Gli autori desiderano ringraziare i membri passati della Fotografico laboratorio: Sam-Kyu Kim, Kathryn Plichta e Victoria Miranda-Thompson per il loro contributo al miglioramento di molti di questi protocolli. Noi riconosciamo il sostegno della National Science Foundation (sovvenzioni IOS-0840932 e IOS-0.724.978) a SP Fotografici.

1. Soil Sample Collection <ol><li> Si consideri che copre una superficie minima di 2-4 m 2 per ogni sito di campionamento. </li><li> Raccogliere campioni di terreno a una profondità di almeno 15 cm (Figura 4). </li><li> Prendere almeno 5 campioni casuali all&#39;interno di questa area. </li><li> Prendere 3 sottocampioni per campione. Seconda dell&#39;obiettivo dello studio, sia combinare sottocampioni o mantenerli separati. </li><li> Collocare ogni campione in un sacchetto di plastica. Considerare doppio insaccamento per evitare perdite di campioni (Figura 4). </li><li> Campioni etichetta con un pennarello indelebile. Includere le seguenti informazioni da ogni sito di campionamento: Informazioni sul sito (compresi località / zona / nome e GPS informazioni coordinare posto), la data, l&#39;habitat, la vegetazione associata, temperatura, altitudine, ecc NOTA: Se necessario, raccogliere e / o registrare la presenza di insetti o altri invertebrati raccolti con il campione per la successiva identificazione. Essi possono rappresentare potenziali naturpadroni di casa al EPN. </li><li> Strumenti di raccolta Pulire accuratamente tra i campioni di lavaggio con acqua e / o disinfezione con il 70% di etanolo o soluzione di candeggina allo 0,5%. </li><li> I campioni in un dispositivo di raffreddamento (8 - 15 ° C) durante il trasporto al laboratorio. </li><li> Prendete una porzione del campione di suolo per l&#39;analisi per ottenere informazioni sulla composizione del suolo, tessitura, umidità, conducibilità elettrica o di altri parametri pedologici desiderati. </li></ol> . 2 Isolamento Nematode da campioni di suolo: Tecnica Insect-baiting <ol><li> Rimuovere eventuali detriti (per esempio., Pietre, pezzi di legno o di corteccia, foglie, ecc.) Raccolti con i vostri campioni per evitare la contaminazione con microrganismi saprobico. </li><li> Aggiungere acqua per inumidire il terreno e agevolare la circolazione dei nematodi. NOTA: Utilizzare un flacone spray per aumentare lentamente il livello di umidità del suolo. </li><li> Posizionare circa 200 a 250 ml di terreno umido in un contenitore di plastica pulito con un coperchio. </li><li> Aggiungi esche insetti. Considerare 5 a 10 insetti alla superficie del suolo di ciascun campione (Figura 5). </li><li> Coprire il contenitore con un coperchio e girare contenitori a testa in giù. </li><li> Mantenere contenitori in un luogo buio a RT (solito, la 22 - 25 ° C). </li><li> Controllare contenitori ogni 2-3 giorni, e rimuovere gli insetti morti. NOTA: aggiungere altri insetti sani per ogni contenitore per un ulteriore adescamento del campione di terreno. </li><li> Rimuovere gli insetti morti dai contenitori. NOTA: Cadavers con un marrone o ocra colorazione di solito sono parassitati da steinernematids, mentre il rosso di cadaveri viola scuro mattoni sono parassitato da heterorhabditids. </li><li> Sciacquare cadaveri in acqua sterile. </li><li> Mettere cadaveri in una trappola bianco modificato per il recupero del nematode progenie (vedi procedura riportata di seguito). </li></ol> 3. Nematode Recovery da cadaveri infetti: Modificato Trappola bianca <ol><li> Posizionare la parte superiore di un diametro di 50 (o 60) mm. Capsula Petri all&#39;interno di una parabola più grande (100 mm). </li><li> Impostare un peccatocarta da filtro circolare gle (Whatman # 1) all&#39;interno del piatto più piccolo. </li><li> Posizionare cadaveri sulla carta da filtro del piatto più piccolo assicurandosi cadaveri non si toccano a vicenda per evitare qualsiasi contaminazione. </li><li> Riempire l&#39;esterno (grande) Scatola di Petri con ca. 20 ml di acqua distillata sterile. Non aggiungere acqua nel piatto che contiene i cadaveri. </li><li> Coprire il grande piatto Petri e il suo contenuto con il coperchio (Figura 6). </li><li> Etichettare i piatti di conseguenza. Aggiungere le seguenti informazioni: nome Nematode (specie / isolato), la data di infezione (infezione data è stata fissata) e quella trappola (questa è la data della trappola è impostato). </li><li> Tenere trappola a RT finché non si verifica emergere di stadi giovanili infettive (IJ). Questo processo può richiedere tra i 10 - 25 giorni a seconda delle specie e / o varietà considerata nematodi. </li><li> Acqua raccolta con IJs rimuovendo il più grande piatto della trappola e versando acqua con nematodi in un bicchiere. </li><li> Consentire nematodi decantare sul fondo della bEaker. Questo processo può richiedere alcuni minuti. </li><li> Versare acqua accuratamente, assicurandosi nematodi rimangono nel fondo del bicchiere. </li><li> Risciacquare nematodi con l&#39;aggiunta di più acqua e permettendo nematodi a decantare. Questa fase può essere ripetuta 2 - 3 volte finché l&#39;acqua è pulita. </li><li> Posizionare sospensione nematode in un pallone di coltura tissutale (250 ml). Mantenere la concentrazione della sospensione di 1.000 - 3.000 nematodi / ml. </li><li> Boccetta Store con sospensione nematode in una stanza fredda o in incubatrice tra i 10 - 20 ° C. NOTA: controllare boccette memorizzati periodicamente durata di EPN è variabile. Di solito steinernematids possono essere conservati per 6-12 mesi senza il bisogno di subcoltura, che heterorhabditids possono richiedere più controlli periodici. </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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Questa manifestazione è la prima delle due presentazioni che descrivono una serie di tecniche che sono comunemente usati per lo studio EPN e dei loro batteri simbionti. Queste tecniche rappresentano passaggi fondamentali per la creazione di successo delle culture EPN in laboratorio e costituiscono anche la base per altri biotest che impiegano EPN come organismi modello per la ricerca. Vari sondaggi del terreno di EPN hanno dimostrato che la loro abbondanza e la distribuzione può variare tr...

Molti nematodi sono associati con gli insetti, e le loro interazioni ospite variano da vantaggioso per dannosa. Qui, ci concentriamo su un gruppo di nematodi che sono patogeni di insetti, noto anche come nematodi entomopatogeni (o EPN). Due famiglie di nematodi appartengono a questo gruppo di nematodi: Steinernematidae e Heterorhabditidae 11,13. Il loro effetto patogeno è infatti conferita dalla loro interazione con anaerobi facoltativi batteri enterici (batteri Xenorhabdus associano con steinernematids e batteri Photorhabdus associare a heterorhabditids) 2. I batteri sono vectored da un host ad un altro insetto da parte l&#39;unica tappa libero nematode vita, il giovane infettivo terzo stadio (noto anche come dauer giovanile, giovanile infettiva o IJ), che vive nel terreno. Una volta dentro l&#39;insetto, i nematodi rilasciano i batteri nella emolinfa dell&#39;insetto, che uccidono l&#39;insetto ospite da una massiccia setticemia. I batteri anchedegradare i tessuti dell&#39;insetto e diventano fonte di cibo dei nematodi &#39;, consentendo loro di maturare e moltiplicarsi. Di solito, una o due generazioni di nematodi adulti sono prodotte all&#39;interno del cadavere insetto. La progenie degli ultimi riassocia generazione adulta con poche cellule batteriche in più specializzarsi modo che il loro precedente rapporto nutrizionale, e coltivare queste cellule nei loro intestini mentre si muovono fuori dalla carcassa insetti nel terreno dove potranno attendere un altro insetto di parassitare 6,11,14 (Figura 1). Sin dalla loro scoperta nel 1927, EPN sono stati considerati importanti alternative ai pesticidi chimici in quanto possono parassitare una vasta gamma di insetti che sono parassiti agricoli 3,4,5. Tuttavia, negli ultimi due decenni, questi nematodi e dei loro batteri simbionti sono stati riconosciuti come sistema modello malleabile per studiare aspetti biologici, ecologici ed evolutivi di ospite-microbo interactions 14. L&#39;obiettivo di questa presentazione è quello di mostrare i metodi e le tecniche più comunemente utilizzate per il campionamento del suolo e l&#39;isolamento di EPN. Una varietà di strumenti è disponibile e può essere utilizzato per raccogliere campioni di terreno, tra cui: defittonatrici suolo, cazzuole, trivelle, trivelle, tubi di campionamento, pale a mano, tra gli altri (Figura 2). A seconda dello scopo dello studio, due strategie di campionamento possono essere considerati: a) stratificato, b) campionamento casuale (Figura 3). Il campionamento stratificato è solitamente utilizzato come parte di uno studio intensivo in una zona specifica o demarked in un determinato periodo di tempo. In generale, un transetto è demarked e campioni di suolo sono presi a intervalli stabiliti nel transetto per un periodo di tempo (Figura 3A) 15. Il campionamento casuale viene generalmente impiegato quando concentrandosi su una vasta area. Questa strategia di campionamento è stato utilizzato per studiare la diversità di EPN fROM una vasta area geografica o regione (Figura 3B) 12. Una serie di fattori può essere considerato in funzione del focus dello studio tra cui una vasta gamma di altezze, texture del suolo e degli habitat (ad es., Campi coltivati, boschi, pascoli, parchi, spiagge, aree ripariali, ecc.) Mostriamo la tecnica dell&#39;insetto adescamento, che è stato originariamente descritto da biancheria da letto e Akhurst 1 e rappresenta un semplice e selettivo alternativa per il recupero EPN da campioni di suolo. Questa è una procedura selettiva che si basa sul presupposto che i nematodi (fase IJ) saranno attratti a un host insetto e parassitano esso. Questa tecnica può anche essere considerato per l&#39;isolamento di altri patogeni insetti quali funghi e batteri 8,10. Abbiamo anche dimostrare la tecnica trappola bianco modificato, che viene utilizzato per il recupero nematode progenie di insetti infetti padroni di casa 7,14. Si tratta di un sforzo incontratohod che offre il vantaggio di recuperare un &#39;clean&#39; nematode progenie priva di detriti dai cadaveri di insetti degradanti. Infine, le tecniche descritte ed illustrate in questo articolo corrispondono a quelli concepiti e / o eseguite nel nostro laboratorio così come quelli descritti da vari colleghi e collaboratori.

<table><tbody><tr><td>&lt;em&gt;Galleria mellonella&lt;/em&gt;</td><td>Timberline</td><td>http://www.timberlinefisheries.com/ProductDetails.asp?ProductCode=WAXLG</td><td>For insect baiting technique</td></tr><tr><td>Filter paper, 55 mm Grade 1 Cellulose</td><td>Whatman/VWR</td><td>28450-048</td><td>Infection chamber and White trap assembly</td></tr><tr><td>60 x 15 mm Petri dishes</td><td>VWR</td><td>25384-092</td><td>Infection chamber and White trap assembly</td></tr><tr><td>100 x 15 mm Petri dishes</td><td>VWR</td><td>25384-088</td><td>Infection chamber and White trap assembly</td></tr><tr><td>ZIPloc plastic bags</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Mechanical pipettor P1000, P200 ml</td><td>VWR</td><td>89130-562; 89130-566&amp;nbsp;</td><td>Infection chamber and White trap assembly</td></tr><tr><td>Pippetor tips 1000ml, 200 ml</td><td>VWR</td><td>16466-004 ; 53510</td><td>Infection chamber and White trap assembly</td></tr><tr><td>Bleach</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling , disinfecting</td></tr><tr><td>Sodium hypochlorite</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling , disinfecting</td></tr><tr><td>70% Ethyl alochol</td><td>AAPER</td><td>17212945</td><td>Soil sampling , disinfecting</td></tr><tr><td>Shovels</td><td>Ace Hardware or local hardware store</td><td>N/A</td><td>Soil sampling</td></tr><tr><td>Tubular soil sampler</td><td>Accuproducts</td><td></td><td>Soil sampling</td></tr><tr><td>Soil probe, long handle</td><td>Nupla&amp;nbsp;</td><td>PRB4T 69401 Classic</td><td>Soil sampling</td></tr><tr><td>Measuring tape</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Flagging tape, various colors</td><td>Ace Hardware or local hardware store</td><td></td><td>Soil sampling</td></tr><tr><td>Tissue culture flasks</td><td>VWR</td><td>Falcon, 29185-304&amp;nbsp;</td><td>White trap, collection of EPN</td></tr><tr><td>Wash bottles, polyethylene, wide mouth</td><td>VWR</td><td>16650-107</td><td>White trap, collection of EPN</td></tr></tbody></table>

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

Nematodi entomopatogeni (aka EPN) rappresentano un gruppo di nematodi del suolo abitare che parassitano una vasta gamma di insetti. Questi nematodi appartengono a due famiglie: Steinernematidae e Heterorhabditidae. Fino ad oggi, più di 70 specie sono state descritte nel Steinernematidae e ci sono circa 20 specie in Heterorhabditidae. I nematodi hanno una collaborazione mutualistica con i batteri Enterobacteriaceae e insieme agiscono come un complesso potente insetticida che uccide una vasta gamma di specie di insetti. Qui, ci concentriamo sulle tecniche più comuni considerati per la raccolta EPN dal suolo. La seconda parte di questa presentazione si concentra sulla tecnica insetti adescamento, un approccio ampiamente utilizzato per l&#39;isolamento di EPN dai campioni di suolo, e la tecnica trappola bianco modificato che viene utilizzato per il recupero di questi nematodi da insetti infetti. Questi metodi e le tecniche sono passaggi fondamentali per la creazione di successo di culto EPNUres in laboratorio e anche la base per altri testi biologici che considerano questi nematodi come organismi modello per la ricerca in altre discipline biologiche. Le tecniche mostrate in questa presentazione corrispondono a quelle eseguite e / o creazione di membri della SP Archivio laboratorio così come quelli descritti da vari autori.

Soil Sample Collection Terza fase larve infettive di EPN sono le uniche fasi di vita liberi nel ciclo di vita di questi nematodi &#39;, che risiedono nel terreno (Figura 1). Pertanto, la raccolta di campioni di suolo è un metodo molto efficace per il recupero di questi nematodi. Figura 4 mostra un profilo del terreno che indica la profondità alla quale devono essere prelevati campioni. Preservare l&#39;umidità di un campione è un fattore chiave per la so...

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Soil Sampling and Isolation of Entomopathogenic Nematodes Steinernematidae, Heterorhabditidae

Nematodi entomopatogeni sono nematodi del suolo abitare che parassitano una vasta gamma di insetti. Dimostriamo metodi di campionamento utilizzati per l&#39;isolamento di questi nematodi dal terreno utilizzando due tecniche: l&#39;adescamento insetto e la trappola bianco modificato, per il recupero dei nematodi dai campioni di suolo e cadaveri insetti infetti, rispettivamente.

Campionamento e Isolamento di Nematodi entomopatogeni Suolo (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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Soil Sampling and Isolation of Entomopathogenic Nematodes (Steinernematidae, Heterorhabditidae)

24.9K Views.  University of Arizona.  Nematodi entomopatogeni sono nematodi del suolo abitare che parassitano una vasta gamma di insetti. Dimostriamo metodi di campionamento utilizzati per l'isolamento di questi nematodi dal terreno utilizzando due tecniche: l'adescamento insetto e la trappola bianco modificato, per il recupero dei nematodi dai campioni di suolo e cadaveri insetti infetti, rispettivamente. 

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.

Progettazione e funzionamento di un continuo<sup&gt; 13</sup&gt; C e<sup&gt; 15</sup&gt; N Etichettatura Camera di Uniform o differenziale, metaboliche e strutturali, Flora Isotope Labeling

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

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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.

Integrato Campo Lysimetry e Porewater campionamento per la valutazione della chimica Mobilità nel suolo e alla vegetazione Fondata

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

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

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

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

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

Sviluppo di<em&gt; Metarhizium anisopliae</em&gt; Come micosintetizzatore: dall&#39;isolamento al campo

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

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

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

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

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

In vivo e in vitro Allevamento di Nematodi entomopatogeni (Steinernematidae e Heterorhabditidae)

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

Bioingegneria

Rapid Isolation of Wild Nematodes by Baermann Funnel

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

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

Rapido isolamento dei nematodi selvatici di Baermann Funnel

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

Genetica

Culturing and Genetically Manipulating Entomopathogenic Nematodes

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

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

Coltivare e manipolare geneticamente i nematodi entomopatogeni

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

Immunologia e infezioni

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

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

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

Isolamento e selezione di funghi entomopatogeni da campioni di suolo e valutazione della virulenza fungina contro gli insetti nocivi

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

Biologia

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

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

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

Confronto dei metodi per isolare i funghi entomopatogeni da campioni di suolo

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

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

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

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

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

Un approccio altamente scalabile per eseguire indagini ecologiche sui nematodi della caenorhabdite selfing

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

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

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

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

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

Isolamento e caratterizzazione del microbiota naturale del nematode modello Caenorhabditis elegans

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

Campionamento e Isolamento di Nematodi entomopatogeni Suolo (Steinernematidae, Heterorhabditidae)

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