This work was funded by the National Natural Science Foundation of China (32370135), the Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-CSAL-202302), the Science and Technology Project of Jiangsu Vocational College of Agriculture and Forestry (2021kj29).

1. Sterile hydroponic A. thaliana cultivation
<ol>
	<li>Prepare A. thaliana seedlings.
	<ol>
		<li>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.</li>
		<li>Pour the prepared sterilization medium into sterile square Petri dishes (13 cm x 13 cm) before solidification. Avoid air drying to maintain humidity.</li>
		<li>Immerse A. thaliana seeds in a 2 mL microcentrifuge tube filled with 1 mL of sterile water at 4 &#176;C for over 12 h, and then sterilize the seeds with 1 mL of 2% (vol/vol) NaClO for 2 min.</li>
		<li>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.</li>
		<li>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 &#176;C and humidity at 40%-60%.</li>
	</ol>
	</li>
	<li>Transplant A. thaliana.
	<ol>
		<li>Cut 5 mm holes on 40 &#181;m pore size cell stainer and sterile them.</li>
		<li>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.</li>
		<li>Place the 6-well plates in a light incubator (Figure 1A) and incubate for 10 days.</li>
	</ol>
	</li>
</ol>
2. Bacteria cultivation and inoculation
<ol>
	<li>Prepare bacterial suspension for inoculation.
	<ol>
		<li>For Bacillus velezensis, inoculate bacterial cells into sterile LB medium and incubate at 37 &#176;C with shaking at 170 rpm until it reaches OD600 of 0.8-1.2.</li>
		<li>Collect the bacterial cells by centrifugation at 6000 x g for 2 min and resuspend the cells in PBS buffer (2 mM KH2PO4,&#160;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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Measuring bacteria colonized on roots
<ol>
	<li>Harvest the root tissue and plate the colonized cells. 
	NOTE: All steps should be conducted under gnotobiotic condition
	<ol>
		<li>Weigh the 2 mL tubes and record the weight as W0.</li>
		<li>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.</li>
		<li>Put the root into the weighed microcentrifuge tube, weigh the roots together with the tube, and record it as W1.</li>
		<li>Add 1 mL of PBS to each tube and vortex the tubes for 8 min at the maximum speed.</li>
		<li>Dilute the suspensions with the collected root-colonized bacterial cells to 1 x 10-1-1 x 10-4&#160;(according to the bacteria colonization ability). Spread the diluted bacterial suspensions on plates containing LB agar medium and incubate at 37 &#176;C for 10 h.</li>
	</ol>
	</li>
	<li>Count the colonies and normalize the data.
	<ol>
		<li>Count the bacterial colonies on LB plates.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>

Rhizobacterial Colonization Detection on &lt;i&gt;Arabidopsis thaliana&lt;/i&gt;

<ol>
	<li>Wang, B., Wan, C., Zeng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+on+cotton+plants+with+a+GFP+labeled+strain+of+Bacillus+axarquiensis.">Colonization on cotton plants with a GFP labeled strain of Bacillus axarquiensis.</a> Curr Microbiol. 77 (10), 3085-3094 (2020).</li><li>Zhai, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+tool+for+production+of+GFP-expressing+Rhodopseudomonaspalustris+for+visualization+of+bacterial+colonization.">A genetic tool for production of GFP-expressing Rhodopseudomonaspalustris for visualization of bacterial colonization.</a> AMB Express. 9 (1), 141 (2019).</li><li>Synek, L., Rawat, A., L'Haridon, F., Weisskopf, L., Saad, M. M., Hirt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+strategies+of+plant+colonization+by+beneficial+endophytic+Enterobacter+sp.+SA187.">Multiple strategies of plant colonization by beneficial endophytic Enterobacter sp. SA187.</a> Environ Microbiol. 23 (10), 6223-6240 (2021).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+velezensis+tolerance+to+the+induced+oxidative+stress+in+root+colonization+contributed+by+the+two-component+regulatory+system+sensor+ResE.">Bacillus velezensis tolerance to the induced oxidative stress in root colonization contributed by the two-component regulatory system sensor ResE.</a> Plant Cell Environ. 44 (9), 3094-3102 (2021).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+commensal+type+VII+secretion+system+causes+iron+leakage+from+roots+to+promote+colonization.">Plant commensal type VII secretion system causes iron leakage from roots to promote colonization.</a> Nat Microbiol. 8 (8), 1434-1449 (2023).</li><li>Feng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+chemotaxis+compounds+in+root+exudates+and+their+sensing+chemoreceptors+in+plant-growth-promoting+Rhizobacteria+Bacillus+amyloliquefaciens+SQR9.">Identification of chemotaxis compounds in root exudates and their sensing chemoreceptors in plant-growth-promoting Rhizobacteria Bacillus amyloliquefaciens SQR9.</a> Mol Plant Microbe Interact. 31, 995-1005 (2018).</li><li>Woo, S. L., Hermosa, R., Lorito, M., Monte, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichoderma:+a+multipurpose,+plant-beneficial+microorganism+for+eco-sustainable+agriculture.">Trichoderma: a multipurpose, plant-beneficial microorganism for eco-sustainable agriculture.</a> Nat Rev Microbiol. 21 (5), 312-326 (2023).</li><li>Nongkhlaw, F. M., Joshi, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+study+on+colonization+and+antimicrobial+property+of+endophytic+bacteria+associated+with+ethnomedicinal+plants+of+Meghalaya.">Microscopic study on colonization and antimicrobial property of endophytic bacteria associated with ethnomedicinal plants of Meghalaya.</a> J Microsc Ultrastruct. 5 (3), 132-139 (2017).</li><li>Ravelo-Ortega, G., Raya-Gonz&#225;lez, J., L&#243;pez-Bucio, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+from+rhizosphere+microbes+that+promote+plant+growth.">Compounds from rhizosphere microbes that promote plant growth.</a> Curr Opin Plant Biol. 73, 1369-5266 (2023).</li><li>Schulz-Bohm, K., Gerards, S., Hundscheid, M., Melenhorst, J., de Boer, W., Garbeva, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calling+from+distance:+attraction+of+soil+bacteria+by+plant+root+volatiles.">Calling from distance: attraction of soil bacteria by plant root volatiles.</a> ISME J. 12 (5), 1252-1262 (2018).</li><li>Sharifi, R., Lee, S. M., Ryu, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe-induced+plant+volatiles.">Microbe-induced plant volatiles.</a> New Phytol. 220 (3), 684-691 (2018).</li><li>Eckshtain-Levi, N., Harris, S. L., Roscios, R. Q., Shank, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+community+members+increase+Bacillus+subtilis+maintenance+on+the+roots+of+Arabidopsis+thaliana.">Bacterial community members increase Bacillus subtilis maintenance on the roots of Arabidopsis thaliana.</a> Phytobiomes J. 4, 303-313 (2020).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+colonization+by+beneficial+rhizobacteria.">Root colonization by beneficial rhizobacteria.</a> FEMS Microbiol Rev. 48, (2024).</li><li>Yahya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+root+exudation+and+architecture+for+improved+growth+of+wheat+mediated+by+phosphate+solubilizing+bacteria.">Differential root exudation and architecture for improved growth of wheat mediated by phosphate solubilizing bacteria.</a> Front Microbiol. 12, 744094 (2021).</li><li>Husna, K. B. -. E., Won, M. -. H., Jeong, M. -. I., Oh, K. -. K., Park, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+genomic+insight+of+surfactin-producing+Bacillus+velezensis+and+its+biocontrol+potential+against+pathogenic+contamination+in+lettuce+hydroponics.">Characterization and genomic insight of surfactin-producing Bacillus velezensis and its biocontrol potential against pathogenic contamination in lettuce hydroponics.</a> Environ Sci Pollut Res Int. 30 (58), 121487-121500 (2023).</li></ol>

The authors declare that they have no conflicts of interest with the contents of this article.

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...

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 instruments1,2. 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 calculated4,5. 
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 &#176;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter cell stainer</td>
			<td>Solarbio</td>
			<td>F8200-40&#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader&#160;</td>
			<td>Tecan</td>
			<td>Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr>
			<td>Murashige and Skoog medium</td>
			<td>Hopebio</td>
			<td>HB8469-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaClO</td>
			<td>Alfa</td>
			<td>L14709</td>
			<td></td>
		</tr>
		<tr>
			<td>Phytagel</td>
			<td>Sigma-Aldrich</td>
			<td>P8169</td>
			<td></td>
		</tr>
		<tr>
			<td>Square petri dish</td>
			<td>Ruiai Zhengte</td>
			<td>PYM-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genie2</td>
			<td>Scientific Industries</td>
			<td>G560E</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring bacterial colonization on arabidopsis thaliana roots in hydroponic condition

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 &#176;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.

Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Our research focuses on the interaction of beneficial rhizobacteria with plant roots. We are looking for the key mechanisms to enhance the colonization of beneficial bacteria, thus improving the efficiency and the stability of microbial fertilizers in the field. The methods for bacterial colonization of plant roots have been extensively studied, but there are still problems.For example, it's hard to measure the colonization on different sites distinctively, In the future we think applying microscope and high throughput counting will help to solve the problems. Our protocol is highly applicable with good repeatability, and it is more suitable for accurate rhizosphere bacteria colonization determination.

To test the accuracy of the bacteria colonization ability detected by this method in the A. thaliana&#160;rhizosphere, we inoculated Bacillus velezensis SQR9 WT and a derived mutant &#916;8mcp into A. thaliana rhizosphere separately. The &#916;8mcp 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 assa...

Watch this Scientific Journal Video about Author Spotlight: Enhancing Rhizobacteria Colonization on Plant Roots for Improved Microbial Fertilizer Efficiency at JoVE.com

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.

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...

measuring-bacterial-colonization-on-arabidopsis-thaliana-roots

Research

JoVE Journal

Environment

Hydroponic Cultivation of Arabidopsis thaliana Seedlings for Bacterial Inoculations

Optimizing Bacillus velezensis Cultivation and Inoculation for Arabidopsis thaliana Seedling Growth Enhancement

Quantitative Assessment of Bacillus velezensis Colonization on Arabidopsis thaliana Roots

424 Views.  Chinese Academy of Agricultural Sciences.  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.

Rhizobacterial Colonization Detection on Arabidopsis thaliana

Podsumowanie