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>
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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?...

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><tbody><tr><td>&lt;em&gt;Galleria mellonella&lt;/em&gt;</td><td>Timberline</td><td>http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td><td>For insect baiting technique</td></tr><tr><td>Filter paper, 55 mm Grade 1 Cellulose</td><td>Whatman/VWR</td><td>28450-048</td><td>Infection chamber and White trap assembly</td></tr><tr><td>60 x 15 mm Petri dishes</td><td>VWR</td><td>25384-092</td><td>Infection chamber and White trap assembly</td></tr><tr><td>100 x 15 mm Petri dishes</td><td>VWR</td><td>25384-088</td><td>Infection chamber and White trap assembly</td></tr><tr><td>ZIPloc plastic bags</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Mechanical pipettor P1000, P200 ml</td><td>VWR</td><td>89130-562; 89130-566&amp;nbsp;</td><td>Infection chamber and White trap assembly</td></tr><tr><td>Pippetor tips 1000ml, 200 ml</td><td>VWR</td><td>16466-004 ; 53510</td><td>Infection chamber and White trap assembly</td></tr><tr><td>Bleach</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling , disinfecting</td></tr><tr><td>Sodium hypochlorite</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling , disinfecting</td></tr><tr><td>70% Ethyl alochol</td><td>AAPER</td><td>17212945</td><td>Soil sampling , disinfecting</td></tr><tr><td>Shovels</td><td>Ace Hardware or local hardware store</td><td>N/A</td><td>Soil sampling</td></tr><tr><td>Tubular soil sampler</td><td>Accuproducts</td><td></td><td>Soil sampling</td></tr><tr><td>Soil probe, long handle</td><td>Nupla&amp;nbsp;</td><td>PRB4T 69401 Classic</td><td>Soil sampling</td></tr><tr><td>Measuring tape</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Flagging tape, various colors</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Tissue culture flasks</td><td>VWR</td><td>Falcon, 29185-304&amp;nbsp;</td><td>White trap, collection of EPN</td></tr><tr><td>Wash bottles, polyethylene, wide mouth</td><td>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）?...

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 ...

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

24.9K 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 ...

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance

Here, we report the different stages involved in the knowledge-based development of an effective mycoinsecticide, including the isolation, identification, screening, and selection of the "best-fit" entomopathogenic fungus, Metarhizium anisopliae, for the control of insect pests in agriculture.

的发展<em&gt; Metarhizium anisopliae</em&gt;作为防腐剂:从隔离到现场表现

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and ...

In vivo and In vitro Rearing of Entomopathogenic Nematodes (Steinernematidae and Heterorhabditidae)

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont&#8211;free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock&#8217;s Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

In vivo and In vitro Rearing of Entomopathogenic Nematodes Steinernematidae and Heterorhabditidae

The goal of this presentation is to demonstrate in vivo and in vitro techniques for the rearing of entomopathogenic nematodes. In vivo methods consider the rearing of these nematodes with an insect host, whereas the in vitro methods utilize rich agar media.

在体内和昆虫病原线虫体外培育（ 斯氏和异小 ）

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the ...

生物工程

Rapid Isolation of Wild Nematodes by Baermann Funnel

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild nematodes in their natural environments are valuable for understanding many aspects of biology, including the selective regimes in which distinctive genomic and phenotypic characters evolve, the genetic basis for complex trait variation, and the natural genetic diversity fundamental to all animal populations. This manuscript describes a simple and efficient method for extracting nematodes from their natural substrates, including rotting fruits, flowers, fungi, leaf litter, and soil. The Baermann funnel method, a classical nematology technique, selectively isolates active nematodes from their substrates. Because it recovers nearly all active worms from the sample, the Baermann funnel technique allows for the recovery of rare and slow-growing genotypes that co-occur with abundant and fast-growing genotypes, which might be missed in extraction methods that involve multiple generations of reproduction. The technique is also well suited to addressing metagenetic, population-genetic, and ecological questions. It captures the entire population in a sample simultaneously, allowing an unbiased view of the natural distribution of ages, sexes, and genotypes. The protocol allows for deployment at scale in the field, rapidly converting substrates into worm plates, and the authors have validated it through fieldwork on multiple continents.

This protocol outlines a method for efficiently extracting live nematodes from natural substrates in the field.

由Baermann Funnel快速分离野生线虫

Beyond being robust experimental model organisms, Caenorhabditis elegans and its relatives are also real animals that live in nature. Studies of wild ...

遗传学

Culturing and Genetically Manipulating Entomopathogenic Nematodes

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic of their life cycle is the mutualistic association with the bacteria Photorhabdus and Xenorhabdus, respectively. The nematode parasites are able to locate and enter suitable insect hosts, subvert the insect immune response, and multiply efficiently to produce the next generation that will actively hunt new insect prey to infect. Due to the properties of their life cycle, entomopathogenic nematodes are popular biological control agents, which are used in combination with insecticides to control destructive agricultural insect pests. Simultaneously, these parasitic nematodes represent a research tool to analyze nematode pathogenicity and host anti-nematode responses. This research is aided by the recent development of genetic techniques and transcriptomic approaches for understanding the role of nematode secreted molecules during infection. Here, a detailed protocol on maintaining entomopathogenic nematodes and using a gene knockdown procedure is provided. These methodologies further promote the functional characterization of entomopathogenic nematode infection factors.

Entomopathogenic nematodes live in symbiosis with bacteria and together they successfully infect insects by undermining their innate immune system. To promote research on the genetic basis of nematode infection, methods for maintaining and genetically manipulating entomopathogenic nematodes are described.

培养和遗传操纵昆虫致病线虫

Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema are obligate parasites of insects that live in the soil. The main characteristic ...

免疫与感染

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

生物学

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

从土壤样品中分离昆虫致病真菌的方法比较

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

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

的发展

在体内和昆虫病原线虫体外培育（斯氏和异小）

由Baermann Funnel快速分离野生线虫

培养和遗传操纵昆虫致病线虫

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

从土壤样品中分离昆虫致病真菌的方法比较

一种高度可扩展的方法，用于对自发性Caenorhabditis 线虫进行生态调查

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

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

的发展

在体内和昆虫病原线虫体外培育（ 斯氏和异小 ）

由Baermann Funnel快速分离野生线虫

培养和遗传操纵昆虫致病线虫

土壤样本中昆虫病原真菌的分离与选择及真菌对害虫的毒力评价

从土壤样品中分离昆虫致病真菌的方法比较

一种高度可扩展的方法，用于对自发 性Caenorhabditis 线虫进行生态调查

模型线虫秀丽隐杆线虫天然微生物群的分离和表征

土壤采样及昆虫病原线虫的分离（Steinernematidae，异小）

在体内和昆虫病原线虫体外培育（斯氏和异小）

一种高度可扩展的方法，用于对自发性Caenorhabditis 线虫进行生态调查