We thank Eli Jensen, Stefano Padilla, and Kasey Yturralde for assistance, and Karen London and anonymous reviewers of the manuscript. We thank Jake Baker and Karla Torres for video footage. Funding was provided to R.W.H by the NAU School of Forestry and NAU Technology and Research Initiative Fund (TRIF).

1.选择韧皮部和去除树<ol><li>选择具有特定特征的一棵树。收集松树韧皮部（ 即在松属树木），因为他们有一个独特的韧皮层，也就是几毫米厚。18,27一旦树位于具有数较低的树枝，检查是否有其他缺陷，如昆虫的袭击和/或病原体。另外，用韧皮部与其他针叶树如云杉树的韧皮部三明治。9其他树种，可适合韧皮部切除，如硬木。 注：针叶树大冠通常具有最厚的韧皮组织。最大化韧皮部的量，最好是切成具有在躯干（伯乐）几乎没有缺陷和分支的树。松树是自我修剪工作最好的。韧皮部通常在生长季节最厚，并且比在地面附近较厚越往上躯干。韧皮部有时难以祛瘀E从在秋季或冬季的树木。 </li><li>砍一棵树下来，或者使用最近削减日志来获得韧皮组织。选择一个方向坠落，这将最大限度地减少对树木的破坏，以及附近的树木。切枝断的树干，允许在韧皮部搬迁到树皮的材料更容易获得。 </li><li>一旦树被切断（砍伐;丢弃在地上）开始刮树皮关闭后备箱用锋利的刀片拉伸（ 图1A）。刮树皮的区域断，直至达到韧皮层。 注：韧皮层典型地是在颜色（ 例如，奶油色）和潮湿的打火机，而外皮是烘干机中，较暗的颜色（ 图1B）。请特别注意不要刮伤韧皮部。树皮的面积刮下取决于所需的韧皮部件（多个）的大小。 </li><li>树皮被删除后，切断韧皮部片的轮廓用锋利的刀。确保刀割一路木质部（ 图2A）。 </li><li>以除去韧皮部，用手指仔细剥离背面的韧皮部开始在韧皮部片的一个角。用小刀将协助刮韧皮部关木质部。继续拉动韧皮部关断，直到整片被除去。 注：典型的韧皮部剥离最简单的夏季树。对于韧皮部是非常难以去除，一铲形工具可以在努力提供帮助。 </li><li>立即将韧皮部片进入无菌袋。为了达到最佳效果，真空密封袋（ 图2B），或者，如果使用保鲜袋包，从塑料袋中取出所有的空气。这增加了韧皮部的寿命。或者，把多件韧皮部的一个袋子。在密闭的塑料袋店韧皮部稍高于冰点（1〜10℃），以保持其新鲜度。 </li></ol> 2.创建韧皮部三明治<ol><li>切成两个相等的部分透明的丙烯酸，聚碳酸酯或类似的硬的，透明的材料（ 例如玻璃）稍微大的r除片韧皮部的（ 图2C）。圆形的丙烯酸类的边缘，以防止角部从撕开封口膜密封。该协议使用英寸厚的透明压克力1/8。 注：该切割块的大小取决于受试生物体的需要和研究的长度。例如，一对小蠹虫的利用韧皮部的4分米2个多月的时期，但只需要1分米2，如果研究以在几天内发生。 </li><li>钻一个孔的丙烯酸系片之一以允许研究有机体（多个）的条目。孔的尺寸和数量取决于目标（ 图2D）。 </li><li>之前，将所述丙烯酸系片之间的韧皮部，灭菌丙烯酸系表面（具有&gt; 70％的乙醇），或者，如果使用新的丙烯酸，除去保护膜。 </li><li>把一块新鲜的韧皮部的无菌压克力片之间。东方丙烯酸系片与韧皮部的内侧或外侧的孔（多个），whichever被需要的。通常情况下，面对的韧皮部（一侧有树皮上）朝向所述入口孔的外侧。 </li></ol> 3.密封的韧皮部三明治<ol><li>以创建围绕韧皮部三明治的临时密封，使用2英寸周围韧皮三明治（ 图2C）的边缘拉宽封口膜条带。另外，使用聚偏二氯乙烯保鲜膜密封的边缘。6下一步，将一个夹子上的夹层两侧挤压丙烯酸的韧皮部。确保整个表面被夹紧，以防止丙烯酸类和韧皮部（ 图2C）之间的空气空间。如果没有正确夹紧，标本可能丙烯酸和韧皮部之间移动。 </li><li>创建一个半永久密封添加非胶合的环氧树脂或凡士林周围的韧皮部。确保该材料完全包围韧皮部。接下来，用夹子或螺钉与螺栓（可能需要钻孔的时间提前）举行的丙烯酸紧张的韧皮部。三明治只有保持活力长达一两个月。 </li><li>根据研究生物的氧需求，在三明治中的一个或多个侧面添加的空气过滤器。这将允许空气进入韧皮部三明治但限制从韧皮部失水。我们使用简单的木炭过滤器，也减少真菌和细菌污染的风险。 </li><li>对于需要进入和退出的意愿研究标本，更换丙烯酸片用木材或类似材料的昆虫能够钻入之一。这是用于观察木蛀虫，特别重要的，因为后幼虫状态在韧皮层完成时，他们然后钻入木质部。 </li><li>为了防止测试对象从离开所述入口孔，将小培养皿（或其它的目的，磁带）上的孔中，堵逸出。因为驻留在这些空间中的生物体是使习惯到低亮度水平，则可能有必要将三明治在黑暗的房间或盒，或代替邻在上面Paque上料挡住光线。 </li></ol> 4.观察生物体在韧皮部三明治<ol><li>引入研究标本进入韧皮部三明治（ 图2D）的进入口。使用下一个红光或白光设定设定在一个低水平（ 图2E）解剖显微镜观察标本。 </li><li>至韧皮部夹层内记录样本的活动或生长附加的照相机或摄像机的显微镜。附上特殊摄像机到显微镜目镜（ 图2E和F）。对于非常小的生物，如螨类，线虫和​​pseudoscorpions，使用附连到显微镜的高清晰度视频摄像机。 </li><li>录制的声音，插入麦克风入韧皮部三明治的入口孔或通过韧皮部夹层的侧面。因为进入孔是小的，用一个小的麦克风等的驻极体电容传声器。 </li><li>从侧面或表面记录韧皮部三明治使用的压电元件（ 图3B）。要回放的声音，使用相同的压电元件或附加一个触觉传感器（励磁），以丙烯酸类的顶部或底部的表面上。 </li></ol>

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No conflicts of interest declared

韧皮部三明治允许节肢动物，微生物和其他小生物栖息韧皮部组织的介绍和观察。1,7,8,9,17,18这种技术导致了新的发现，并更好地理解行为，生命历史特征，开发，并在树韧皮部生物体的相互作用。1,5,10夹心这里描述的协议是过去的设计中的一种混合，并且将提供一种容易地构成以最小的设备和材料的一种经济的三明治。玻璃，丙烯酸树脂或聚碳酸酯片可以重复使用，并且仅消耗材料是石蜡膜和韧皮部。 虽然在协议中的所有步骤都是重要的，一定的步骤应遵循严格，以确保最高程度的成功。首先，树应该位于具有树干的部分游离的分支，或相对自由。有许多分支的树都难以剃除，并会ÿ屈服一些可行的韧皮部片，去掉了韧皮部时，每个分支必须被削减左右。接着，以最小化韧皮部的空气暴露是重要的。我们很快将每个韧皮部片在去除后袋。一旦三到六个片被除去，它们被转移到真空密封袋;我们使用一个功率变换器以运行该缩放器关闭该领域的车辆电池。最后，消毒丙烯酸片，一般清洁时，执行此协议将减少夹心真菌的生长。这对于延长观察尤为重要。 如上文提到的，有韧皮夹心装置的局限性。由于韧皮层是薄的，只有很小的生物体，通常保留在韧皮部可以被引入到夹心。较大的昆虫，例如木蛀虫（ 即Buprestid，天牛种）可以被引入，并观察它们的生命周期的早期阶段。这一时间通常限制在2到3周;这点后，幼虫需要木质部木孔成化蛹。相反，对于小蠹虫，特别是那些在属的Ips，一个完整的周期可观察到，包括交配，卵孵化，喂养，化蛹和羽化成成虫。在这一点上，夹心的生存能力通常用尽由于干燥和真菌的生长。28另外，这种装置不允许昆虫自由地和自然地定殖或退出韧皮部三明治。27 我们的协议是柔性的尺寸，形状和韧皮部的使用类型的条件。简短的研究需要较少韧皮部材料和夹心尺寸可以相应地缩放。许多针叶树种已被用来作为供体韧皮部的韧皮部三明治（ 例如，黄松12，道格拉斯冷杉2，云杉29，火炬松27，长叶松27）。在夹心材料也可以改变;为前充足，玻璃板可以用来代替丙烯酸和环氧树脂或胶带代替封口膜。 该协议的最困难的部分是韧皮部删除过程。树木彼此相邻可以不同多么困难的韧皮部是去除。当一棵树有困难的韧皮部，耐心是关键。在这些情况下，小心地运行硬盘木质部和韧皮部海绵之间的刀。这个过程从字面上感觉像剥皮的树。 掌握基本的夹层技术后，改建为协议可以帮助满足特定的需求。例如，通过珩磨除去韧皮部所需的技术中，较大块可以被删除，并用于创建较大的三明治。另外，改变可制成以满足特定的仪器， 例如 ，额外的孔并监控化学辐射（图3A），或者记录或重放的声音（图3B）。修改可以在允许的韧皮部的半永久性保存及其器官主义，或生物体以后可以让免费的临时观察。

针叶树的韧皮部和树皮组织是主机数以千计的生物。 Phloeophagy，在内部树皮的韧皮部组织的喂养，通常是用树皮甲虫，woodborers等几个无脊椎动物和微生物类群的生活在树木相关的习惯，23树皮甲虫（鞘翅目：象虫科）制定和韧皮部内居住的除外当成年人寻求新的宿主树木的短周期。31蠹虫已被广泛研究，由于在树上18,19的经济影响，但树材料内的昆虫直接行为观察受到了限制。4此外，画廊由蠹虫构造成为栖息地的物种万千。11广大真菌30日，3细菌，螨虫10,21，和16,19线虫以及其他捕食性和寄生节肢动物22,24居住在韧皮部的材料。这里提供的技术允许直接观察树皮甲虫，螨虫，木蛀虫，通常生活在皮层下的环境。轻微改动的协议，可以向研究真菌和细菌。 树组织内树皮甲虫和相关生物利用进行了研究，“韧皮部三明治。”早期使用这种技术可以在文献约会被发现回到1933年，当它被用来观察花旗松甲虫的幼虫龄期（ 蠹pseudotsugae）。2韧皮部夹层经历了许多派生了不同材质变得可用。最初，该装置包括一块韧皮部置于两个板玻璃，由弹性带压在一起之间的第2以后，夹具，胶带，胶水，塑料和其它材料已在夹层结构的构造中使用。13,14- ，15,17,26,28这里所描述的协议提供了改进了一些过去的设计中。例如，在过去，测试物种进入夹层的侧面，在玻璃或塑料制成的板之间。这限制画廊的建设一个方向。在顶板的使用条目孔允许对试验物种发起天然库结构有更大的自由度。所提出的协议的另一个优点是它的简单设计，它可以很容易地构造有一些工具。用韧皮部三明治已经允许的取食行为，繁殖，发展直接观察和生物的相互作用，否则不可能有1,5，22这个方法也是一个极好的工具K-12教育和科学程序和显示。 有几个细微之处中创建一个韧皮部三明治，很难从解释或手稿未报告。我们认为，生产韧皮部三明治的视觉（ 即，视频）的描述是必要的，将是有价值的科学家和有兴趣学习phloeophagous生物教育工作者。我们的协议提供了一种简单和廉价的方式来观察节肢动物，微生物和居住在韧皮部组织中的其他生物。

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technique for studying arthropod and microbial communities within tree tissues

Phloem tissues of pine are habitats for many thousands of organisms. Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear young, feed, or hide from natural enemies or harsh environmental conditions outside of the tree. Organisms that persist within the phloem habitat are difficult to observe given their location under bark. We provide a technique to preserve intact phloem and prepare it for experimentation with invertebrates and microorganisms. The apparatus is called a &#8216;phloem sandwich&#8217; and allows for the introduction and observation of arthropods, microbes, and other organisms. This technique has resulted in a better understanding of the feeding behaviors, life-history traits, reproduction, development, and interactions of organisms within tree phloem. The strengths of this technique include the use of inexpensive materials, variability in sandwich size, flexibility to re-open the sandwich or introduce multiple organisms through drilled holes, and the preservation and maintenance of phloem integrity. The phloem sandwich is an excellent educational tool for scientific discovery in both K-12 science courses and university research laboratories.

上述协议将使研究者观察到，生活在一个树的树皮下方的隐蔽环境微生物。为了说明使用采用此方法。12在这个实验中这种技术，我们描述了具有代表性的研究，从我们的实验室，韧皮部三明治，观察对生殖输出，隧道的距离，和树皮甲虫的生存声学处理的影响（ 图。图2E和2F）。直接观察甲虫，成为可能，利用韧皮部三明治，显示了一些有趣的发现。首先，我们通过一个标记每24h丙烯酸标志着甲虫“位置记录每天的掘进距离。这个过程跨越透露，将一直藏而不韧皮部夹层声音处理显著差异。接下来，我们观察到在产蛋与特定的声音处理的减少。韧皮部夹层的移动特性允许电子的gg观测要在解剖显微镜下，而不会干扰甲虫完成。在这些观察，我们捕捉到与连接到显微镜高清摄像头甲虫产卵行为。其他值得注意的意见包括队友的杀​​戮和飞行特别声学处理相关的启动。韧皮部夹心法是对我们的树皮甲虫的反应声学处理的研究。这些发现，这将是不可能的，观察一棵树的树皮底下，要制定管理方案为打击树皮甲虫爆发宝贵的贡献。 <img alt="图1" src="/files/ftp_upload/50793/50793fig1highres.jpg" /> 图1. A）工具需要从树上取下树皮。件1和2是用来刮去树皮从树上的拉片。项目3（斧形）和图4（拉锯）是有用的，除去的分支附近的剃须面积。B）使用战平刀片从树上取下树皮。注意微红树皮下的韧皮部的光色。 <img alt="图2" src="/files/ftp_upload/50793/50793fig2highres.jpg" /> 图2. A）去除树皮后，韧皮部被刮去树。B）存放在真空密封袋。C）韧皮部三明治夹抱着丙烯酸拼凑，封口膜四周边缘，以防止污染和韧皮部的干燥新鲜的韧皮部。ð ）树皮甲虫洞附近钻亚克力。E）用显微镜观察韧皮部三明治。韧皮部内夹心小蠹虫的F）的视频显示。 <img alt="图3" src="/files/ftp_upload/50793/50793fig3highres.jpg" /> 图3。 <st荣&gt; A）从韧皮部夹层甲虫信息素的收购。空气通过含有超Q吸收剂。B）的记录和播放声音经由压电换能器（图片中央），以山松甲虫内韧皮部三明治管被拉出。

Watch this Scientific Journal Video about Technique for Studying Arthropod and Microbial Communities within Tree Tissues at JoVE.com

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

技术树内组织学习节肢动物和微生物群落

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

technique-for-studying-arthropod-microbial-communities-within-tree

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10.3K 次观看.  Northern Arizona University. We provide a technique to preserve intact tree phloem and prepare it for observation. We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues. 

视频: Technique for Studying Arthropod and Microbial Communities within Tree Tissues

Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep), peak current density (jp), formal potential (Ef) and peak separation (&#916;Ep). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.

Chronoamperometric growth of anodic electroactive microbial biofilms in a fed-batch reactor using a three-electrode setup, controlled by a potentiostat, is demonstrated. The extracellular electron transfer is characterized using cyclic voltammetry in the presence of the electron donor and in the absence of the electron donor. Fundamental data analysis is demonstrated.

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup ...

科研

环境科学

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

成绩单及代谢物谱烟草树和杨树的评价作为原料的生物基工业

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

A Technical Perspective in Modern Tree-ring Research - How to Overcome Dendroecological and Wood Anatomical Challenges

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to environmental changes and to finally reconstruct such changes. This is done by analyzing growth variations back in time and correlating various plant-specific parameters to (for example) temperature records. Integrating wood anatomical parameters in these analyses would strengthen reconstructions, even down to intra-annual resolution. We therefore present a protocol on how to sample, prepare, and analyze wooden specimen for common macroscopic analyses, but also for subsequent microscopic analyses. Furthermore we introduce a potential solution for analyzing digital images generated from common small and large specimens to support time-series analyses. The protocol presents the basic steps as they currently can be used. Beyond this, there is an ongoing need for the improvement of existing techniques, and development of new techniques, to record and quantify past and ongoing environmental processes. Traditional wood anatomical research needs to be expanded to include ecological information to this field of research. This would support dendro-scientists who intend to analyze new parameters and develop new methodologies to understand the short and long term effects of specific environmental factors on the anatomy of woody plants.

Here we present a protocol outlining how to sample wooden specimens for the overall assessment of their growth structures. Macro- and microscopic preparation and visualization techniques necessary to generate well-replicated and highly resolved wood anatomical and dendroecological dataset, are described are described.

现代树轮研究技术的角度 - 如何克服Dendroecological和木解剖挑战

Dendroecological research uses information stored in tree rings to understand how single trees and even entire forest ecosystems responded to ...

Watershed Planning within a Quantitative Scenario Analysis Framework

There is a critical need for tools and methodologies capable of managing aquatic systems within heavily impacted watersheds. Current efforts often fall short as a result of an inability to quantify and predict complex cumulative effects of current and future land use scenarios at relevant spatial scales. The goal of this manuscript is to provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework. Sites are first selected for inclusion within the watershed assessment by identifying sites that fall along independent gradients and combinations of known stressors. Field and laboratory techniques are then used to obtain data on the physical, chemical, and biological effects of multiple land use activities. Multiple linear regression analysis is then used to produce landscape-based cumulative effects models for predicting aquatic conditions. Lastly, methods for incorporating cumulative effects models within a scenario analysis framework for guiding management and regulatory decisions (e.g., permitting and mitigation) within actively developing watersheds are discussed and demonstrated for 2 sub-watersheds within the mountaintop mining region of central Appalachia. The watershed assessment and management approach provided herein enables resource managers to facilitate economic and development activity while protecting aquatic resources and producing opportunity for net ecological benefits through targeted remediation.

There is a critical need for tools and methodologies capable of managing aquatic systems in the face of uncertain future conditions. We provide methods for conducting a targeted watershed assessment that enables resource managers to produce landscape-based cumulative effects models for use within a scenario analysis management framework.

定量的情景分析框架内流域规划

There is a critical need for tools and methodologies capable of managing aquatic systems within heavily impacted watersheds. Current efforts often fall ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

多年生草试验中土壤、根际和根系微生物群落的分离与分析

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Assessing the Particulate Matter Removal Abilities of Tree Leaves

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on collecting various sized particulate matter (PM) retained on leaf surfaces. We further characterized the retention efficiency of leaves to various sized PM, which will help to assess the abilities of urban trees to remove PM from ambient air quantitatively. 
Taking three broadleaf tree species (Ginkgo biloba, Sophora japonica, and Salix babylonica) and two needleleaf tree species (Pinus tabuliformis and Sabina chinensis) as the research objects, leaf samples were collected 4 days (short PM retention period) and 14 days (long PM retention period) after the latest rainfall. PM retained on the leaf surfaces was collected by means of WC, BC, and UC in sequence. Then, retention efficiencies of leaves (AEleaf) to three types of the various sized PM, including easily removable PM (ERP), difficult-to-remove PM (DRP), and totally removable PM (TRP), were calculated. Only around 23%-45% of the total PM retained on leaves could be cleaned off and collected by WC. When the leaves were cleaned through WC+BC, the underestimation of the PM retention capacity of different tree species was in the range of 29%-46% for various sized PM. Almost all PM retained on leaves could be removed if UC was supplemented to WC+BC. 
In conclusion, if the UC was complemented after the conventional cleaning methods, more PM on leaf surfaces could be eluted and collected. The procedure developed in this study can be used for assessing the PM removal abilities of different tree species.

The ultrasonic cleaning method was applied to elute the particulate matter (PM) retained on leaf surfaces after PM was eluted by the conventional cleaning methods (water cleaning only or water cleaning plus brush cleaning). The methodology can help to improve the estimation accuracy for PM retention capacity of leaves.

树叶颗粒物去除能力的评价

Based on the conventional cleaning methods (water cleaning (WC) + brush cleaning (BC)), this study evaluated the influence of ultrasonic cleaning (UC) on ...

Characterizing Microbiome Dynamics &#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical applications driven by microorganisms. Next generation sequencing methods are widely utilized to resolve microbiomes, but they are generally resource and time intensive and deliver mostly qualitative information. Flow cytometric microbiome analysis does not suffer from those disadvantages and can provide relative subcommunity abundances and absolute cell numbers at-line. Although it does not deliver direct phylogenetic information, it can enhance the analysis depth and resolution of sequencing approaches. In sharp contrast to medical applications in both research and routine settings, flow cytometry is still not widely used for microbiome analysis. Missing information on sample preparation and data analysis pipelines may create an entry barrier for the researchers facing microbiome analysis challenges that would often be textbook flow cytometry applications. Here, we present three comprehensive workflows for pure cultures, complex communities in clear medium and complex communities in challenging matrices, respectively. We describe individual sampling and fixation procedures and optimized staining protocols for the respective sample sets. We elaborate the cytometric analysis with a complex research centered and an application focused bench top device, describe the cell sorting procedure and suggest data analysis packages. We furthermore propose important experimental controls and apply the presented workflows to the respective sample sets.

Characterizing Microbiome Dynamics &amp;#8211; Flow Cytometry Based Workflows from Pure Cultures to Natural Communities

Flow cytometric analysis has proven valuable for investigating pure cultures and monitoring microbial community dynamics. We present three comprehensive workflows, from sampling to data analysis, for pure cultures and complex communities in clear medium as well as in challenging matrices, respectively.

微生物动力学的特征-基于流式细胞仪的工作流从纯文化到自然群落

The investigation of pure cultures and monitoring of microbial community dynamics is vital to understand and control natural ecosystems and technical ...

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.

自动 3D 光学相干断层扫描，以在大型空间尺度上阐明生物膜变形

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...