Bu protokollerin birçok iyileştirme katkıları için Sam-Kyu Kim, Kathryn Plichta ve Victoria Miranda-Thompson: Yazarlar Stock laboratuar geçmiş üyelerine teşekkür etmek istiyorum. Biz Ulusal Bilim Vakfı desteği kabul SP Stoku (hibe IOS-0840932 ve IOS-0724978).

1.. Toprak Örnek Toplama <ol><li> Her örnekleme site için 4 m 2 - 2 minimum alanı kapsayan düşünün. </li><li> En az 15 cm derinlikte (Şekil 4) de toprak örnekleri toplamak. </li><li> Bu alanda en az 5 rastgele numune almak. </li><li> Örnek başına 3 Alt-numûnelerin al. , Çalışmanın amacı bağlı numûnelerin birleştirmek veya onları ayrı tutmak ya. </li><li> Bir plastik torba içinde her örnek yerleştirin. Numunelerin sızmasını (Şekil 4) önlemek için çift torbalama düşünün. </li><li> Su geçirmez bir işaretleyici ile etiket örnekleri. Vb (yerellik / alan / site adı ve GPS koordinat bilgileri dahil) Site bilgileri, tarih, habitat, ilişkili bitki örtüsü, sıcaklık, yükseklik,: Her örnekleme sitesinden aşağıdaki bilgileri içerir NOT: Varsa, böcekler ya da sonraki tanımlama için numune toplanan diğer omurgasız varlığını toplamak ve / veya kaydedin. Bunlar potansiyel natur temsil edebilirarkadaşları EPN ana. </li><li> Bol su ile yıkama ve / veya% 70 etanol ya da% 0.5 &#39;lik ağartıcı çözeltisi ile dezenfekte edilmesi ile örnekler arasında temiz toplama araçları. </li><li> Laboratuara taşınması sırasında - (15 ° C 8) bir soğutucu örnekleri tutun. </li><li> Toprak yapısı, doku, nem, elektrik iletkenliği veya diğer istenen toprak parametreleri hakkında bilgi elde etmek için analiz için toprak numunesinin bir kısmını al. </li></ol> . Toprak Örneklerinin 2 Nematod İzolasyon: Böcek-kızdırma Tekniği <ol><li> Herhangi bir enkaz kaldırmak (yani., Kayalar, vb ahşap veya ağaç kabuğu, yaprak, parçaları.) Saprobik mikroorganizma ile kirlenmesini önlemek için örnekleri ile toplanır. </li><li> Toprak nemlendirmek ve nematod hareketini kolaylaştırmak için su ilave edilir. Not: yavaşça toprağın nem seviyesini artırmak için bir sprey şişesi kullanın. </li><li> Bir kapak ile temiz bir plastik kap içinde nemli toprağın yaklaşık 200-250 ml yerleştirin. </li><li> Böcek yemler Ekle. Her bir numunenin toprak yüzeyine (Şekil 5) 5-10 böcekler düşünün. </li><li> Bir kapakla örtün ve kabı ters kapları çevirin. </li><li> (- 25 ° C, genellikle 22), karanlıkta ve oda sıcaklığında muhafaza kapları. </li><li> 3 gün ve ölü böcekler çıkarın - her 2 kapları atın. NOT: toprak numunesinin daha fazla kızdırma için her kaba ilave sağlıklı böcekleri ekleyin. </li><li> Kaplardan ölü böcekler çıkarın. NOT: Koyu mor kadavra kırmızı tuğla heterorhabditids tarafından parazitlenmiş oysa bir kahverengi ya da koyu sarı renk ile Cadavers genellikle steinernematids tarafından asalaklanmış edilir. </li><li> Steril su içinde kadavra durulayın. </li><li> (Aşağıdaki prosedüre bakınız) nematod döl yeniden elde edilmesi için bir modifiye edilmiş beyaz kapanında kadavra yerleştirin. </li></ol> . Enfekte Disseksiyon 3 Nematod Kurtarma: Modifiye Beyaz Trap <ol><li> 50 (veya 60) mm çap üst yerleştirin. Daha büyük bir tabak (100 mm) iç petri. </li><li> Bir günah Setküçük bir tabak içinde gle dairesel filtre kağıdı (Whatman # 1). </li><li> Emin kadavra yapma küçük çanak filtre kağıdının üzerine yerleştirin kadavra herhangi bir bulaşmayı önlemek için birbirlerine dokunmayın. </li><li> Ca ile dış (daha büyük) Petri çanağı doldurun. Steril damıtılmış su, 20 ml. Kadavra tutan çanak su katmayın. </li><li> Büyük Petri kabı ve kapağı ile içeriği (Şekil 6) kapsamaktadır. </li><li> Buna göre yemekler etiketleyin. Nematod adı (tür / izole), enfeksiyon tarih (tarih enfeksiyon kuruldu) ve tuzak tarih (bu tuzak kurmak tarihidir): Aşağıdaki bilgileri ekleyin. </li><li> Enfektif genç safhalardan (IJs) ortaya çıkışı oluşana kadar oda sıcaklığında tuzak tutun. Nematod türlerinin ve / veya kabul gerginlik bağlı olarak 25 gün - Bu işlem 10 arasında sürebilir. </li><li> Tuzak büyük çanak kaldırılması ve bir behere nematod ile su dökerek IJs ile hasat su. </li><li> Nematodlar b dibine süzün izin verEaker. Bu işlem birkaç dakika sürebilir. </li><li> Emin nematodlar beher dibinde kalacak yapma, dikkatli bir şekilde su dökün. </li><li> Daha fazla su ilave edilir ve nematod boşaltmak için izin vererek nematodları durulayın. Su temiz olana kadar 3 kez - Bu adım 2 tekrar edilebilir. </li><li> Bir doku kültür şişesine (250 mi) içinde süspansiyon nematod yerleştirin. 3000 nematod / ml - süspansiyonun konsantrasyonu 1.000 tutun. </li><li> C. ° 20 - soğuk bir odada veya inkübatör nematod süspansiyon ile mağaza şişesi 10 arasında NOT: EPN raf ömrü değişkendir periyodik olarak saklanan şişeler kontrol edin. Heterorhabditids daha periyodik olarak check-up gerektirebilir oysa, alt kültür gerek olmadan 12 ay - Ortalama steinernematids 6 saklanabilir. </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>

Çıkar çatışması ilan etti.

Bu gösteri genellikle EPN ve simbiyotik bakteri eğitim için kullanılan tekniklerin bir dizi tanımlamak iki sunumlar ilk. Bu teknikler laboratuvarda EPN kültürlerin başarılı kurulması için önemli adımlar temsil ve aynı zamanda araştırma için bir model organizma EPN kullanır diğer biyoanalizlere için temelini oluşturur. EPN Çeşitli zemin etütleri onların bolluk ve dağıtım mevsim, habitatlar ve coğrafi bölgelere 4, 5,9,15 arasında farklılık olduğunu...

Birçok nematod böcekler ile ilişkili, ve ev sahibi ilişkileri yararlı gelen zararlı değişir edilir. Burada, aynı zamanda entomopatojenik nematod (veya EPN) olarak bilinen böcek patojen nematodlara, bir grup üzerinde odaklanır. Steinernematidae ve Heterorhabditidae 11,13: İki nematod aile nematod bu gruba aittir. Onların patojenik etkisi fakültatif anaerobik enterik bakteriler (Wenorhabdus bakteri heterorhabditids ile ilişkilendirmek steinernematids ve Fotorhabdus bakteri ile ilişkilendirmek) 2 ile etkileşimi tarafından kazandırılan aslında. Bakteri toprakta yaşayan tek serbest yaşayan nematod aşamasında, (ayrıca Dauer yavru, enfektif yavru veya IJ olarak da bilinir) üçüncü aşama enfektif yavru, tek böceğin diğerine vectored edilir. Bir kez böcek içinde, nematodlar büyük septisemi ile böcek ev sahibi öldüren, böceğin hemolimf içine bakteri bırakın. Ayrıca, bakterilerböceğin dokularını aşağılamak ve onları olgun ve çoğalmaya izin nematodların &#39;besin kaynağı olur. Genellikle, yetişkin nematod bir veya iki kuşak böcek kadavranın içinde üretilmektedir. Birkaç bakteri hücreleri ile son nesil yetişkin yeniden ilişkilendirir soy, daha önceki beslenme ilişki daha şekilde uzmanlaşmış ve onlar parasitize başka böcek bekleyecek toprağa böcek karkas dışarı hareket olarak bağırsaklarında bu hücreleri beslemek 6,11,14 (Şekil 1). Onlar tarım zararlıları 3,4,5 olan böceklerin geniş bir yelpazede parasitize gibi 1927 yılında keşfedilmesinden bu yana, EPN&#39;dir kimyasal pestisitlerin değerli alternatifleri kabul edilmiştir. Ancak, son iki yılda, bu nematodlar ve simbiyotik bakterilerin konak-mikrop ETKİLEŞİMİ biyolojik, ekolojik ve evrimsel yönlerini incelemek için uysal bir model sistem olarak kabul edilmiştirns 14. Bu sunumun amacı yöntemleri ve en yaygın toprak örneklemesi ve EPN izolasyonu için kullanılan teknikleri göstermektir. Çeşitli araçlar mevcuttur ve dahil olmak üzere toprak örnekleri toplamak için kullanılabilir: Toprak corers, mala, post-delik kazma, burgular, örnekleme tüpleri, el kürek, diğerleri arasında (Şekil 2). Çalışmanın amacına bağlı olarak, iki örnekleme stratejileri olarak kabul edilebilir: a) tabakalı, b) rastgele örnekleme (Şekil 3). Tabakalı örnekleme genellikle belirli bir zaman süresi boyunca belirli bir ya da demarked alanda yoğun bir çalışmanın bir parçası olarak kullanılır. Genel olarak, bir transect demarked ve toprak örnekleri belirli bir süre (Şekil 3A) üzerinde 15 transect belirtilen aralıklarla alınır. Geniş bir alanda odaklanarak zaman rastgele örnekleme genellikle kullanılır. Bu numune alma stratejisi EPN f çeşitliliğini incelemek için kullanılmıştırgeniş bir coğrafi alana veya bölge (Şekil 3B) 12 rom. Bir dizi faktör yüksekliklerde, toprak dokular ve habitatları (örn.., Ekili alanlar, ormanlar, meralar, parklar, kıyılarda, nehir kıyısı alanları, vb) çeşitli bir yelpazede de dahil olmak üzere çalışmanın odak bağlı olarak kabul edilebilir. Başlangıçta yatak ve Akhurst 1 tarafından tarif edildiği ve toprak numunelerinden EPN geri kazanılması için basit ve seçici bir alternatif temsil eder böcek yem tekniği göstermektedir. Bu nematod (IJ aşama) bir böceğin çekti ve onu parasitize olacağını öncül dayalı bir seçici işlemdir. Bu teknik aynı zamanda, diğer böcek mantar gibi patojenlerin ve bakterilerin izolasyonu için 8,10 olarak kabul edilebilir. Ayrıca enfekte olmuş böcek ana 7,14 ikinci nematod döl almak için kullanılan modifiye edilmiş beyaz tuzak tekniği göstermektedir. Bu araya bir zahmetsizaşağılayıcı böcek kadavra enkaz ücretsiz &#39;temiz&#39; bir nematod döl alma avantajı sunuyor hod. Son olarak, tarif edilen ve bu makalede gösterilen teknikler tasarlanmış ve / veya laboratuarda yanı sıra çeşitli arkadaşları ve arkadaşları tarafından tarif edilenlere gerçekleştirilen edilenlere tekabül eder.

<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)

Entomopatojenik nematodlar (aka EPN) böceklerin geniş bir yelpazede parasitize toprakta yaşayan nematod bir grubu temsil eder. Steinernematidae ve Heterorhabditidae: Bu nematod iki aileye aittir. Şimdiye kadar, 70&#39;in üzerinde tür Steinernematidae tarif edilmiştir ve yaklaşık 20 tür Heterorhabditidae de vardır. Nematodlar Enterobacteriaceae bakteri ile bir ortaklık mutualistik ve birlikte böcek türlerinin geniş bir yelpazede öldüren kuvvetli bir insektisid kompleksi olarak hareket ederler. Bu yazıda, topraktan EPN toplamak için düşünülen en yaygın teknikler üzerinde durulacak. Bu sunumun ikinci bölümü böcek-kızdırma tekniği, toprak örneklerinden EPN izolasyonu için yaygın olarak kullanılan bir yaklaşım, ve enfekte olmuş böcekler bu nematod kurtarma için kullanılan modifiye Beyaz tuzak tekniği üzerinde duruluyor. Bu yöntemler ve teknikler EPN kültünün başarılı kurulması için önemli adımlarLaboratuvarda ayrıca ures diğer biyolojik disiplinlerde araştırma için bir model organizma bu nematod dikkate diğer biyoanalizlere için temelini oluşturur. Bu sunumda gösterilen teknikler SP Stock laboratuar üyelerinin yanı sıra, çeşitli yazarlar tarafından tarif edilenler ile gerçekleştirilebilir ve / ya da tasarlanmış karşılık gelir.

Toprak Örnek Toplama EPN Üçüncü aşama enfektif gençler toprakta (Şekil 1) ikamet bu Nematodlar &#39;yaşam döngüsü içinde sadece özgür yaşam evreleri vardır. Bu nedenle toprak örnekleri toplayarak bu nematod kurtarmak için çok etkili bir yöntemdir. 4 örnek alınması gereken hangi derinliğini gösteren bir toprak profili göstermektedir. Bir numunenin nem korunması nematodların hayatta kalmak için önemli bir faktör...

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

Entomopatojenik nematodlar böcek geniş bir yelpazede parasitize toprakta yaşayan yuvarlak solucan bulunmaktadır. Sırasıyla toprak örneklerinin ve enfekte olmuş böcek kadavra, nematod geri kazanılması için böcek kızdırma ve modifiye edilmiş beyaz tuzak: iki teknikler kullanılarak topraktan bu nematod izolasyonu için kullanılan örnekleme yöntemlerini göstermektedir.

Toprak Örnekleme ve Entomopatojenik Nematodlar İzolasyonu (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 Görüntüleme.  University of Arizona.  Entomopatojenik nematodlar böcek geniş bir yelpazede parasitize toprakta yaşayan yuvarlak solucan bulunmaktadır. Sırasıyla toprak örneklerinin ve enfekte olmuş böcek kadavra, nematod geri kazanılması için böcek kızdırma ve modifiye edilmiş beyaz tuzak: iki teknikler kullanılarak topraktan bu nematod izolasyonu için kullanılan örnekleme yöntemlerini göstermektedir. 

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.

Sürekli Tasarımı ve İşletilmesi<sup&gt; 13</sup&gt; C ve<sup&gt; 15</supÜniformalı veya Diferansiyel için&gt; N Etiketleme Odası, Metabolik ve Yapısal, Bitki İzotop Etiketleme

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

Çevre

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.

Entegre Saha Lysimetry ve gözenek suyu Örnekleme Topraklarda Kimyasal Hareketlilik Değerlendirilmesi ve kurulan Bitki Örtüsü

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.

Geliştirilmesi<em&gt; Metarhizium anisopliae</em&gt; Bir Mycoinsecticide Olarak: İzolasyondan Saha Performansına

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 ve in vitro Entomopatojenik Nematodlar Yetiştiriciliğinin olarak (Steinernematidae ve Heterohabditidae)

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

Biyomühendislik

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.

Baermann Huni tarafından Vahşi Nematodların Hızlı İzolasyonu

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

Genetik

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.

Entomopatojenik Nematodların Kültürlenmesi ve Genetik Olarak Manipüle Edilmesi

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

İmmünoloji ve Enfeksiyon

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.

Toprak Örneklerinden İntomopatojenik Mantarların İzolasyonu ve Seçimi ve Böcek Zararlılarına Karşı Mantar Virülansının Değerlendirilmesi

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

Biyoloji

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).

Entomopatojenik mantarları toprak örneklerinden izole etme yöntemlerinin karşılaştırılması

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.

Kendinden Çıkan Caenorhabditis Nematodlarının Ekolojik Araştırmalarını Gerçekleştirmek için Yüksek Oranda Ölçeklenebilir Bir Yaklaşım

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.

Model Nematod Caenorhabditis elegans'ın Doğal Mikrobiyotasının İzolasyonu ve Karakterizasyonu

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

Toprak Örnekleme ve Entomopatojenik Nematodlar İzolasyonu (Steinernematidae, Heterorhabditidae)

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