这项工作得到了中国国家自然科学基金 （42307173 和 42107328） 和国家重点研发计划 （2022YFD1500202 和 2022YFF1001800） 的财政支持。P.W， Z.X 设计了这项研究。P.W、B.X、Z.C、J.X 和 N.Z 分析了数据并创建了数字。P.W 写了手稿的初稿。Z.X.、R.Z. 和 Q.S. 修订了手稿。

该协议通常适用于来自各种植物的根际土壤微生物群。这里以 cucumber 为例。材料 表中列出了用于研究的试剂和设备的详细信息。
1. 细菌分离
<ol><li>根际土壤隔离<ol><li>黄瓜培养<ol><li>用 75% 乙醇和 2% 次氯酸钠 （NaClO） 溶液对黄瓜籽进行消毒，然后用无菌水冲洗11.</li>
			<li>在无菌条件下发芽种子，并选择那些达到双叶阶段12 的种子。随后，将持续发芽的黄瓜幼苗移植到装有 2 公斤黑土的花盆中。 注：采用东北不同产地的两种黑土，以减少系统误差。</li>
		</ol></li>
		<li>土壤悬浮液制备<ol><li>使用以下配方制备 PBS-S 缓冲液：6.33 g NaH2PO4·H2O、16.5 g Na2HPO4·7H2O、200 μL Silwet L-77 和 1 L 蒸馏水。</li>
			<li>当幼苗达到四叶阶段12 时，倒置花盆并使用消毒手套将根与 2 毫米厚的根际土壤隔离。</li>
			<li>将根放入 50 mL 离心管中的 30 mL PBS-S 缓冲液中。摇动 20 分钟后，通过 100 μm 尼龙细胞过滤器过滤悬浮液以去除根和大沉淀物。</li>
			<li>将滤液转移到另一个 50 mL 离心管中，在室温下以 3200 x g 离心 15 分钟，然后暂时储存在 4 °C。</li>
		</ol></li>
	</ol></li>
	<li>根际土壤细菌细胞的提取。 注：该方法采用差速离心法13，尽管 Nycodenz 密度梯度离心法14 也适用于此步骤。<ol><li>用 0.2% Na4P2O7 溶液将土壤悬浮液稀释至 500 mL，并以 1,000 x g 离心 10 分钟，以初步将生物体从土壤中分离出来。</li>
		<li>重新匀浆沉淀，以相同的低速离心 60 秒，并重复该过程 3 次。合并上清液，以 12,000 x g 离心 20 分钟，然后弃去上清液。</li>
		<li>将沉淀物重悬于 50 mL 的 PBS-S 缓冲液中，构成根际土壤细菌细胞悬液。</li>
	</ol></li>
</ol>2. 根际生物膜微生物群的测序和鉴定
<ol><li>培养基制备 注：胰蛋白酶大豆肉汤 （TSB） 和最低盐甘油谷氨酸酯 （MSgg） 培养基可在市场上买到。以下配方仅供参考。<ol><li>使用以下配方制备 TSB 培养基：15 μg/mL 胰蛋白胨、5 μg/mL 大豆蛋白胨、5 μg/mL NaCl，pH = 7。</li>
		<li>使用以下配方制备 MSgg 培养基：5 mM KH2PO4、100 mM 3-（N-吗啉代）丙烷磺酸 （MOPS）、2 mM MgCl2、700 μM CaCl2、50 μM MnCl2、50 μM FeCl3、1 μM ZnCl2、2 μM 硫胺素、0.5% 甘油、0.5% 谷氨酸、50 μg/mL 色氨酸、50 μg/mL 苯丙氨酸、 pH 值 = 7。</li>
		<li>要制备 TSB-MSgg 培养基，请将 TSB 培养基与 MSgg 培养基以 1：1 的比例 （v/v） 混合15。</li>
	</ol></li>
	<li>Pellicle 生物膜共培养<ol><li>将细胞悬液在 TSB 培养基中于 30 °C 孵育过夜（通常为 12 小时）以激活细菌。</li>
		<li>将 100 μm 尼龙细胞过滤器放在 24 孔板上。在 TSB 培养基中孵育细菌并设置三次重复。在 30 °C 下培养表膜生物膜 36 小时。 注意：如果最后一步的表膜生物膜没有得到很好的培养，TSB-MSgg 培养基是 TSB 培养基的有效替代品。</li>
		<li>取下含有薄膜生物膜的过滤器。将 2 mL PBS-S 缓冲液添加到新的 24 孔细胞板中，将 24 孔过滤器放入其中以洗涤生物膜，并去除游离细胞。重复洗涤过程 3 次。</li>
	</ol></li>
	<li>表膜生物膜 DNA 的提取<ol><li>按照制造商的说明使用 DNA 试剂盒提取生物膜基因组。</li>
	</ol></li>
	<li>高通量测序 注意：生物技术公司可以帮助大多数实验室完成测序实验。如果需要剩余溶液中的微生物组成，请在此步骤中对其进行测序。测序步骤因仪器而异。请按照制造商的说明进行操作。步骤 2.4.1 仅供参考。<ol><li>基于具有全长 16S rRNA 基因序列的数据库扩增 16S rRNA 基因的 V3-V4 区域。根据每个样品的最小序列数16 创建物种 OTU。对物种分类注释进行聚类，并从每个样本中获取群落的组成。</li>
		<li>根据测序数据，根据其相对丰度选择排名前五的菌株。</li>
	</ol></li>
	<li>高通量细菌分离和培养 注：此步骤是根据最新的微生物学和生物信息学方法17 设计的。稀释涂层板方法可用于细菌分离，但与高通量技术相比，它更耗时且针对性更差。<ol><li>混合培养的生物膜。使用有限稀释以确保 24 孔板中不超过 30% 的孔显示细菌生长。 注：要确定最合适的稀释度，请使用各种稀释率进行初步实验。最佳稀释度应每毫升含有不超过两个可培养细菌，代表细菌在不到 30% 的孔中孵育。</li>
		<li>扩增细菌的 16S rRNA 基因，同时为孔和板添加标记，并使用高通量 Illumina 平台17 对其进行测序。</li>
		<li>使用市售软件进行生物信息学分析，以获得每种培养物的纯度和物种分类信息。</li>
		<li>通过连续线培养培养前五名丰度纯细菌，并使用甘油在 -80 °C 下保存细菌。</li>
	</ol></li>
</ol>3. 合成微生物群落的构建
<ol><li>重建的生物膜培养物 注：详情请参考 任 et al.18。5 个菌株对应 31 个合成组合，包括 5 个单物种、10 个双物种、10 个三物种、5 个四物种和 1 个五物种联盟。<ol><li>在 TSB 培养基中孵育 5 种菌株，直到它们的 OD600 达到 1 以激活它们。</li>
		<li>用 TSB 培养基准备几个 24 孔板，并在其上放置 100 μm 尼龙细胞过滤器。 注：如果上一步的生物膜没有得到很好的培养，TSB-MSgg 培养基是 TSB 培养基的有效替代品。</li>
		<li>在培养基中孵育合成群落的 31 种组合。确保每个孔中细菌的接种数一致。为每个组合设置 3 次重复。</li>
		<li>在 30 °C 下培养生物膜 36 小时。</li>
	</ol></li>
	<li>表膜生物膜的定量<ol><li>按照步骤 2.2.3 中的相同过程进行操作。</li>
		<li>将生物膜转移到含有 180 μL 结晶紫溶液的新 24 孔板中，并染色 20 分钟。</li>
		<li>将染色样品转移到另一个含有 200 μL 96% 乙醇的 24 孔板中，洗脱 30 分钟，并测量 OD590。</li>
	</ol></li>
</ol>4. Pellicle 细胞数量定量
注：为了确定每种物种的比例以及表膜和剩余溶液中的细胞计数，有必要进行定量 PCR。有关详细信息，请参阅 Sun et al.16。
<ol><li>为选定的合成微生物群落设计菌株特异性引物。</li>
	<li>使用 Roary 进行基因组比较并找出每个分离株的菌株特异性单拷贝基因。确保引物靶向这些基因。</li>
	<li>将扩增的片段与 PMD19T 质粒连接。使用含有相应片段的质粒作为模板生成标准曲线。</li>
	<li>按照步骤 2.2 中提到的相同步骤进行操作。</li>
	<li>按如下方式制备反应组分：7.2 μL H2O、10 μL 2x qPCR Master mix、0.4 μL 10 μM 每种引物和 2 μL 模板 DNA。</li>
	<li>在以下条件下使用实时 PCR 仪器进行 PCR：95 °C 10 分钟，95 °C 40 个循环 30 秒，60 °C 45 秒，然后是标准熔解曲线段。</li>
	<li>每次处理进行 6 次生物学重复。</li>
</ol>

从根际土壤中开发协同多物种生物膜

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作者没有需要披露的利益冲突。

按照该方案，基于来自不同植物的根际土壤中的微生物群构建了一系列健壮的合成多物种生物膜群落。使用定量 PCR 技术，可以清楚地破译每个群落的组成。生物膜的生物量表明它们潜在代谢相互作用的强度，尽管这里无法揭示合作背后的各种特性和机制19。鉴于自然微生物群落的规模和复杂性，合成群落 （SynComs） 代表了克服现有障碍的模型系统14。该协议强?...

生物膜代表附着在表面或与界面相连的复杂微生物群落。人们普遍认为，在各种环境（例如自然栖息地、临床环境和工业环境）中遇到的大多数细菌都在生物膜群落中持续存在1。在土壤中，根际（包括根表面及其相邻的 2 毫米厚区域）形成了一个生态位，养分利用率更高。特定细菌已经进化出利用这一生态位的策略，促进微生物增殖并促进根际生物膜群落的形成2。
根际微生物被认为是植物的“第二个基因组”3。植物生长促进微生物 （PGPM） 与植物共生，提供显着的生长促进和生物控制功能4。一方面，PGPM 可以为植物根系提供养分，增强养分吸收，抵御病原体，并降解根际土壤中的污染物5。另一方面，PGPM 利用植物根系分泌物作为生长和进化的营养来源，从而通过自身的新陈代谢影响根际土壤和根系。值得注意的是，所有这些功能的先决条件是 PGPM 在植物根际的有效定植，形成稳定的生物膜6。
根际生物膜影响根际微生物的组装过程，根际微生物组装的时空特征与多物种生物膜的相互作用密切相关7。它是植物与微生物组相互作用的枢纽，为它们提供稳定可控的生态位，以便它们有效互动。因此，研究根际生物膜也是深入了解植物与微生物之间相互作用的重要方向。
然而，目前对根际多物种生物膜的研究很少。微生物组组成的高度复杂性使得回答围绕自然微生物群落的基本生态问题变得具有挑战性8。在实验过程中，需要考虑土壤类型、植物类型、微生物群落数量众多等诸多条件。各种因素限制了根际多物种生物膜的研究。
为了进一步研究根际土壤中的多物种生物膜群落，例如种间协同相互作用或代谢物交叉喂养8，最好在实验室条件下人工构建一个简化的、有代表性的、稳定的和可处理的合成微生物群落 9,10。在这里，标准化方案结合了几种最新的微生物学和生物信息学技术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 % Na4P2O7 solution</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>20041418</td>
			<td>Used to dilute the soil suspension.</td>
		</tr>
		<tr>
			<td>100 &#956;m Nylon Cell Filter</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>F613463-0001</td>
			<td>Used to filter culture solution and separate pellicle biofilm.</td>
		</tr>
		<tr>
			<td>2% NaClO solution</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>L03336903</td>
			<td>Used to sterilize the seeds.</td>
		</tr>
		<tr>
			<td>24-well Plate</td>
			<td>Merck &#38; Co., Inc.</td>
			<td>PSRP010</td>
			<td>Used for micobial cultivation and pellicle biofilm separation.</td>
		</tr>
		<tr>
			<td>3-(N-morpholino) propane sulfonic acid (MOPS)</td>
			<td>Bioolook Scientific Research Special</td>
			<td>2360010</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Beijing Su-bio Biotech Co., Ltd.</td>
			<td>62.547.254</td>
			<td>Properly-sized centrifuge tubes for our protocol.</td>
		</tr>
		<tr>
			<td>75% C2H5OH solution</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>801769680</td>
			<td>Used to sterilize the seeds.</td>
		</tr>
		<tr>
			<td>Acinetobacter baumannii XL-1</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Strains isolated from rhizosphere soil.</td>
		</tr>
		<tr>
			<td>Bacillus velezensis SQR9</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Strains isolated from rhizosphere soil.</td>
		</tr>
		<tr>
			<td>Bacteria DNA Kit</td>
			<td>Nanjing Dinsi Biotechnology Co.,Ltd.</td>
			<td>D3350020000K04U021</td>
			<td>Used to extract bacterial DNA.</td>
		</tr>
		<tr>
			<td>Black Soil I</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Black soil from Jilin Province.</td>
		</tr>
		<tr>
			<td>Black Soil II</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Black soil from Heilongjiang Province.</td>
		</tr>
		<tr>
			<td>Burkholderia cenocepacia XL-2</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Strains isolated from rhizosphere soil.</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Xilong Scientific Co.,Ltd.</td>
			<td>10035048</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>G508009-0001</td>
			<td>High-speed centrifuge.</td>
		</tr>
		<tr>
			<td>ChamQ SYBR qPCR Master Mix</td>
			<td>Vazyme Biotech Co.,Ltd.</td>
			<td>Q311-02</td>
			<td>Used for DNA amplification in qPCR.</td>
		</tr>
		<tr>
			<td>Clean Bench</td>
			<td>Suzhou Antai Airtech Co., Ltd.</td>
			<td>NB026143</td>
			<td>Used for aseptic operation.</td>
		</tr>
		<tr>
			<td>Constant Temperature Incubator</td>
			<td>ShangHai CIMO Medical Instrument Co.,Ltd</td>
			<td>GNP-9080BS-<img alt="Equation 1" src="/files/ftp_upload/66926/66926eq01.jpg" style="margin: auto;" /></td>
			<td>Used for microbial cultivation.</td>
		</tr>
		<tr>
			<td>Cristal Violet Dye</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>A600331</td>
			<td>Used for pellicle biofilm staining and quantifivation.</td>
		</tr>
		<tr>
			<td>Cucumber Seed</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>&#34;Jinchun I&#34; cucumber seed.</td>
		</tr>
		<tr>
			<td>Culturome v 1.0</td>
			<td>The Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences</td>
			<td>N/A</td>
			<td>Used for high-throughput rhizosphere soil bacterial cultivation and identification</td>
		</tr>
		<tr>
			<td>FeCl3</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10011918</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Fridge</td>
			<td>Hefei Midea Refrigerator Co.,Ltd.</td>
			<td>BCD-556WKPM(Q)</td>
			<td>Used for strain preservation.</td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Bioolook Scientific Research Special</td>
			<td>2180020</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>10010618</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Hydroponic Planting Basket</td>
			<td>Yulv Furniture Store</td>
			<td>6526262626</td>
			<td>Used for cucumber hydroponics.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Xilong Scientific Co.,Ltd.</td>
			<td>10017618</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Xilong Scientific Co.,Ltd.</td>
			<td>7791186</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Xilong Scientific Co.,Ltd.</td>
			<td>10400101</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Msgg Medium</td>
			<td>Shanghai Bioesn Biotechnology Co.,Ltd.</td>
			<td>BES20791KB</td>
			<td>Pellicle biofilm culture medium.</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>Merck &#38; Co., Inc.</td>
			<td>S9390-100G</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>NaCI</td>
			<td>Chinasun Specialty Products co., Ltd.</td>
			<td>HS0416</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;H2O</td>
			<td>Merck &#38; Co., Inc.</td>
			<td>S9638-25G</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>PBS-S Buffer</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>E607008-0001</td>
			<td>Basic buffer for living tissues.</td>
		</tr>
		<tr>
			<td>Phenylalanine</td>
			<td>RYON</td>
			<td>RT3486L005</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Beijing Labgic Technology Co.,Ltd.</td>
			<td>BS-1000-T</td>
			<td>Used for strain inoculation.</td>
		</tr>
		<tr>
			<td>PMD19T Plasmid</td>
			<td>Takara Biomedical Technology (Beijing) Co.,Ltd.</td>
			<td>D102A</td>
			<td>Used to generate qPCR standard curves.</td>
		</tr>
		<tr>
			<td>Pot</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>YHHWS2401</td>
			<td>Used for cucumber soil culture.</td>
		</tr>
		<tr>
			<td>Primer for qPCR</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>N/A</td>
			<td>Used for DNA amplification in qPCR.</td>
		</tr>
		<tr>
			<td>Real-Time PCR Instrument</td>
			<td>Thermo Fisher Scientific (China) Co.,Ltd.</td>
			<td>4484073</td>
			<td>Used to perform qPCR on selected pellicle biofilm.</td>
		</tr>
		<tr>
			<td>Roary</td>
			<td>The Wellcome Trust SangerInstitute</td>
			<td>N/A</td>
			<td>Used to design primers of qPCR</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Shanghai Zhichu Instrument Co.,Ltd</td>
			<td>ZQTY-50S</td>
			<td>Used for solution mixing and microbial cultivation.</td>
		</tr>
		<tr>
			<td>Silwet L-77</td>
			<td>Cytiva Bio-technology(Hangzhou) Co., Ltd.</td>
			<td>SL77080596</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>Soy peptone</td>
			<td>Qingdao Hi-tech Industrial Park Hope Bio-technology Co., Ltd</td>
			<td>HB8275</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Shanghai Yidian Analysis Instrument Co.,Ltd.</td>
			<td>76713100010</td>
			<td>Used for pellicle biofilm cristal violet quantifivation.</td>
		</tr>
		<tr>
			<td>Sterile Water</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd.</td>
			<td>SW150302</td>
			<td>Used to wash the pellicle biofilm.</td>
		</tr>
		<tr>
			<td>Sterilized Nitrile Gloves</td>
			<td>Beijing Labgic Technology Co.,Ltd.</td>
			<td>223016852LLZA</td>
			<td>Used for basic experimental operations.</td>
		</tr>
		<tr>
			<td>Template DNA</td>
			<td>Sangon Biotech (Shanghai) Co.,Ltd.</td>
			<td>N/A</td>
			<td>Used for DNA amplification in qPCR.</td>
		</tr>
		<tr>
			<td>Thiamine</td>
			<td>Bioolook Scientific Research Special</td>
			<td>2180020</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>LP0042B</td>
			<td>Used to prepare TSB media.</td>
		</tr>
		<tr>
			<td>Tryptophan</td>
			<td>Bioolook Scientific Research Special</td>
			<td>2190020</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
		<tr>
			<td>TSB Medium</td>
			<td>Guangdong Huankai Microbial Sci. &#38; Tech. Co.,Ltd.</td>
			<td>024048</td>
			<td>Broad-spectrum bacterial medium.</td>
		</tr>
		<tr>
			<td>TSB-Msgg Meidium</td>
			<td>Nanjing Agricultural University</td>
			<td>N/A</td>
			<td>Mixed medium for pellicle biofilm culture with wider applicability.</td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Nanjing Chemical Reagent Co.,Ltd</td>
			<td>C0310520123</td>
			<td>Used to prepare MSgg media.</td>
		</tr>
	</tbody>
</table>

an approach to constructing multispecies biofilm communities from rhizosphere soil

多物种生物膜是自然界中细菌自然存在的主要生活方式，包括根际土壤，尽管目前对它的了解有限。在这里，我们提供了一种快速建立协同多物种生物膜群落的方法。第一步是使用差速离心法从根际土壤中提取细胞。之后，这些土壤细胞接种到培养基中以形成薄膜生物膜。孵育 36 小时后，使用 16S rRNA 基因扩增子测序方法测定生物膜的细菌组成和下面的溶液。同时，使用有限稀释法从表膜生物膜中进行高通量细菌分离。然后，在 16S rRNA 基因扩增子测序数据 （表膜生物膜样本） 中选择丰度最高的前 5 个细菌分类群，以进一步用于构建多物种生物膜群落。使用 24 孔板快速建立 5 个细菌分类群的所有组合，通过结晶紫染色测定选择具有最强生物膜形成能力的生物膜，并通过 qPCR 定量。最后，通过上述方法获得了最强大的合成细菌多物种生物膜群落。该方法为开展根际多物种生物膜研究以及确定代表性群落为研究控制这些物种之间相互作用的原则提供了信息指导。

Biofilms represent intricate microbial communities either affixed to surfaces or linked with interfaces. There is broad acknowledgment that the majority of bacteria encountered in various environments, such as natural habitats, clinical settings, and industrial contexts, endure within biofilm communities1. In soil, the rhizosphere&#8212;encompassing the root surface and its adjacent 2 mm thick region&#8212;forms an ecological niche with heightened nutrient availability. Specific bacteria have evolved strategies to exploit this niche, fostering microbial proliferation and fostering the formation of biofilm communities in the rhizosphere2.
Rhizosphere microbes are considered the &#39;second genome&#39; of plants3. Plant growth-promoting microorganisms (PGPM) engage in mutualistic symbiosis with plants, providing significant growth promotion and biocontrol functions4. On the one hand, PGPM can supply nutrients to plant roots, enhance nutrient uptake, defend against pathogens, and degrade pollutants in the rhizosphere soil5. On the other hand, PGPM utilizes plant root exudates as a nutrient source for growth and evolution, thereby influencing the rhizosphere soil and root system through their own metabolism. It is worth noting that the prerequisite for all these functions is the effective colonization of PGPM in the plant rhizosphere, forming stable biofilms6.
Rhizosphere biofilm affects the assembly process of rhizosphere microbes, and the temporal and spatial characteristics of rhizosphere microbe assembly are closely related to the interaction of multispecies biofilm7. It serves as the hub of plant-microbiome interactions, providing a stable and controllable niche for them&#160;to interact effectively. Therefore, studying rhizosphere biofilm is also an important direction to gain insight into the interaction between plants and microbes.
However, there is currently little research on rhizosphere multispecies biofilms. The high complexity of microbiome composition makes it challenging to answer fundamental ecological questions surrounding natural microbial communities8. In the process of experiments, many conditions, such as soil type, plant type, and the large number of microbial communities, need to be considered. Various factors limit the research of rhizosphere multispecies biofilms.
To further investigate multispecies biofilm communities from rhizosphere soil, such as interspecies synergistic interactions or metabolite cross-feeding8, it is desirable to artificially construct a simplified, representative, stable, and tractable synthetic microbial community under laboratory conditions9,10. Here, a standardized protocol combines several of the latest microbiological and bioinformatic techniques.

作者聚焦：开发合成微生物群落以生产第二代生物肥料

一种从根际土壤构建多物种生物膜群落的方法

按照上述程序，观察到黄瓜根际土壤微生物群的生物膜形成能力的显着梯度（图 1）。生物膜扩增子测序证实了表膜中的物种（图 2）。基于测序数据的热图，选择了表膜中丰度大于剩余溶液中丰度的前五种细菌物种（图 3）。这里以一组典型的合成群落为例。结晶紫染色和定量 PCR 直接可视化表膜生物量的差异和群落中每个物种?...

本文介绍了一种从各种根际土壤建立协同多物种生物膜群落的快速和标准化程序。这是一种独特的协议，旨在探测和模拟复杂的根际土壤微生物群。

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current ...

an-approach-to-constructing-multispecies-biofilm-communities-from

Research

JoVE Journal

Biology

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

504 次观看.  Nanjing Agricultural University. 在我们的研究中，我们专注于根际细菌和生物肥料，我们的目标是开发一个合成细菌群落，用作第二代生物肥料。我们对根际细菌的研究重点已从单一菌株转移到群落水平。然而，没有建立合成细菌群落的标准方法。目前，微生物组分析预测的合成细菌群落的相容性和定植能力仍然存在不确定性。因此，它也是第二代生物肥料发展的一大瓶颈。...

视频: 作者聚焦：开发合成微生物群落以生产第二代生物肥料

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.

A rapid and standardized procedure for establishing synergistic multispecies biofilm communities from various rhizosphere soils is presented here. It is a unique protocol designed to probe and simulate the complex rhizosphere soil microbiota.