作者要感谢过去的库存实验室的成员:萨姆 - 圭金，凯瑟琳Plichta和维多利亚米兰达 - 汤普森他们对许多这些协议的改进作出贡献。我们承认美国国家科学基金会的支持（资助IOS-0840932和IOS-0724978），以SP的股票。

1，土壤样品采集<ol><li>考虑覆盖的2的最小面积- 4 平方公尺的每个采样点。 </li><li>收集的土壤样品在一个深度至少为15厘米（ 图4）。 </li><li>至少需要5个随机样本在此区域内。 </li><li>取每个样品3子样本。根据研究的目的，无论是结合子样本，或让他们分开。 </li><li>将每个样品在一个塑料袋。考虑双套袋，以避免样品泄漏（ 图4）。 </li><li>标签样本具有防水标志。包括从每个采样点的以下信息:网站信息（包括本地/区域/网站名称和GPS坐标信息），日期，生地，相关的植被，温度，海拔等。 注意:如果适用，收集和/或记录昆虫或与样品用于后续识别收集的其他无脊椎动物的存在。他们可能是潜在的自然色人EPN主机。 </li><li>通过用水充分洗涤和/或用70％乙醇或0.5％的漂白剂溶液消毒他们的样品之间干净的收集工具。 </li><li>运输到实验室中 - （15℃8）保持样品冷却器。 </li><li>对分析的土壤样品的一部分以获得对土壤成分，质地，水分，导电性或其它期望的土壤参数的信息。 </li></ol> 2，从土壤样品线虫隔离:昆虫引诱技术<ol><li>取出所有碎片（ 即 。，岩石，木材或树皮，树叶等的作品。）收集与您的样品，以避免污染人工林则与微生物。 </li><li>加水以润湿土壤和促进线虫的运动。 注:使用喷雾瓶，慢慢增加了土壤的湿度水平。 </li><li>将约200至250 mL潮湿的土壤中一个带盖子的干净的塑料容器。 </li><li>添加昆虫诱饵。考虑5至10只昆虫，以每个样品的土壤表面（ 图5）。 </li><li>盖上容器盖，把容器倒置。 </li><li>保持容器在黑暗中，并在室温（通常为22 - 25℃）。 </li><li>检查集装箱每2 - 3天，除去死虫。 注:添加额外的健康昆虫每个容器的土壤样品的进一步引诱。 </li><li>从容器中取出死虫。注:标本组用褐色或赭石色通常由steine​​rnematids寄生，而砖红色至暗紫色的尸体被heterorhabditids寄生。 </li><li>在无菌水冲洗尸体。 </li><li>尸体放置在修改后的白陷阱线虫后代的恢复（参见下面的步骤）。 </li></ol> 3，线虫恢复从受感染的标本组:改良白色陷阱<ol><li>放置一个50（或60）毫米直径的顶部。陪替氏培养皿内的一个较大的盘（100毫米）。 </li><li>设置一个罪GLE圆形滤纸（Whatman公司＃1）内的小盘子。 </li><li>对较小的盘子，确保尸体的滤纸地方尸体不互相接触，以避免任何污染。 </li><li>与CA填写外（大）培养皿。将20ml无菌蒸馏水。请勿在保存尸体的菜加水。 </li><li>覆盖大培养皿，其与盖的内容（ 图6）。 </li><li>相应地标注菜肴。添加以下信息:线虫名（种/株），感染日期（日期感染被设置）和陷阱的日期（这是陷阱被设置的日期）。 </li><li>陷阱保持在室温，直到感染幼年阶段（IJS）出现时。这个过程可能10之间取 - 26天视线虫种类和/或应变考虑。 </li><li>通过删除陷阱较大菜和浇水线虫放入烧杯中水的收获与IJS。 </li><li>允许线虫倾析将b的底部伊克。这个过程可能需要几分钟。 </li><li>小心地倒入水中，并确保线虫保留在烧杯的底部。 </li><li>通过加入更多的水，并允许线虫倒出洗净线虫。这个步骤可以重复2 - 3次，直到水是干净的。 </li><li>放置线虫悬浮液在组织培养瓶（250毫升）。保持悬浮浓度为1,000 - 3,000线虫/毫升。 </li><li>商店烧瓶线虫悬浮在冷室或培养箱中在10 - 20°C。 注:定期检查贮存瓶作为EPN的保质期是可变的。通常steine​​rnematids可存放6 - 12个月不传代培养的需要，而heterorhabditids可能需要更多的定期检查。 </li></ol>

<ol>
	<li>Bedding, R. A., Akhurst, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+the+detection+of+insect+parasitic+rhabditid+nematodes+in+soil.">A simple technique for the detection of insect parasitic rhabditid nematodes in soil.</a> Nematologica. 21, 109-110 (1975).</li><li>Boemare, N. E., Akhurst, R. A., Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K. -. H., Stackebrandt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Genera Photorhabdus and Xenorhabdus. In The Prokaryotes. , 473-488 (2007).</li><li>Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Entomopathogenic Nematology. , (2002).</li><li>Gaugler, R., Kaya, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Entomopathogenic Nematode s in Biological Control. , (1990).</li><li>Hazir, S., Keskin, N., Stock, S. P., Kaya, H. K., &#214;zcan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+distribution+of+entomopathogenic+nematodes+(Rhabditida:+Steinernematidae+and+Heterorhabditidae)+in+Turkey.">Diversity and distribution of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Turkey.</a> Biodiversit., &amp; Conservation. 12, 375-386 (2003).</li><li>Kaya, H. K., Gaugler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entomopathogenic+nematodes.">Entomopathogenic nematodes.</a> Annual Review of Entomology. 38, 181-206 (1993).</li><li>Kaya, H. K., Stock, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Techniques in insect nematology. In Manual of techniques in insect pathology. , 281-324 (1997).</li><li>Lacey, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of techniques in invertebrate pathology. , (2012).</li><li>McCoy, C. 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没有利益冲突的声明。

该演示是第一次两个报告描述了若干常用于研究EPN及其共生菌技术。这些技术代表了成功建立EPN文化在实验室的关键步骤，也是形成的基础，雇用EPN作为模式生物用于研究其他生物测定。 EPN的各种土壤调查结果显示，他们的丰度和分布可以跨季节，生境和地理区域4，5,9,15有所不同。因素如土壤质地，水分含量和温度，以及主机的可用性可能在它们的分布起着关键的作用。因此，至关重要的是，在这项统计调查的初步数据。一旦抽取样本，重要的是他们尽快处理。然而，如果这不是一个可能性，它们应该被存储在冷室或其它适当的温度控制单元。不同温度下可以是考虑用于存储昆虫诱饵容器取决于样品的起源。通常情况下，更低的温度（ 例如 ，15℃）被认为是温和的样本和样本的热带地区，温暖的温度（ 例如 ，30℃）对拟。 昆虫诱饵技术是最常用的方法从土壤样品恢复EPN的。这是一个方便的策略，允许EPN的在实验室中进行检索。大蜡螟末龄幼虫， 大蜡螟 （鳞翅目:螟蛾科）通常被认为是;然而，其他的昆虫宿主（ 即蟋蟀，面包虫，甲虫幼虫等 ）和/或适宜的阶段可以考虑，并建议使用。一些研究已经表明， 拱廊诱饵方法的单个循环提取往往不足以用于确定EPN的特定样品中存在或为从土壤样品中提取所有的EPN的S15。 12,15 40％ -通常情况下，回收率20之间变化。 另一种土壤样品在实验室的诱饵是引诱原位的考虑。这是放置在采样点昆虫诱饵。在这种情况下，昆虫被放置在笼中（通常由金属或塑料网），并留在划出站点在研究区为给定的时间周期。与昆虫的笼子，然后检索和检查EPN感染。然而，这种方法是劳动密集且耗时15。 线虫捕捉技术最初是由白10的设计，后来被卡亚和库存6修改。该技术利用线虫的吸引力，以水（ 即，正hygrotropism）以及成功恢复EPN的自由昆虫组织和/或碎屑的感染阶段。

许多线虫与昆虫有关，其宿主相互作用的范围从有利到不利的。在此，我们专注于一组的线虫是昆虫病原体，也被称为昆虫病原线虫（或EPN）。两种线虫的家庭属于这个组线虫:线虫和异11,13。其致病作用实际上是通过它们与兼性厌氧的肠道细菌（ 致病细菌与steinernematids和光杆状菌联想到heterorhabditids关联）2相互作用所赋予的。该细菌通过唯一的自由生活线虫阶段，第三阶段的感染幼年（也称为永久性幼虫少年，感染幼年或IJ），它生活在土壤中的向量从一个昆虫宿主到另一个。一旦昆虫体内，线虫释放细菌进入昆虫的血淋巴，后者通过大规模败血症的昆虫宿主。此外，细菌还降低昆虫的组织，成为线虫的食物来源，使他们走向成熟和繁殖。通常，一个或两个世代成​​年线虫是昆虫的尸体内产生。最后成年一代重新关联一些细菌细胞中的后代一个更专业的方式比他们以前的营养关系，并培养这些细胞在它们的肠子，因为他们从昆虫尸体搬出到土壤中，他们将等待另一个昆虫寄生6,11,14（ 图1）。 由于他们的发现于1927年，EPN被认为是有价值的替代化学杀虫剂，因为它们可以寄生在多种昆虫是农业害虫3,4,5。然而，在过去的二十年里，这些线虫及其共生菌已被确认为研究宿主 - 微生物interactio的生物学，生态学和进化方面的可塑性模型系统NS 14。 这个演讲的目的是显示的方法和技术最常用的土壤取样和EPN的隔离。各种工具可用，并且可以用于采集土壤样本，包括:土壤取样器，铲子，后孔挖掘机，螺旋钻，取样管，手铲，等等（ 图2）。 取决于研究的目的，两个采样策略可以考虑:1）分层，二）随机抽样（ 图3）。分层抽样通常用作在超过给定的时间周期的特定或区域划出了深入的研究的一部分。在一般情况下，一个样被划出和土壤采样在规定的时间间隔中的样条在一段时间（ 图3A）15。专注于一个广泛的区域时，随机抽样，一般使用。这种取样策略已经用于研究EPN f的多样性ROM中的较大的地理区域或地区（ 图3B）12。许多因素可以根据研究，包括海拔，土壤质地和栖息地（ 例如 。，耕地，森林，草场，公园，海岸，河岸地区等）的多元化的重点加以考虑。 我们展示了昆虫引诱技术，它最初是由床上用品和Akhurst 1描述和表示从土壤样品中恢复EPN一个简单的和有选择性的选择。这是基于这样的前提，该线虫（IJ阶段）将被吸引到昆虫宿主和寄生它选择性步骤。这种技术也可以考虑用于其他昆虫病原体如真菌，细菌和8,10隔离。 我们还证明其用于从受感染的昆虫宿主7,14检索线虫后代改性白阱技术。这是满足一个轻松HOD，提供检索一个&#39;干净&#39;线虫后代免于降解昆虫尸体碎片的优势。 最后，描述和本文中所述的技术对应于那些构思和/或在我们的实验室，以及那些由不同的同事和合作者描述进行。

<table border="0" cellpadding="0" cellspacing="0" width="1119">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Name of Reagent/ Equipment</td>
			<td style="width:309px;">Company</td>
			<td style="width:241px;">Catalog Number</td>
			<td style="width:299px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:271px;">Galleria mellonella</td>
			<td style="width:309px;">Timberline</td>
			<td style="width:241px;">http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td>
			<td>for insect baiting technique</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:271px;">Filter Paper, 55mm Grade 1 Cellulose</td>
			<td style="width:309px;">Whatman/VWR</td>
			<td>28450-048</td>
			<td>infection chamber and White trap assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">60 x 15 mm Petri Dishes</td>
			<td style="width:309px;">VWR</td>
			<td>25384-092</td>
			<td>infection chamber and White trap assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">100 x 15 Petri Dishes</td>
			<td style="width:309px;">VWR</td>
			<td style="width:241px;">25384-088</td>
			<td>infection chamber and White trap assembly</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">ZIPloc Plastic Bags</td>
			<td style="width:309px;">Ace Hardware or local hardware store</td>
			<td style="width:241px;">N/A</td>
			<td>soil sampling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:271px;">Mechanical Pipettor P1000, P200 ml</td>
			<td style="width:309px;">VWR</td>
			<td>89130-562; 89130-566&#160;</td>
			<td>infection chamber and White trap assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">pippetor tips 1000ml, 200 ml</td>
			<td style="width:309px;">VWR</td>
			<td style="width:241px;">16466-004 ; 53510</td>
			<td>infection chamber and White trap assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Bleach</td>
			<td style="width:309px;">Ace Hardware or local hardware store</td>
			<td>N/A</td>
			<td>soil sampling , desisnfecting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium Hypochlorite</td>
			<td style="width:309px;">Ace Hardware or local hardware store</td>
			<td>N/A</td>
			<td>soil sampling , desisnfecting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">70% Ethyl alochol</td>
			<td style="width:309px;">AAPER</td>
			<td>17212945</td>
			<td>soil sampling , desisnfecting</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:271px;">Shovels</td>
			<td style="width:309px;">Ace Hardware or local hardware store</td>
			<td style="width:241px;">N/A</td>
			<td>soil sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tubular soil sampler</td>
			<td>Accuproducts</td>
			<td style="width:241px;">N/A</td>
			<td>soil sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Soil Probe, long handle</td>
			<td>Nupla&#160;</td>
			<td style="width:241px;">PRB4T 69401 Classic</td>
			<td>soil sampling</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Measuring tape</td>
			<td style="width:309px;">Ace Hardware or local hardware store</td>
			<td style="width:241px;">N/A</td>
			<td>soil sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Flagging tape, various colors</td>
			<td style="width:309px;">Ace Hardware or local hardware store</td>
			<td style="width:241px;">N/A</td>
			<td>soil sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">tissue culture flasks</td>
			<td style="width:309px;">VWR</td>
			<td style="width:241px;">Falcon, 29185-304&#160;</td>
			<td>White trap, collection of EPN</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:271px;">wash bottles, Polyethylene, Wide Mouth</td>
			<td style="width:309px;">VWR</td>
			<td>16650-107</td>
			<td>White trap, collection of EPN</td>
		</tr>
	</tbody>
</table>

soil sampling and isolation of entomopathogenic nematodes (steinernematidae, heterorhabditidae)

昆虫病原线虫（又名EPN）代表一组土栖线虫的寄生多种昆虫的。这些线虫属两个家庭:线虫和异。到现在为止，有超过70种已在Steine​​rnematidae描述，并有大约20种的异小。线虫有肠杆菌科细菌互利共生的伙伴关系和他们一起作为一个有效的杀虫复杂，杀灭多种昆虫种类。 在此，我们重点考虑从土壤中采集EPN中的最常用的技术。这个演讲的第二部分着重于昆虫引诱技术，为EPN从土壤样品中分离一种广泛使用的方法，以及用于这些线虫从感染虫的复苏修改白色陷阱技术。这些方法和技术是成功建立EPN邪教的关键步骤在实验室中，也URES形成的基础，即认为这些线虫作为模式生物的研究在其他生物学科等生物测定。在此演示文稿中显示的技术对应于那些由SP联合实验室成员，以及那些由不同的作者进行描述和/或设计。

土壤样品采集 EPN的第三阶段感染性幼虫是在这些线虫的生活周期（ 图1），其驻留在土壤中的唯一的自由生活阶段。因此，采集的土壤样品，是要收回这些线虫的一种非常有效的方法， 图4示出了表示在该样品应采取深处的土壤剖面。保存的样品的水分对于线虫的存活的关键因素。因此，要保持土壤样品中的塑料袋和过境实验室（ 图4）中维护它们在凉爽的温度是很重要的。 线虫恢复在自然界中，发现昆虫通过EPN自然寄生的机会都小于3％，除非有发现一种流行病和昆虫与EPN为患更大的样本。因此，土壤样品与昆虫的引诱是一种方便的APPRoach以增强EPN从它们的天然生境中恢复图5描绘了土壤诱饵技术。在这种情况下，用螺丝顶的250毫升塑料容器中使用。约250g土壤被放置在容器中，5 - 10 G。蜡螟幼虫加到土壤的表面（ 图5右）。 5天之后，该容器被打开以检索死昆虫与EPN感染的迹象。请注意线虫幼虫感染用红色着色（ 图5左）。 图6A示出了修改后的白阱的示意图。 图6B显示了设置了陷阱。请注意，EPN感染的尸体不相互接触和滤纸在小培养皿保持干燥。 图6C表示从尸体IJS出现。注意线虫形成在陪替氏培养皿，由于它们移动到&#39;路径&#39;（IJS）病房的水。取决于线虫物种和还依赖于它的白阱保持在温度和湿度的时间从尸体IJ出现是可变的。 图6D示出IJS已经在这个陷阱中较大的盘子的水。观察水中的线虫看起来干净，无虫组织。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/52083/52083fig1highres.jpg" width="500" /> 昆虫病原线虫的图1。生命周期。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52083/52083fig2highres.jpg" width="500" /> 图2。铲，挖掘机及用于收集土壤样本等设备。（一）经典点铲（B）手铲。<html> （三）从左至右依次为:Viehmeyer管，抹子，OAKFIELD管，土壤取样器。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52083/52083fig3highres.jpg" width="500" /> 图3。土壤采样策略（A）分层（B）随机。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/52083/52083fig4highres.jpg" width="500" /> 土壤采样策略。图片左边的图4。示意图显示表明在该样本应采取所需深度的土壤剖面。右侧的图像显示了在一个塑料袋用适当的标签土壤样品。 3/52083fig5highres.jpg"宽度="500"/&gt; 土壤样品与昆虫幼虫。图片右侧的图5。:0084显示健康的幼虫，而在左边的图像显示死亡幼虫5天引诱期后。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/52083/52083fig6highres.jpg" width="500" /> 图6。示意图（A）和修改后的白陷阱的照片（BD）。 （二）陷阱设置（C）接近受感染的尸体呈现IJ出现，（D）特写显示IJS的大量出现，从尸体入水。

Watch this Scientific Journal Video about Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae at JoVE.com

Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

昆虫病原线虫是土栖线虫的寄生多种昆虫。我们展示了用于这些线虫使用两种技术的土壤隔离采样方式:昆虫引诱和修改后的白的陷阱，对于线虫的土壤样品和感染的昆虫尸体，分别回收。

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

Entomopathogenic nematodes (a.k.a. EPN) represent a group of soil-inhabiting nematodes that parasitize a wide range of insects. These nematodes belong to ...

soil-sampling-isolation-entomopathogenic-nematodes-steinernematidae

Nanyang Technological University

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JoVE Journal

Environment

Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

24.8K 次观看.  University of Arizona. 昆虫病原线虫是土栖线虫的寄生多种昆虫。我们展示了用于这些线虫使用两种技术的土壤隔离采样方式:昆虫引诱和修改后的白的陷阱，对于线虫的土壤样品和感染的昆虫尸体，分别回收。 

视频: Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

设计一个连续和操作<sup&gt; 13</sup&gt; C和<sup&gt; 15</sup&gt;Ñ贴标商会统一或差分，代谢和结构，植物同位素标记

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

科研

环境科学

Integrated Field Lysimetry and Porewater Sampling for Evaluation of Chemical Mobility in Soils and Established Vegetation

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals is often not well understood. Here we demonstrate an integrated field lysimetry and porewater sampling method for evaluating the mobility of chemicals applied to soils and established vegetation. Lysimeters, open columns made of metal or plastic, are driven into bareground or vegetated soils. Porewater samplers, which are commercially available and use vacuum to collect percolating soil water, are installed at predetermined depths within the lysimeters. At prearranged times following chemical application to experimental plots, porewater is collected, and lysimeters, containing soil and vegetation, are exhumed. By analyzing chemical concentrations in the lysimeter soil, vegetation, and porewater, downward leaching rates, soil retention capacities, and plant uptake for the chemical of interest may be quantified. Because field lysimetry and porewater sampling are conducted under natural environmental conditions and with minimal soil disturbance, derived results project real-case scenarios and provide valuable information for chemical management. As chemicals are increasingly applied to land worldwide, the described techniques may be utilized to determine whether applied chemicals pose adverse effects to human health or the environment.

Field lysimetry and porewater sampling allow researchers to evaluate the fate of chemicals applied to soils and established vegetation. The goal of this protocol is to demonstrate how to install required instrumentation and collect samples for chemical analysis during integrated field lysimetry and porewater sampling experiments.

综合外地Lysimetry和孔隙水采样的化学活动性土壤的评价和植被成立

Potentially toxic chemicals are routinely applied to land to meet growing demands on waste management and food production, but the fate of these chemicals ...

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.

An Optimized Enrichment Technique for the Isolation of Arthrobacter Bacteriophage Species from Soil Sample Isolates

We present an enrichment protocol for the isolation of bacteriophages infecting bacteria in the Arthrobacter genus. This enrichment protocol produces fast and reproducible results for the isolation and amplification of Arthrobacter phages from soil isolates.

一种优化富集技术的隔离<em&gt;节杆菌</em&gt;噬菌体种从土壤样品隔离

Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of ...

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays 1. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs 2, with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

Behavioral assays provide powerful tools for understanding neuronal function. Here we present several protocols for quantifying predatory feeding behavior found in the model nematode Pristionchus pacificus and its relatives. Additionally, we provide methods for analyzing predatory feeding adaptations including mouth structures and teeth.

测定掠夺取食行为<em&gt; Pristionchus</em&gt;及其他线虫

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

一个插角Thermogradient表来控制土壤温度为评估植物生长和监测土壤过程

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Wastewater Irrigation Impacts on Soil Hydraulic Conductivity: Coupled Field Sampling and Laboratory Determination of Saturated Hydraulic Conductivity

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. Rather than discharging treated wastewater to a stream, and thereby directly impacting the stream quality, the effluent is applied to forested and cropped land managed by the University. Concerns related to reductions in soil hydraulic conductivity occur when considering wastewater reuse. The methodology described in this manuscript, matching soil sample size with the size of the laboratory-based hydraulic conductivity measurement apparatus, provides the benefits of a relatively rapid collection of samples with the benefits of controlled laboratory boundary conditions. The results suggest that there may have been some impact of wastewater reuse on the soil&#39;s ability to transmit water at deeper depths in the depressional areas of the site. Most of the reductions in the soil hydraulic conductivity in the depressions appear to be related to the depth from which the sample was collected, and by inference, associated with the soil structural and textural differences.

Here we present a methodology which matches a soil sample size and a hydraulic conductivity measurement device to prevent the so-called wall flow along the inside of the soil container from being erroneously included in water flow measurements. Its use is demonstrated with samples collected from a wastewater irrigation site.

污水灌溉对土壤水力电导率的影响: 耦合场取样与饱和水力电导率的实验室测定

Since the early 1960s, an alternative wastewater discharge practice at The Pennsylvania State University has been researched and its impacts monitored. ...

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 ...

Use of Principal Components for Scaling Up Topographic Models to Map Soil Redistribution and Soil Organic Carbon

Landscape topography is a critical factor affecting soil formation and plays an important role in determining soil properties on the earth surface, as it regulates the gravity-driven soil movement induced by runoff and tillage activities. The recent application of Light Detection and Ranging (LiDAR) data holds promise for generating high spatial resolution topographic metrics that can be used to investigate soil property variability. In this study, fifteen topographic metrics derived from LiDAR data were used to investigate topographic impacts on redistribution of soil and spatial distribution of soil organic carbon (SOC). Specifically, we explored the use of topographic principal components (TPCs) for characterizing topography metrics and stepwise principal component regression (SPCR) to develop topography-based soil erosion and SOC models at site and watershed scales. Performance of SPCR models was evaluated against stepwise ordinary least square regression (SOLSR) models. Results showed that SPCR models outperformed SOLSR models in predicting soil redistribution rates and SOC density at different spatial scales. Use of TPCs removes potential collinearity between individual input variables, and dimensionality reduction by principal component analysis (PCA) diminishes the risk of overfitting the prediction models. This study proposes a new approach for modeling soil redistribution across various spatial scales. For one application, access to private lands is often limited, and the need to extrapolate findings from representative study sites to larger settings that include private lands can be important.

Landscape processes are critical components of soil formation and play important roles in determining soil properties and spatial structure in landscapes. We propose a new approach using stepwise principal component regression to predict soil redistribution and soil organic carbon across various spatial scales.

利用主成分扩展地形模型绘制土壤再分配和土壤有机碳

Landscape topography is a critical factor affecting soil formation and plays an important role in determining soil properties on the earth surface, as it ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

微图设计及植物和土壤样品制备，进行15次氮气分析

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

摘要

探索更多视频

此视频中的章节

设计一个连续和操作

综合外地Lysimetry和孔隙水采样的化学活动性土壤的评价和植被成立

一种优化富集技术的隔离

测定掠夺取食行为

一个插角Thermogradient表来控制土壤温度为评估植物生长和监测土壤过程

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

污水灌溉对土壤水力电导率的影响: 耦合场取样与饱和水力电导率的实验室测定

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

利用主成分扩展地形模型绘制土壤再分配和土壤有机碳

微图设计及植物和土壤样品制备，^进行15次氮气分析

土壤采样及昆虫病原线虫的分离（Steine​​rnematidae，异小）

摘要

探索更多视频

此视频中的章节

设计一个连续和操作

综合外地Lysimetry和孔隙水采样的化学活动性土壤的评价和植被成立

一种优化富集技术的隔离

测定掠夺取食行为

一个插角Thermogradient表来控制土壤温度为评估植物生长和监测土壤过程

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

污水灌溉对土壤水力电导率的影响: 耦合场取样与饱和水力电导率的实验室测定

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

利用主成分扩展地形模型绘制土壤再分配和土壤有机碳

微图设计及植物和土壤样品制备，进行15次氮气分析

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

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