optimizing bacillus velezensis cultivation and inoculation for arabidopsis thaliana seedling growth enhancement

シロイヌナズナにおける根圏細菌コロニー形成の検出

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.

著者スポットライト:微生物肥料効率を改善するための植物根の根茎形成の強化

水耕栽培状態における シロイヌナズ ナの根の細菌コロニー形成の測定

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.

シロイヌナズナ苗の成長促進のためのBacillus velezensisの栽培と接種の最適化

Bacillus velezensis細胞を無菌のLuria-Bertani培地に接種し、600ナノメートルの光学密度が0.8〜1.2に達するまで、170RPMで振とうしながら摂氏37度でインキュベートします。細胞懸濁液を6, 000 Gで2分間遠心分離し、ペレットをPBS緩衝液に再懸濁します。最後の洗浄後、ペレットを新しい半強度MS培地に再懸濁し、600ナノメートルで1の最終光学密度にします。光学濃度が0.01に達するまで、細菌培養物を100倍に希釈します。希釈した細菌懸濁液をシロイヌナズナの苗を含む6ウェルプレートに接種し、ライトインキュベーターで2日間培養します。

optimizing-bacillus-velezensis-cultivation-inoculation-for

Research

JoVE Journal

Environment

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

90 ビュー.  Chinese Academy of Agricultural Sciences. Bacillus velezensis細胞を無菌のLuria-Bertani培地に接種し、600ナノメートルの光学密度が0.8〜1.2に達するまで、170RPMで振とうしながら摂氏37度でインキュベートします。細胞懸濁液を6, 000 Gで2分間遠心分離し、ペレットをPBS緩衝液に再懸濁します。最後の洗浄後、ペレットを新しい半強度MS培地に再懸濁し、600ナノメートルで1の最終光学密度にします。光学濃度が0.01に達?...

ビデオ: シロイヌナズナ苗の成長促進のためのBacillus velezensisの栽培と接種の最適化

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.

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 ...