorescence microscopy or laser confocal instruments^1,2. Those strategies can well reflect bacterial colonization in situ³, but they are not as accurate as traditional plate counting methods in quantification. Besides, due to the limitation of only displaying partial root zones under the microscope, sometimes it can be influenced by subjective bias.

Here, we describe a quantitative method, which includes collecting the colonized bacterial cells and counting the bacterial CFUs on a plate. This method is based on dilution and plating by which the colonized strains that were stripped from plant roots can be counted, and the total colonized bacteria number on the root can be calculated^4,5.

First, A. thaliana was cultured in hydroponic conditions, and then bacterial cells were inoculated in the rhizosphere at a final concentration of 0.01 OD₆₀₀. The infected root tissues were harvested 2 days post-inoculation and washed in sterile water to remove the uncolonized bacterial cells. Further, bacterial cells colonized on the root were collected, diluted in phosphate-buffered saline (PBS) buffer, and plated onto a Luria-Bertani (LB) agar medium. After incubation at 37 °C for 10 h, single colonies on LB plates were counted and normalized to determine the bacterial cells colonized on roots.

This method is highly applicable, has good repeatability, and is more suitable for accurate rhizosphere bacterial colonization determination.

Protocol

1. Sterile hydroponic A. thaliana cultivation

1. Prepare A. thaliana seedlings.
   1. Prepare culture A. thaliana seedlings medium, which consists of 1/2 MS medium (Murashige and Skoog) with 2% (wt/vol) sucrose and 0.9% (wt/vol) agar.
   2. Pour the prepared sterilization medium into sterile square Petri dishes (13 cm x 13 cm) before solidification. Avoid air drying to maintain humidity.
   3. Immerse A. thaliana seeds in a 2 mL microcentrifuge tube filled with 1 mL of sterile water at 4 °C for over 12 h, and then sterilize the seeds with 1 mL of 2% (vol/vol) NaClO for 2 min.
   4. Thoroughly wash the seeds with sterile water six times to remove the NaClO solution. Sow the seeds on Petri dishes with MS agar medium with a sterile 1 mL tip.
   5. Seal the Petri dishes with breathable medical tape and place them vertically in a light incubator to grow for 1 week. Set the light incubator day/night ratio to 16 h/8 h, maintain the temperature at 23 °C and humidity at 40%-60%.
2. Transplant A. thaliana.
   1. Cut 5 mm holes on 40 µm pore size cell stainer and sterile them.
   2. Transplant three 7-day-old A. thaliana plants through the holes of a cell strainer and put them into a 6-well plate containing 3 mL of sterile liquid 1/2 MS medium with 0.5% sucrose.
   3. Place the 6-well plates in a light incubator (Figure 1A) and incubate for 10 days.

2. Bacteria cultivation and inoculation

1. Prepare bacterial suspension for inoculation.
   1. For Bacillus velezensis, inoculate bacterial cells into sterile LB medium and incubate at 37 °C with shaking at 170 rpm until it reaches OD₆₀₀ of 0.8-1.2.
   2. Collect the bacterial cells by centrifugation at 6000 x g for 2 min and resuspend the cells in PBS buffer (2 mM KH₂PO₄, 8 mM Na₂HPO₄, 136 mM NaCl, 2.6 mM KCl). Repeat the centrifuging and resuspending steps 3 times to remove the LB medium and the bacterial metabolites.
   3. Suspend the bacteria cells in a fresh 1/2 MS medium at a final concentration of OD₆₀₀ 0.01. Inoculate the bacteria suspensions to the 6-well plates that grow A. thaliana seedlings, place the 6-well plates in the light incubator, and cultivate them for 2 days.

3. Measuring bacteria colonized on roots

1. Harvest the root tissue and plate the colonized cells.
   NOTE: All steps should be conducted under gnotobiotic condition
   1. Weigh the 2 mL tubes and record the weight as W₀.
   2. Harvest the co-cultured A. thaliana root tissues and wash them in sterile water 3 times. Place the root on filter paper to remove the excess water.
   3. Put the root into the weighed microcentrifuge tube, weigh the roots together with the tube, and record it as W₁.
   4. Add 1 mL of PBS to each tube and vortex the tubes for 8 min at the maximum speed.
   5. Dilute the suspensions with the collected root-colonized bacterial cells to 1 x 10^-1-1 x 10^-4 (according to the bacteria colonization ability). Spread the diluted bacterial suspensions on plates containing LB agar medium and incubate at 37 °C for 10 h.
2. Count the colonies and normalize the data.
   1. Count the bacterial colonies on LB plates.
   2. Calculate the CFUs according to the corresponding dilution and normalize the data with root fresh weight (W₁-W₀). The final results indicate the count of bacterial cells colonized per gram of root at 2 days post-inoculation (Figure 1B).

Results

To test the accuracy of the bacteria colonization ability detected by this method in the A. thaliana rhizosphere, we inoculated Bacillus velezensis SQR9 WT and a derived mutant Δ8mcp into A. thaliana rhizosphere separately. The Δ8mcp is a mutant that lacks all the chemoreceptor encoding genes, and it has a significantly decreased colonization⁶. We measured their colonization at 2 days post-inoculation by the present root colonization assay. The results showed a significant root colonization reduction of Δ8mcp, indicating that this condition and method effectively measure the bacteria colonization (Figure 1C).

Figure 1: Growing A. thaliana and the root colonization of Bacillus velezensis SQR9 and Δ8mcp. (A) The top view of 7-day-old A. thaliana growing in 6-well plates with cell stainer in hydroponic condition. (B) The side view of 7-day-old A. thaliana growing in 6-well plates, each well containing 3 seedlings. (C) Colonization of B. velezensis wild-type SQR9 and Δ8mcp measured using the presented assay. Six replicates were included, and the data are presented as mean ± SEM. Please click here to view a larger version of this figure.

Discussion

To achieve good reproducibility, there are four critical steps for the colonization detection process of this protocol. First, it is necessary to ensure that the number of inoculated bacteria cells is exactly the same in each experiment. Second, controlling the uncolonized bacteria cleaning intensity with sterile water is also necessary. Third, every sample dilution process needs to be vortexed before being performed to let the sample be in a complete mixing state to avoid the absorption errors due to the characteristics of bacteria that are easy to sink in the microcentrifuge tube. Therefore, we recommend using a vortex instrument to mix the bacterial suspension before each absorption. Fourth, asepsis should be strictly ensured during all operations using this method to avoid any possibility of contamination.

This method is used to determine the bacteria colonized on the root surface. Except root surface bacteria, endophytic bacteria, and fungi also colonize the plant rhizosphere⁷. The colonization ability of endophytic bacteria can still be detected by using the protocol described in this paper, but the root tissue should be ground to release endophytic bacteria. However, the endophytic fungi are different from bacteria. Most of the mainstream methods use transmission electron microscopy (TEM) to observe the growth of hyphae, which is a qualitative detection method similar to fluorescent labeling bacteria for visualization⁸.

When comparing the colonization of different strains, they should not be inoculated in one 6-well plate to avoid the influence of the bacterial volatile compounds⁹ and plant volatile compounds^10,11. We chose 2-day post-inoculation for detecting root because the colonization of SQR9 reached the maximum between 2 and 4 days; the time for detecting the bacterial colonization can be adjusted according to the strains.

Compared with other described methods, this method is accurate and easy to operate. For example, the method described by Noam et al. has the solid-grown arabidopsis inoculated with bacteria immediately when transplanted to hydroponic condition^12,13. Here, we propose the bacteria should be inoculated to the rhizosphere after several days of transplanting to avoid the influence of plant adaption to the changed growth environment. The method described in this study is also good for plants to release root exudate, which affects colonization.

To let the plant shoots grow separately in the air and completely away from the liquid immersion, we made some minor modifications and processing on the cell stainer to make them available for this hydroponic method. Moreover, this method is more suitable for accurate bacterial colonization determination in small quantities but not for large quantities of root exudates collected in hydroculture¹⁴. It is important to note that this method is modified based previously published studies^12,14,15.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well plate	Corning	3516
Filter cell stainer	Solarbio	F8200-40µm
Microplate reader	Tecan	Infinite M200 PRO
Murashige and Skoog medium	Hopebio	HB8469-5
NaClO	Alfa	L14709
Phytagel	Sigma-Aldrich	P8169
Square petri dish	Ruiai Zhengte	PYM-130
Vortex Genie2	Scientific Industries	G560E