Os autores gostariam de agradecer aos membros anteriores do Banco de laboratório: Sam-Kyu Kim, Kathryn Plichta e Victoria Miranda-Thompson por suas contribuições para a melhoria de muitos destes protocolos. Agradecemos o apoio da National Science Foundation (subvenções IOS-0840932 e IOS-0724978) a SP estoque.

1. Coleta de Amostras de solo <ol><li> Considere que cobre uma área mínima de 2 - 4 m 2 para cada local de amostragem. </li><li> Recolha de amostras de solo a uma profundidade de pelo menos 15 cm (Figura 4). </li><li> Tome pelo menos 5 amostras aleatórias dentro desta área. </li><li> Tome 3 subamostras por amostra. Dependendo do objetivo do estudo, ou combinar as subamostras ou mantê-los separados. </li><li> Coloque cada amostra em um saco plástico. Considere duplo ensacamento para evitar vazamento de amostras (Figura 4). </li><li> Amostras de etiquetas com um marcador à prova d&#39;água. Inclua as seguintes informações de cada local de amostragem: informações do site (incluindo localidade / área / nome do site e GPS coordenar informações), data, habitat, a vegetação associada, temperatura, altitude, etc NOTA: Se for o caso, recolher e / ou registrar a presença de insetos e outros invertebrados coletados com a amostra para posterior identificação. Eles podem representar potencial naturhospedeiros al EPN. </li><li> Ferramentas de recolha limpos entre as amostras mediante a lavagem com água e / ou desinfectar as com etanol a 70% ou solução de lixívia a 0,5%. </li><li> Manter as amostras em um refrigerador (8 - 15 ° C) durante o transporte para o laboratório. </li><li> Pegue uma porção da amostra de solo para análise para obter informações sobre a composição do solo, textura, umidade, condutividade elétrica ou outros parâmetros do solo desejados. </li></ol> . 2 Isolamento Nematóides de amostras de solo: Técnica do Inseto-baiting <ol><li> Remova todos os detritos (ie., Rochas, pedaços de madeira ou casca, folhas, etc.), Coletados com suas amostras para evitar a contaminação com microorganismos saprobic. </li><li> Adicionar água para umedecer o solo e facilitar a circulação de nematóides. NOTA: Use um frasco de spray para aumentar lentamente o nível de umidade do solo. </li><li> Colocar aproximadamente 200 a 250 ml de solo húmido num recipiente de plástico limpo com uma tampa. </li><li> Adicionar iscas de insetos. Considere 5 a 10 insectos para a superfície do solo de cada amostra (Figura 5). </li><li> Cobrir o recipiente com tampa e ligar recipientes de cabeça para baixo. </li><li> Manter recipientes no escuro e à temperatura ambiente (normalmente 22-25 ° C). </li><li> Verifique os recipientes a cada 2 - 3 dias, e remover insetos mortos. NOTA: adicionar insetos saudáveis ​​adicionais para cada recipiente para posterior baiting da amostra de solo. </li><li> Remover insetos mortos dos recipientes. NOTA: Cadáveres com um marrom ou ocre coloração geralmente são parasitados por steinernematids, enquanto tijolo vermelho para cadáveres roxo escuro são parasitados por heterorhabditids. </li><li> Enxágüe cadáveres em água estéril. </li><li> Coloque cadáveres em uma armadilha Branco modificado para recuperação de nematóide progênie (ver procedimento abaixo). </li></ol> . 3 Nematode recuperação de cadáveres infectados: Modificado Armadilha Branco <ol><li> Coloque a parte superior de um diâmetro 50 (ou 60) mm. Placa de Petri dentro de um prato grande (100 mm). </li><li> Definir um pecadopapel de filtro circular gle (Whatman # 1) no interior do prato menor. </li><li> Coloque cadáveres sobre o papel de filtro do prato menor certificando-se de cadáveres não se tocam para evitar qualquer contaminação. </li><li> Encha o exterior (maior) placa de Petri com ca. 20 ml de água destilada estéril. Não adicionar água no prato que contém os cadáveres. </li><li> Cobrir a grande placa de Petri e seu conteúdo, com a tampa (Figura 6). </li><li> Rotular pratos de acordo. Adicione as seguintes informações: Nome do Nemátodo (espécie / isolado), a data de infecção (infecção data foi definida) e data armadilha (esta é a data em que a armadilha está configurado). </li><li> Mantenha a armadilha RT até a emergência dos estágios juvenis infectantes (IJS) ocorre. Este processo pode levar entre 10 - 25 dias, dependendo da espécie e / ou tensão considerado nematóides. </li><li> Colheita de água com IJs removendo o maior prato da armadilha e despejar água com nematóides para um copo. </li><li> Permitir nemátodos decantar para a parte inferior da bEaker. Este processo pode demorar alguns minutos. </li><li> Despeje água cuidadosamente, certificando-se de nematóides permanecem no fundo do copo. </li><li> Lavar nematóides, adicionando mais água e permitindo que os nemátodos para decantar. Este passo pode ser repetido 2-3 vezes, até que a água é limpa. </li><li> Colocar a suspensão de nemátodos num balão de cultura de tecidos (250 ml). Manter a concentração da suspensão a 1.000 - 3.000 nemátodos / ml. </li><li> Frasco loja com suspensão de nematóides em uma sala fria ou em estufa entre 10-20 ° C. NOTA: verifique frascos armazenados periodicamente como a vida de prateleira de EPN é variável. Normalmente steinernematids pode ser armazenado por 6-12 meses, sem a necessidade de subcultura, enquanto heterorhabditids pode exigir mais check-ups periódicos. </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>

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Esta demonstração é a primeira de duas apresentações que descrevem um número de técnicas que são comummente usados ​​para estudar EPN e suas bactérias simbióticas. Estas técnicas representam passos fundamentais para o sucesso da criação culturas EPN em laboratório e também servir de base para outros bioensaios que empregam EPN como organismos modelo para a pesquisa. Vários levantamentos de solos de EPN têm mostrado que a sua abundância e distribuição pode variar entre...

Muitos nematóides estão associados com insetos, e suas interações hospedeiro variam de benéfico para prejudicial. Aqui, vamos nos concentrar em um grupo de nematóides que são patógenos de insetos, também conhecido como nematóides entomopatogênicos (ou EPN). Duas famílias de nematóides pertencem a este grupo de nematóides: Steinernematidae e Heterorhabditidae 11,13. O seu efeito patogênico é de fato conferida pela sua interação com anaeróbios facultativos bactérias entéricas (bactérias Xenorhabdus associar steinernematids e bactérias Photorhabdus associar heterorhabditids) 2. As bactérias são vetorizado de um hospedeiro a outro por insetos a única fase livre nematóide de vida, a terceira fase juvenil infecciosa (também conhecida como juvenil Dauer, juvenil infecciosa ou IJ), que vive no solo. Uma vez dentro do inseto, os nematóides liberar as bactérias na hemolinfa do inseto, que matam o inseto hospedeiro por septicemia maciça. As bactérias tambémdegradam os tecidos do insecto e se tornar fonte de alimento dos nematóides, permitindo-lhes a amadurecer e multiplicar. Normalmente, uma ou duas gerações de nematóides adultos são produzidos dentro do cadáver do inseto. A progênie dos últimos reassociates geração de adultos com algumas células bacterianas em um mais especializar maneira que sua relação nutricional anterior e cultivar essas células em seus intestinos que se deslocam para fora do inseto carcaça no solo onde irão aguardar outro inseto parasitar 6,11,14 (Figura 1). Desde sua descoberta em 1927, EPN foram consideradas alternativas valiosas para pesticidas químicos como eles podem parasitar uma grande variedade de insetos que são pragas agrícolas 3,4,5. No entanto, ao longo das últimas duas décadas, esses nematóides e suas bactérias simbióticas ter sido reconhecido como um sistema modelo para o estudo de aspectos maleável biológicos, ecológicos e evolutivos de host-micróbio interactions 14. O objetivo desta apresentação é mostrar os métodos e técnicas mais comumente utilizadas para a amostragem do solo e isolamento de EPN. Uma variedade de ferramentas está disponível e pode ser usado para a coleta de amostras de solo, incluindo: corers solo, espátulas, escavadoras pós-buracos, brocas, tubos de amostragem, pás de mão, entre outros (Figura 2). Dependendo do objectivo do estudo, duas estratégias de amostragem pode ser considerada: a) estratificado, b) amostragem (Figura 3). A amostragem estratificada é normalmente usado como parte de um estudo intensivo de uma área específica ou demarcada sobre um dado período de tempo. Em geral, uma seção transversal é demarcado e amostras de solo são tomadas a intervalos previstos na transecção ao longo de um período de tempo (Figura 3A) 15. A amostragem aleatória é geralmente empregada quando o foco em uma área extensa. Esta estratégia de amostragem foi usado para estudar a diversidade de EPN fesde uma grande área geográfica ou região (Figura 3B) 12. Uma série de fatores podem ser considerados, dependendo do foco do estudo, incluindo uma grande variedade de elevações, texturas de solo e habitats (por exemplo., Campos cultivados, florestas, pastos, parques, praias, matas ciliares, etc.) Mostramos a técnica de atracção de insectos, que foi originalmente descrito por Bedding e Akhurst 1 e representa uma alternativa simples e selectiva para a recuperação a partir de amostras de solo de EPN. Este é um processo selectiva que é baseado na premissa de que os nemátodos (estádio IJ) será atraído para uma série de insectos e que parasitam. Esta técnica também pode ser considerada para o isolamento de outros agentes patogénicos de insectos, tais como fungos, bactérias e 8,10. Também demonstramos a técnica armadilha branco modificado que é utilizado para a recuperação de nemátodo progenitura de hospedeiros infectados insectos 7,14. Este é um esforço method que oferece a vantagem de recuperar uma &quot;limpa&quot; nematóide progênie livre de restos de cadáveres de insetos degradantes. Finalmente, as técnicas descritas e mostradas neste artigo correspondem aos concebido e / ou realizados no nosso laboratório, bem como os descritos por vários colegas e 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)

Nematóides entomopatogênicos (aka EPN) representam um grupo de nematóides que vivem no solo que parasitam uma grande variedade de insetos. Esses nematóides pertencem a duas famílias: Steinernematidae e Heterorhabditidae. Até agora, mais de 70 espécies já foram descritas no Steinernematidae e existem cerca de 20 espécies no Heterorhabditidae. Os nematóides têm uma parceria de mutualismo com bactérias Enterobacteriaceae e, juntos, eles agem como um complexo potente inseticida que mata uma grande variedade de espécies de insetos. Aqui, vamos nos concentrar nas técnicas mais comuns considerados para a recolha de EPN do solo. A segunda parte desta apresentação centra-se na técnica de atracção de insectos, uma abordagem amplamente utilizada para o isolamento de EPN de amostras de solo, e a técnica de armadilha branco modificado, que é utilizado para a recuperação destes nematóides de insectos infectadas. Estes métodos e técnicas são passos fundamentais para o sucesso da criação EPN cultUres em laboratório e também servir de base para outros bioensaios que consideram estes nematóides como organismos modelo para a pesquisa em outras disciplinas biológicas. As técnicas indicadas nesta apresentação correspondem às realizadas e / ou concebida por membros da SP laboratório, bem como os descritos por vários autores.

Coleta de Amostras de solo Em estágio Terceiro juvenis infectantes de EPN são os únicos estágios gratuitos de vida no ciclo de vida desses nematóides que residem no solo (Figura 1). Portanto, a recolha de amostras de solo é um método muito eficiente para recuperar estes nemátodos. Figura 4 mostra um perfil de solo que indica a profundidade a que devem ser recolhidas amostras. Preservar a umidade de uma amostra é um fator chave para a sobrevivência ...

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Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Nematóides entomopatogênicos são vermes que vivem no solo que parasitam uma grande variedade de insetos. Demonstramos métodos de amostragem utilizados para o isolamento destes nematóides do solo utilizando duas técnicas: a iscagem inseto ea armadilha Branco modificado, para a recuperação de nematóides de amostras de solo e cadáveres de insetos infectados, respectivamente.

Amostragem do solo e isolamento de nematóides entomopatogênicos (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)

24.7K visualizações.  University of Arizona.  Nematóides entomopatogênicos são vermes que vivem no solo que parasitam uma grande variedade de insetos. Demonstramos métodos de amostragem utilizados para o isolamento destes nematóides do solo utilizando duas técnicas: a iscagem inseto ea armadilha Branco modificado, para a recuperação de nematóides de amostras de solo e cadáveres de insetos infectados, respectivamente. 

Vídeo: 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.

Projeto e operação de uma contínua<sup&gt; 13</sup&gt; C e<sup&gt; 15</sup&gt; N Labeling Câmara de uniforme ou Diferencial, metabólica e estrutural, Fábrica de Marcação por Isótopo

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

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.

Integrado Campo Lysimetry e Porewater Amostragem para Avaliação de Química Mobilidade em Solos e Vegetação Fundada

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.

Uma técnica de enriquecimento Aperfeiçoado para o isolamento de<em&gt; Arthrobacter</em&gt; Bacteriophage Espécie de amostra de solo Isolados

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.

Ensaiar comportamentos alimentares predadores em<em&gt; Pristionchus</em&gt; E outros nematóides

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.

A Tabela Gusseted Thermogradient para controlar a temperatura do solo para avaliar o crescimento da planta e monitoramento de processos do solo

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.

Medição e mapeamento de padrões de erosão e deposição relacionadas às concentrações de carbonato de solo sob gestão 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 irrigação de efluentes na condutividade hidráulica do solo: acoplado a amostragem de campo e laboratório determinação da condutividade 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.

Isolamento e análise de comunidades microbianas na rizosfera, solo e raízes em experiências de grama perene

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 principais para ampliação de modelos topográficos para mapear a redistribuição de terra e solo de carbono orgânico

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.

Estimativa do Teor de Celulose Cristalina de Biomassa Vegetal utilizando o 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 ...