quantitative assessment of bacillus velezensis colonization on arabidopsis thaliana roots

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

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.

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.

Quantitative Assessment of Bacillus velezensis Colonization on Arabidopsis thaliana Roots

quantitative-assessment-bacillus-velezensis-colonization-on

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JoVE Journal

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78 조회수.  Chinese Academy of Agricultural Sciences. 시작하려면 2밀리리터 튜브의 무게를 측정하고 초기 무게를 w0로 기록합니다. 공동 배양된 애기장대(Arabidopsis thaliana) 뿌리 조직을 수확하고 멸균수로 세 번 세척합니다. 여과지에 뿌리를 올려 여분의 물을 제거합니다.뿌리를 미리 계량된 마이크로 원심분리기 튜브로 옮깁니다. 튜브로 뿌리의 무게를 측정하고 w1로 기록합니다. 다음으로, 튜브에 PBS 1밀리리터를 넣고 최대...

비디오: Arabidopsis thaliana 뿌리에 대한 Bacillus velezensis 집락화의 정량적 평가

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.

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 ...