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 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)

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 ...

soil-sampling-isolation-entomopathogenic-nematodes-steinernematidae

Research

JoVE Journal

Environment

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

24.7K 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 ...

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.

Yalıtım için bir optimize Zenginleştirme Tekniği<em&gt; Arthrobacter</em&gt; Bakteriyofaj Türlerinin Toprak Numune İzolatlarının gelen

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.

içinde Yırtıcı Beslenme Davranışları tahlil<em&gt; Pristionchus</em&gt; Ve Diğer Nematodlar

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.

Bir Körüklü Thermogradient Tablo Toprak Süreçleri Bitki Büyüme değerlendirilmesi ve İzleme Toprak Sıcaklıklar Denetim

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.

Ölçme ve toprak erozyon ve toprak karbonat konsantrasyonları tarım Yönetimi'nin altında ilgili ifade eşleme

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.

Atıksu Sulaması toprak Hidrolik iletkenlik üzerindeki etkileri: alan örnekleme ve laboratuvar doymuş Hidrolik İletkenlik Tayini

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.

Yalıtım ve mikrobiyal topluluklarda toprak, rizosferde ve kökleri çok yıllık çim deneylerde Analizi

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.

Toprak yeniden dağıtım ve toprak organik karbon eşlemek için topografik modellerini ölçekleme için asıl bileşenlerinin kullanımı

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.

Updegraff Yöntemi Kullanılarak Bitki Biyokütlesinin Kristal Selüloz İçeriğinin Tahmini

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 ...