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 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)

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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Research

JoVE Journal

Environment

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

24.7K 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 ...

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.

Una técnica de enriquecimiento optimizado para el aislamiento de<em&gt; Arthrobacter</em&gt; Bacteriófago Especies de Aísla Muestra de Suelo

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.

El ensayo de los comportamientos de alimentación depredadores en<em&gt; Pristionchus</em&gt; Y otros nematodos

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.

Una tabla de fuelle Thermogradient para controlar las temperaturas del suelo para evaluar el crecimiento de la planta y Seguimiento de los procesos del suelo

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.

Medición y mapeo de patrones de erosión y deposición relacionadas las concentraciones de carbonato de suelo bajo manejo agrícola

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.

Impactos de riego de aguas residuales en la conductividad hidráulica del suelo: unida los muestreo de campo y laboratorio determinación de la conductividad hidráulica saturada

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.

Aislamiento y análisis de comunidades microbianas en el suelo, rizosfera y raíces en los experimentos de pastos perennes

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.

Uso de componentes principales para el escalado de modelos topográficos a mapa de redistribución de la tierra y el suelo de carbono orgánico

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

Estimation of Crystalline Cellulose Content of Plant Biomass using the Updegraff Method

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a significant role in plant growth and development by providing strength, rigidity, rate and direction of cell growth, cell shape maintenance, and protection from biotic and abiotic stressors. The cell wall is primarily composed of cellulose, lignin, hemicellulose and pectin. Recently plant cell walls have been targeted for the second-generation biofuel and bioenergy production. Specifically, the cellulose component of the plant cell wall is used for the production of cellulosic ethanol. Estimation of cellulose content of biomass is critical for fundamental and applied cell wall research. The Updegraff method is simple, robust, and the most widely used method for the estimation of crystalline cellulose content of plant biomass. The alcohol insoluble crude cell wall fraction upon treatment with Updegraff reagent eliminates the hemicellulose and lignin fractions. Later, the Updegraff reagent resistant cellulose fraction is subjected to sulfuric acid treatment to hydrolyze the cellulose homopolymer into monomeric glucose units. A regression line is developed using various concentrations of glucose and used to estimate the amount of the glucose released upon cellulose hydrolysis in the experimental samples. Finally, the cellulose content is estimated based on the amount of glucose monomers by colorimetric anthrone assay.

The Updegraff method is the most widely used method for the cellulose estimation. The main purpose of this demonstration is to provide a detailed Updegraff protocol for estimation of cellulose content in plant biomass samples.

Estimación del contenido de celulosa cristalina de biomasa vegetal utilizando el método Updegraff

Cellulose is the most abundant polymer on Earth generated by photosynthesis and the main load-bearing component of cell walls. The cell wall plays a ...