Los autores desean agradecer a los miembros anteriores de la Bolsa de laboratorio: Sam-Kyu Kim, Kathryn Plichta y Victoria Miranda-Thompson por su contribución a la mejora de muchos de estos protocolos. Queremos agradecer el apoyo de la Fundación Nacional para la Ciencia (subvenciones IOS-0.840.932 e iOS-0724978) a SP Stock.

1. Toma de Muestras de Suelos <ol><li> Considere cubrir una superficie mínima de 2 a 4 m 2 por cada sitio de muestreo. </li><li> Recoger muestras de suelo a una profundidad de al menos 15 cm (Figura 4). </li><li> Tomar por lo menos 5 muestras aleatorias dentro de esta área. </li><li> Tomar 3 submuestras por muestra. Dependiendo del objetivo del estudio, ya sea combinan las submuestras o mantenerlas separadas. </li><li> Colocar cada muestra en una bolsa de plástico. Considere la posibilidad de doble embolsado para evitar fugas de las muestras (Figura 4). </li><li> Muestras de la etiqueta con un marcador indeleble. Incluya la siguiente información de cada sitio de muestreo: Información del sitio (incluyendo localidad / área / nombre del sitio y el GPS las coordenadas información), la fecha, el hábitat, la vegetación asociada, temperatura, altitud, etc NOTA: Si es aplicable, obtener y / o registrar la presencia de insectos u otros invertebrados recolectados con la muestra para su posterior identificación. Pueden representar potencial naturhosts al EPN. </li><li> Herramientas de recogida Limpiar a fondo entre las muestras de lavado con agua y / o desinfección de ellos con etanol al 70% o una solución de cloro al 0,5%. </li><li> Mantener las muestras en un refrigerador (8-15 ° C) durante el transporte al laboratorio. </li><li> Tome una porción de la muestra de suelo para el análisis para obtener información sobre la composición del suelo, la textura, la humedad, conductividad eléctrica o de otros parámetros del suelo deseadas. </li></ol> . 2 Aislamiento de nematodos de muestras de suelo: Insect-cebo Técnica <ol><li> Elimine todos los residuos (es decir., Rocas, trozos de madera o la corteza, las hojas, etc.) Recogidos con sus muestras para evitar la contaminación con microorganismos saprobic. </li><li> Añadir agua para humedecer el suelo y facilitar la circulación de los nematodos. NOTA: Utilice una botella de spray para aumentar poco a poco el nivel de humedad del suelo. </li><li> Coloque aproximadamente 200 a 250 ml de suelo húmedo en un recipiente de plástico limpio con tapa. </li><li> Añadir cebos de insectos. Considere la posibilidad de 5 a 10 insectos a la superficie del suelo de cada muestra (Figura 5). </li><li> Cubra el recipiente con una tapa y voltee los contenedores que al revés. </li><li> Mantener los contenedores en la oscuridad y a temperatura ambiente (generalmente 22-25 ° C). </li><li> Compruebe contenedores cada 2 - 3 días y quitar los insectos muertos. NOTA: agregar insectos sanos adicionales para cada recipiente para su posterior cebo de la muestra de suelo. </li><li> Quite los insectos muertos de los contenedores. NOTA: Los cadáveres con un marrón o coloración ocre son generalmente parasitados por steinernematids, mientras que los de ladrillo rojo a púrpura oscuro cadáveres son parasitados por heterorhabditids. </li><li> Enjuague cadáveres en agua estéril. </li><li> Coloque cadáveres en una trampa Blanco modificada para la recuperación de la progenie nematodo (consulte el siguiente procedimiento). </li></ol> . 3 Nematodo La recuperación de cadáveres infectados: Modificado Trampa Blanco <ol><li> Coloque la parte superior de un diámetro de 50 (o 60) mm. Placa de Petri dentro de un plato más grande (100 mm). </li><li> Establecer un pecadopapel de filtro circular gle (Whatman # 1) en el interior del plato más pequeño. </li><li> Coloque los cadáveres sobre el papel de filtro del plato más pequeño asegurarse de que los cadáveres no se toquen entre sí para evitar cualquier contaminación. </li><li> Llene el exterior (grande) caja de Petri con ca. 20 ml de agua destilada estéril. No agregue agua en el plato que contiene los cadáveres. </li><li> Cubrir la placa de Petri grande y su contenido con la tapa (Figura 6). </li><li> Etiqueta de platos en consecuencia. Agregue la siguiente información: nombre del nematodo (especie / cepa), la fecha de la infección (infección se estableció la fecha) y la fecha de la trampa (esta es la fecha en que la trampa está configurado). </li><li> Mantenga trampa a TA hasta que se produce la emergencia de estados juveniles infectivos (IJs). Este proceso puede tardar entre 10 - 25 días, dependiendo de la especie y / o cepa considerada nematodos. </li><li> Cosecha de agua con JIs mediante la eliminación de la plato más grande de la trampa y vertido de agua con los nematodos en un vaso de precipitados. </li><li> Permitir nematodos decantar a la parte inferior de la bEAKER. Este proceso puede tardar unos minutos. </li><li> Verter el agua cuidadosamente, asegurándose de que los nematodos se mantienen en la parte inferior del vaso de precipitados. </li><li> Enjuague nematodos añadiendo más agua y permitiendo que los nematodos decantar. Este paso se puede repetir 2 - 3 veces hasta que el agua esté limpia. </li><li> Coloque suspensión de nematodos en un matraz de cultivo de tejidos (250 ml). Mantener la concentración de la suspensión a 1.000 - 3.000 nemátodos / ml. </li><li> Frasco tienda con suspensión de nematodos en una habitación fría o en incubadora entre 10 a 20 ° C. NOTA: compruebe frascos almacenados periódicamente a medida que la vida útil de EPN es variable. Por lo general steinernematids se pueden almacenar durante 6 - 12 meses, sin la necesidad de subcultivos, mientras que heterorhabditids pueden requerir más revisiones periódicas. </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. W., Shapiro-Ilan, D., Duncan, L., Nguyen, K. <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+and+other+natural+enemies+as+mortality+factors+for+larvae+of+Diaprepes+abbreviatus+(Coleoptera:+Curculionidae).">Entomopathogenic nematodes and other natural enemies as mortality factors for larvae of Diaprepes abbreviatus (Coleoptera: Curculionidae).</a> Biological Control. 19, 182-190 (2000).</li><li>Meyling, N. V., Eilenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+and+distribution+of+soil+borne+entomopathogenic+fungi+within+a+single+organic+agroecosystem.">Occurrence and distribution of soil borne entomopathogenic fungi within a single organic agroecosystem.</a> Agriculture, ecosystem., &amp; environment. 113, 336-341 (2006).</li><li>Poinar, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Taxonomy and biology of Steinernematidae and Heterorhabditidae. In: Entomopathogenic Nemtaodes in Biological. , 23-61 (1990).</li><li>Stock, S. P., Gress, J. C. <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+phylogenetic+relationships+of+entomopathogenic+nematodes+(Steinernematidae+and+Heterorhabditidae)+from+the+Sky+Islands+of+southern+Arizona.">Diversity and phylogenetic relationships of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) from the Sky Islands of southern Arizona.</a> Journal of invertebrate pathology. 92, 66-72 (2006).</li><li>Stock, S. P., Hunt, D. J., Grewal, P. S., Ehlers, R. U., Shapiro-Ilan, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nematode+morphology+and+systematics.">Nematode morphology and systematics.</a> Nematodes as biological control agents. CAB International Publishing. , 3-43 (2005).</li><li>Stock, S. P., Goodrich&#8211;Blair, H. <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+and+their+bacterial+symbionts:+The+inside+out+of+a+mutualistic+association.">Entomopathogenic nematodes and their bacterial symbionts: The inside out of a mutualistic association.</a> Symbiosis. 46, 65-75 (2008).</li><li>Stuart, R. J., 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=Patchiness+in+populations+of+entomopathogenic+nematodes.">Patchiness in populations of entomopathogenic nematodes.</a> Journal of Invertebrate Pathology. 64, 39-45 (1994).</li><li>White, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+obtaining+infective+nematode+larvae+from+cultures.">A method for obtaining infective nematode larvae from cultures.</a> Science. 66, 302-303 (1927).</li></ol>

No hay conflictos de interés declarado.

Esta demostración es la primera de dos presentaciones que describen una serie de técnicas que se utilizan comúnmente para el estudio de la EPN y sus bacterias simbióticas. Estas técnicas representan pasos fundamentales para el establecimiento exitoso de las culturas EPN en el laboratorio y también constituyen la base para otros bioensayos que emplean EPN como organismos modelo para la investigación. Varios estudios de suelos de EPN han demostrado que su abundancia y distribución pued...

Muchos nematodos están asociadas con los insectos, y sus interacciones huésped ser beneficiosos o perjudiciales. Aquí, nos centramos en un grupo de nematodos que son patógenos de insectos, también conocido como nematodos entomopatógenos (o EPN). Dos familias de nematodos pertenecen a este grupo de nematodos: Steinernematidae y Heterorhabditidae 11,13. Su efecto patógeno es, de hecho, conferida por su interacción con las bacterias entéricas bacterias anaerobias facultativas (Xenorhabdus se asocian con steinernematids y bacterias Photorhabdus asociar con heterorhabditids) 2. Las bacterias se vectored de un insecto huésped a otro por la única etapa de nematodos de vida libre, la infectivo juvenil de tercera etapa (también conocida como juvenil dauer, infectivo juvenil o IJ), que vive en el suelo. Una vez dentro del insecto, los nemátodos liberan las bacterias en la hemolinfa del insecto, que matan el insecto huésped por septicemia masiva. Las bacterias tambiéndegradar los tejidos del insecto y se convierten en fuente de alimento de los nematodos &#39;, lo que les permite madurar y se multiplican. Por lo general, una o dos generaciones de nematodos adultos se producen dentro del cadáver del insecto. La progenie de los últimos reasocia generación adulto con unas pocas células bacterianas en un más especializarse de manera que su relación nutricional anterior, y cultivar estas células en sus intestinos a medida que se mueven hacia fuera de la carcasa de insectos en el suelo donde esperarán otro insecto para parasitar 6,11,14 (Figura 1). Desde su descubrimiento en 1927, EPN se han considerado alternativas valiosas a los plaguicidas químicos, ya que pueden parasitar una amplia gama de insectos que son plagas agrícolas 3,4,5. Sin embargo, en las últimas dos décadas, estos nematodos y sus bacterias simbióticas se han reconocido como un sistema modelo para el estudio maleable aspectos biológicos, ecológicos y evolutivos de INTERACCIÓ host-microbions 14. El objetivo de esta presentación es mostrar los métodos y técnicas más comúnmente utilizadas para el muestreo de suelos y el aislamiento de EPN. Una variedad de herramientas está disponible y se puede utilizar para la recogida de muestras de suelo, incluyendo: corers suelo, paletas, excavadoras post-hole, sinfines, tubos de muestreo, palas de mano, entre otros (Figura 2). Dependiendo del propósito del estudio, dos estrategias de muestreo pueden ser considerados: a) estratificado, b) el muestreo aleatorio (Figura 3). El muestreo estratificado se utiliza por lo general como parte de un estudio intensivo en un área específica o demarcada durante un período determinado de tiempo. En general, un transecto está demarcada y muestras de suelo se toman a intervalos estipulados en el transecto en un período de tiempo (Figura 3) 15. El muestreo aleatorio se emplea generalmente cuando se centra en un área extensa. Esta estrategia de muestreo ha sido utilizado para el estudio de la diversidad de EPN fesde una gran área geográfica o región (Figura 3 B) 12. Una serie de factores se puede considerar en función del objetivo del estudio que incluye una amplia gama de elevaciones, texturas de suelo y los hábitats (por ejemplo., Campos de cultivo, bosques, pastizales, parques, playas, áreas ribereñas, etc.) Se demuestra la técnica de los insectos-cebo, que fue descrito originalmente por ropa de cama y Akhurst 1 y representa una alternativa sencilla y selectiva para la recuperación de la EPN de muestras de suelo. Este es un procedimiento selectivo que se basa en la premisa de que los nematodos (estadio IJ) se sentirán atraídos por un huésped de insectos y parásitos de ella. Esta técnica también se puede considerar para el aislamiento de otros patógenos de insecto tales como hongos, bacterias y 8,10. También demuestran la técnica de trampa Blanco modificada que se utiliza para la recuperación de la progenie de nematodos a partir de huéspedes de insecto infectadas 7,14. Este es un esfuerzo conocidohod que ofrece la ventaja de la recuperación de una &quot;limpia&quot; progenie de nematodos libres de residuos de los cadáveres de insectos degradantes. Por último, las técnicas descritas y mostradas en este artículo corresponden a los concebidos y / o realizado en nuestro laboratorio, así como los descritos por diversos colegas y colaboradores.

<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)

Nematodos entomopatógenos (también conocido como EPN) representan un grupo de nematodos que habitan el suelo que parasitan una amplia gama de insectos. Estos nematodos pertenecen a dos familias: Steinernematidae y Heterorhabditidae. Hasta ahora, más de 70 especies han sido descritas en la Steinernematidae y hay cerca de 20 especies en el Heterorhabditidae. Los nematodos tienen una asociación mutualista con bacterias Enterobacteriaceae y juntos actúan como un complejo insecticida que mata a una amplia gama de especies de insectos. Aquí, nos centramos en las técnicas más comunes que se consideran para la recogida de EPN del suelo. La segunda parte de esta presentación se centra en la técnica de los insectos-cebo, un enfoque ampliamente utilizado para el aislamiento de EPN a partir de muestras de suelo, y la técnica de trampa Blanco modificada que se utiliza para la recuperación de estos nematodos de los insectos infectados. Estos métodos y técnicas son pasos clave para el éxito del establecimiento de EPN cultodas en el laboratorio y también forman la base para otros bioensayos que consideran estos nematodos como organismos modelo para la investigación en otras disciplinas biológicas. Las técnicas mostradas en esta presentación corresponden a las realizadas y / o diseñado por los miembros de SP Stock de laboratorio, así como los descritos por diversos autores.

Recogida de muestras de suelo Tercera etapa juveniles infectivos de EPN son los únicos estadios de vida libre en el ciclo de vida de estos nematodos &#39;que se encuentran en el suelo (Figura 1). Por lo tanto, la recolección de muestras de suelo es un método muy eficiente para recuperarse de estos nematodos. Figura 4 muestra un perfil de suelo que indica la profundidad a la que se deben tomar las muestras. Preservar la humedad de una muestra es un factor ...

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

Nematodos entomopatógenos son gusanos redondos habitan en el suelo que parasitan una amplia gama de insectos. Demostramos métodos de muestreo utilizados para el aislamiento de estos nematodos del suelo mediante dos técnicas: la de cebado de insectos y la trampa de Blanco modificada, para la recuperación de nematodos de muestras de suelo y cadáveres de insecto infectadas, respectivamente.

Muestreo de suelos y aislamiento de nematodos entomopatógenos (Steinernematidae, Heterorhabditidae)

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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Environment

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

24.9K vistas.  University of Arizona.  Nematodos entomopatógenos son gusanos redondos habitan en el suelo que parasitan una amplia gama de insectos. Demostramos métodos de muestreo utilizados para el aislamiento de estos nematodos del suelo mediante dos técnicas: la de cebado de insectos y la trampa de Blanco modificada, para la recuperación de nematodos de muestras de suelo y cadáveres de insecto infectadas, respectivamente. 

Video: 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.

Diseño y aplicación de un continuo<sup&gt; 13</sup&gt; C y<sup&gt; 15</sup&gt; N Cámara Etiquetado de Uniforme o Diferencial, metabólico y estructural, Planta de isótopos de etiquetado

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

Investigación

Medio ambiente

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.

Lysimetry Integrado de campo y toma de muestras de agua intersticial para la Evaluación de la Movilidad de Química en Suelos y Vegetación Establecido

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.

Desarrollo de<em&gt; Metarhizium anisopliae</em&gt; Como Mycoinsecticide: Del aislamiento al funcionamiento del campo

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.

In vivo e in vitro Crianza de nematodos entomopatógenos (Steinernematidae y Heterorhabditidae)

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

Bioingeniería

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.

Aislamiento rápido de nematodos silvestres por Baermann Funnel

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

Genética

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.

Cultivo y manipulación genética de nematodos entomopatógenos

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

Inmunología e Infección

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.

Aislamiento y selección de hongos entomopatógenos a partir de muestras de suelo y evaluación de la virulencia fúngica contra plagas de insectos

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

Biología

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).

Comparación de métodos para aislar hongos entomopatógenos de muestras de suelo

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.

Un enfoque altamente escalable para realizar estudios ecológicos de los nematodos de Caenorhabditis Selfing

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.

Aislamiento y caracterización de la microbiota natural del nematodo modelo 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 ...

Muestreo de suelos y aislamiento de nematodos entomopatógenos (Steinernematidae, Heterorhabditidae)

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