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Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Published: March 1st, 2024

DOI:

10.3791/66241

1State Key Laboratory of Efficient Utilization of Arid and Semi-arid Arable Land in Northern China, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, 2State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, 3Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, 4Jiangsu Engineering and Technology Center for Modern Horticulture, Jiangsu Vocational College of Agriculture and Forestry

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 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.

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 microscop....

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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 m.......

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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 assa.......

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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.......

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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).

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NameCompanyCatalog NumberComments
6-well plateCorning3516
Filter cell stainerSolarbioF8200-40µm
Microplate reader TecanInfinite M200 PRO
Murashige and Skoog mediumHopebioHB8469-5
NaClOAlfaL14709
PhytagelSigma-AldrichP8169
Square petri dishRuiai ZhengtePYM-130
Vortex Genie2Scientific IndustriesG560E

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