这项工作由国家自然科学基金（32370135）、中国农业科学院创新计划（CAAS-CSAL-202302）、江苏省农林职业学院科技项目（2021kj29）资助。

1.无菌水培拟南芥栽培
<ol><li>准备 拟南芥 幼苗。<ol><li>准备培养 拟南芥 幼苗培养基，其由1/2MS培养基（Murashige和Skoog）与2%（wt / vol）蔗糖和0.9%（wt / vol）琼脂组成。</li>
		<li>在凝固之前，将准备好的灭菌培养基倒入无菌方形培养皿（13 cm x 13 cm）中。避免风干以保持湿度。</li>
		<li>将 拟南芥 种子浸入装有1mL无菌水的2mL微量离心管中，在4°C下浸泡12小时以上，然后用1mL 2%（体积/体积）NaClO灭菌种子2分钟。</li>
		<li>用无菌水彻底清洗种子六次，以去除NaClO溶液。用 MS 琼脂培养基将种子播种在培养皿上，吸头为无菌 1 mL。</li>
		<li>用透气的医用胶带密封培养皿，并将它们垂直放置在光照培养箱中生长 1 周。将光照培养箱昼夜比设置为16小时/ 8小时，保持温度为23°C，湿度为40%-60%。</li>
	</ol></li>
	<li>移植拟 南芥。<ol><li>在 40 μm 孔径细胞染色剂上切 5 mm 孔并无菌。</li>
		<li>通过细胞过滤器的孔移植三株 7 天大的 拟南芥 植物，并将它们放入含有 3 mL 无菌液体 1/2 MS 培养基和 0.5% 蔗糖的 6 孔板中。</li>
		<li>将 6 孔板置于光培养箱中（图 1A）并孵育 10 天。</li>
	</ol></li>
</ol>2.细菌培养和接种
<ol><li>准备用于接种的细菌悬浮液。<ol><li>对于 贝氏芽孢杆菌，将细菌细胞接种到无菌LB培养基中，并在37°C下以170rpm振荡孵育，直至达到0.8-1.2的OD600 。</li>
		<li>通过以6000×g离心2分钟收集细菌细胞，并将细胞重悬于PBS缓冲液（2mM KH2PO 4,8mM Na2HPO4,136mM NaCl，2.6mM KCl）中。重复离心和重悬步骤 3 次，以去除 LB 培养基和细菌代谢物。</li>
		<li>将细菌细胞悬浮在新鲜的 1/2 MS 培养基中，终浓度为 OD600 0.01。将细菌悬浮液接种到培养 拟南芥 幼苗的6孔板上，将6孔板置于光照培养箱中，培养2天。</li>
	</ol></li>
</ol>3. 测量定植在根部的细菌
<ol><li>收获根组织并铺板定植细胞。 注意：所有步骤都应在gnotobiotic条件下进行<ol><li>称量 2 mL 试管并将重量记录为 W0。</li>
		<li>收获共培养的 拟南芥 根组织，并在无菌水中洗涤3次。将根放在滤纸上以去除多余的水。</li>
		<li>将根放入称量的微量离心管中，将根与管一起称取，记录为W1。</li>
		<li>向每个试管中加入 1 mL PBS，并以最大速度涡旋试管 8 分钟。</li>
		<li>将含有收集的根定植细菌细胞的悬浮液稀释至 1 x 10-1-1 x 10-4（根据细菌定植能力）。将稀释的细菌悬浮液铺在含有LB琼脂培养基的平板上，并在37°C下孵育10小时。</li>
	</ol></li>
	<li>对菌落进行计数并对数据进行归一化。<ol><li>计算LB平板上的细菌菌落。</li>
		<li>根据相应的稀释度计算CFUs，并用根鲜重（W1-W 0）对数据进行归一化。最终结果表明，接种后2天每克根定植的细菌细胞计数（图1B）。</li>
	</ol></li>
</ol>

拟南芥根际细菌定植检测

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作者声明，他们与本文的内容没有利益冲突。

为了实现良好的可重复性，该协议的定植检测过程有四个关键步骤。首先，必须确保每个实验中接种的细菌细胞数量完全相同。其次，用无菌水控制未定植的细菌清洁强度也是必要的。第三，每个样品稀释过程在进行之前都需要进行涡旋，以使样品处于完全混合状态，以避免由于细菌的特性而导致的吸收误差，这些特性容易在微量离心管中下沉。因此，我们建议在每次吸收前使用涡旋仪混合细菌?...

有定量和定性方法可用于检测单一细菌菌株的根际定植。对于定性方法，应使用组成型表达荧光的菌株，并且应在荧光显微镜或激光共聚焦仪器1,2下检查荧光分布和强度。这些策略可以很好地反映细菌原位定植 3，但它们在定量方面不如传统的平板计数方法准确。此外，由于在显微镜下只能显示部分根区的限制，有时可能会受到主观偏见的影响。
在这里，我们描述了一种定量方法，其中包括收集定植的细菌细胞并计数板上的细菌 CFU。该方法基于稀释和铺板，通过该方法可以计算从植物根部剥离的定植菌株，并且可以计算出根部上的总定植细菌数4,5。
首先，在水培条件下培养 拟南芥 ，然后在根际中以终浓度0.01 OD600接种细菌细胞。接种后 2 天收获感染的根组织，并用无菌水洗涤以去除未定植的细菌细胞。此外，收集定植在根上的细菌细胞，在磷酸盐缓冲盐水 （PBS） 缓冲液中稀释，并接种到 Luria-Bertani （LB） 琼脂培养基上。在37°C孵育10小时后，对LB平板上的单个菌落进行计数并归一化以确定定植在根上的细菌细胞。
该方法适用性强，重复性好，更适用于根际细菌定植的准确测定。

<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

测量 拟南芥 根上的细菌定植是植物-微生物相互作用研究中最常见的实验之一。为了提高可重复性，需要一种标准化的方法来测量细菌在根际中的定植。我们首先在水培条件下培养无菌 拟南芥 ，然后在根际中以 OD600 的终浓度 0.01 接种细菌细胞。接种后 2 天，收获根组织并在无菌水中洗涤 3 次，以去除未定植的细菌细胞。然后称重根部，并通过涡旋收集定植在根部上的细菌细胞。用磷酸盐缓冲盐水 （PBS） 缓冲液以梯度稀释细胞悬液，然后接种到 Luria-Bertani （LB） 琼脂培养基上。将平板在37°C下孵育10小时，然后对LB平板上的单个菌落进行计数并归一化，以指示定植在根上的细菌细胞。该方法用于在单相互作用条件下检测细菌在根际中的定植，具有良好的重现性。

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.

作者聚焦：增强根际细菌在植物根部的定植以提高微生物肥料效率

在水培条件下测量 拟南芥 根上的细菌定植

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.

为了检验该方法在 拟南芥根际中检测细菌定植能力的准确性，我们分别将 芽孢杆 菌SQR9 WT和衍生突变体 Δ8mcp 接种到 拟南芥 根际中。 Δ8mcp 是一种缺乏所有化学感受器编码基因的突变体，并且它的定植显着减少6。我们通过目前的根定植测定法在接种后 2 天测量了它们的定植。结果表明， Δ8mcp的根系定植显著减少，表明该条件和方法有效?...

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

植物促生长根际（PGPR）在根际的定植对其促生长作用至关重要。有必要规范细菌根际定植的检测方法。在这里，我们描述了一种可重复的方法，用于量化细菌在根表面的定植。

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

我们的研究重点是有益根际细菌与植物根系的相互作用。我们正在寻找增强有益菌定植的关键机制，从而提高田间微生物肥料的效率和稳定性。细菌在植物根系定植的方法已经得到了广泛的研究，但仍然存在问题。例如，很难对不同位点的定植进行不同的测量，我们认为在未来应用显微镜和高通量计数将有助于解决问题。我们的方案具有高度适用性和良好的可重复性，更适合于准确的根际细菌定植测定。

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Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

446 次观看.  Chinese Academy of Agricultural Sciences. 我们的研究重点是有益根际细菌与植物根系的相互作用。我们正在寻找增强有益菌定植的关键机制，从而提高田间微生物肥料的效率和稳定性。细菌在植物根系定植的方法已经得到了广泛的研究，但仍然存在问题。例如，很难对不同位点的定植进行不同的测量，我们认为在未来应用显微镜和高通量计数将有助于解决问题。我们的方案具有高度适用性和良好的可重复性，?...

视频: 作者聚焦：增强根际细菌在植物根部的定植以提高微生物肥料效率

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.