これらのプロトコルの多くの改善に貢献し、サム·ギュキム、キャサリンPlichtaビクトリアミランダ·トンプソン：著者は、在庫ラボの過去のメンバーに感謝したいと思います。我々は、SP株式に（補助金のIOS-0840932およびIOS-0724978）国立科学財団の支援を認める。

1。土壌サンプルの採取<ol><li>各サンプリングサイトは4m 2 - 2の最小面積をカバーすることを検討してください。 </li><li>少なくとも15cmの深さ（ 図4）での土壌サンプルを収集します。 </li><li>この領域内に少なくとも5のランダムサンプルを取る。 </li><li>サンプルあたり3サブサンプルを取る。研究の目的は、サブサンプルを組み合わせて、または別のそれらを保つのどちらかによって異なります。 </li><li>ビニール袋に各サンプルを置きます。サンプル（ 図4）の漏れを回避するために二重の袋詰めを考えてみましょう。 </li><li>防水マーカーでラベルサンプル。各サンプリングサイトから以下の情報が含まれる：（ローカリティ/エリア/サイト名やGPSなどの座標情報）サイト情報を、日付、生息地、関連する植生、温度、標高など 注：必要に応じて、その後の識別のための試料を用いて収集した昆虫や他の無脊椎動物の存在を収集し、および/または記録します。彼らは潜在的な漢方を表すことができアルEPNホスト。 </li><li>水で徹底的に洗浄および/または70％エタノールまたは0.5％の漂白剤溶液でそれらを消毒することにより、サンプル間のきれいな収集ツール。 </li><li>研究室への輸送中に - （15°C 8）クーラー内のサンプルを保管してください。 </li><li>土壌の組成、質感、水分、導電性又は他の所望の土壌パラメータに関する情報を得るために分析用の土壌サンプルの一部を取る。 </li></ol> 。土壌サンプルからの2線虫の分離：昆虫苦しませるテクニック<ol><li>ごみを取り除き（ すなわち 、岩などの木材や樹皮、葉の部分。）腐生の微生物による汚染を避けるために、サンプルを用いて収集した。 </li><li>土を湿らせて、線虫の移動を容易にするために水を加える。 NOTE：ゆっくりと土壌の水分レベルを増加させるためにスプレーボトルを使用する。 </li><li>蓋付きの清潔なプラス​​チック容器に湿った土壌の約200〜250ミリリットルを配置します。 </li><li>昆虫の餌を追​​加。各サンプル（ 図5）の土壌表面に5から10昆虫を検討してください。 </li><li>蓋付きの容器をカバーし、上下逆にコンテナを回す。 </li><li> （ - 25°Cの通常22）暗闇の中で、RTでコンテナを維持する。 </li><li> 3日、死んだ虫を削除 - すべての2の容器を確認してください。 注：土壌サンプルをさらに苦しませるために、各コンテナに追加の健全な昆虫を追加します。 </li><li>容器から死んだ虫を取り除く。注：濃い紫色の死体に赤レンガをheterorhabditidsによって寄生されたのに対し、茶色や黄土色に着色した死体は、通常、steine​​rnematidsによって寄生されています。 </li><li>滅菌水に死体を洗浄します。 </li><li> （下記の手順を参照）、線虫の子孫の回収のために変更されたホワイトトラップに死体を置く。 </li></ol>感染した死体から3センチュウ回復：変更されたホワイト罠<ol><li> 50（または60）ミリメートル直径の上に置きます。大きなお皿（100ミリメートル）の内部ペトリ皿。 </li><li> 1罪を設定小さい皿の内側GLE円形の濾紙（ワットマン＃1）。 </li><li>必ず死体を作るより小さな皿のフィルター紙の上に置いて、死体は汚染を避けるために、お互いに手を触れないでください。 </li><li> CAで外側（大きい）ペトリ皿を埋める。滅菌蒸留水20ml。死体を保持している皿に水を追加しないでください。 </li><li>蓋（ 図6）を持つ大規模なシャーレとその内容をカバーしています。 </li><li>それに応じてお料理にラベルを付けます。線虫の名前（種/分離株）、感染日付（日付感染が設定された）とトラップの日付（これはトラップが設定されている日付です）：以下の情報を追加します。 </li><li>感染幼虫期（IJS）の出現が起こるまで、室温でトラップしておいてください。線虫種および/または考慮さ菌株によって25日 - このプロセスは、10の間に取ることができます。 </li><li>トラップの大きなお皿を取り出し、ビーカーに線虫で水を注ぐことによってIJSで収穫水。 </li><li>線虫は、Bの底にデカントすることができますエーカー。このプロセスには数分かかる場合があります。 </li><li>必ず線虫は、ビーカーの底に残っていること、丁寧に水を注ぐ。 </li><li>より多くの水を追加し、線虫をデカントできるようにすることで、線虫を洗浄します。水がきれいになるまで3回 - このステップでは、2を繰り返すことができます。 </li><li>組織培養フラスコ（250ml）に線虫サスペンションを配置します。 3000線虫/ mlに - 1000懸濁液の濃度を保つ。 </li><li> 20℃ - 低温室で、10間のインキュベーター内で線虫懸濁液と店のフラスコ 注：EPNの保存期間が可変であるように、定期的に保存されたフラスコを確認してください。 heterorhabditidsは、より定期的な検診が必要な場合があり、一方、継代培養を必要とせずに、12ヶ月 - 通常steine​​rnematids 6のために保存することができます。 </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>

利害の対立が宣言されていません。

このデモでは、一般的に、EPNとその共生細菌を研究するために使用されている多くの技術を説明する2つのプレゼンテーションの最初のものです。これらの技術は、実験室でのEPN培養の成功確立のための重要なステップを表し、また、研究のためのモデル生物として、EPNを採用し、他のバイオアッセイのための基礎を形成する。 EPNの様々な土壌調査では、その豊かさと分...

多くの線虫、昆虫に関連付けられ、それらの宿主の相互作用が有益から有害な範囲である。ここに、我々はまた、昆虫病原性線虫（あるいはEPN）として知られている昆虫の病原体である線虫のグループに焦点を当てる。 SteinernematidaeとHeterorhabditidae 11,13：二つの線虫の家族が線虫のこのグループに属している。その病原性効果は、実際には通性嫌気性の腸内細菌との相互作用によって付与される（ ラブダス細菌がheterorhabditids関連付けるsteinernematidsとのPhotorhabdus細菌に関連付ける）2。細菌は土壌中に住んでいる唯一の自由生活線虫ステージによって別の昆虫宿主（またdauerの少年、感染少年やIJとして知られている）、3段目の感染性若年性からベクトル化されています。かつて昆虫の中に、線虫は、大規模な敗血症による昆虫宿主を殺す昆虫の血リンパに細菌を放出する。また、細菌昆虫の組織を分解し、それらが成熟し増殖することができ、線虫 &#39;食料源となる。通常、成体線虫の1つまたは2つの世代の昆虫死体内に生成される。より以前の栄養の関係よりも、やり方を専門とし、彼らが寄生する他の昆虫を待ちます土に虫の死骸から外に移動するので、その腸内にこれらの細胞を育てる中で、いくつかの細菌細胞との最後の大人の世代の再アソシエートの子孫6,11,14（ 図1）。 彼らは、農業害虫3,4,5である昆虫の広い範囲に寄生することができるように1927年に発見されているので、EPNは化学農薬への貴重な選択肢と考えられてきた。しかし、過去20年間、これらの線虫とその共生細菌は、宿主微生物の生物生態や進化の側面を研究するための可鍛性のモデル系として認識されているinteractioNS 14。 このプレゼンテーションの目的は、方法及び最も一般的に土壌のサンプリングとEPNの単離のために使用される技術を示すことである。さまざまなツールが利用可能で、含む土壌試料収集するために使用することができます。土壌の芯抜き、こて、ポストホールディガー、オーガー、サンプリングチューブ、ハンドシャベル、とりわけ（ 図2）。 研究の目的に応じて、2つのサンプリング戦略を考慮することができる：a）層状と、b）ランダムサンプリング（ 図3）。層化サンプリングは、通常、所与の期間にわたって特定の又はdemarked領域における集中的な研究の一部として使用される。一般に、トランセクトはdemarkedされ、土壌サンプルを、一定期間（ 図3A）15上トランセクトに規定の間隔で採取する。広範な領域に着目すると、ランダムサンプリングが一般的である。このサンプリング戦略は、EPNのfの多様性を研究するために使用されているROM広い地域または地域（ 図3B）12。多くの要因は、標高、土壌のテクスチャや生息地の多様な範囲（ 例えば 、耕地、森林、牧草地、公園、海岸、水辺エリアなど）を含めた研究の焦点に応じて考えることができる。 私たちは、もともと寝具、Akhurst 1で説明した土壌サンプルからEPNを回復するための簡単かつ選択的な代替手段を表した昆虫苦しませる手法を示している。これは、線虫（IJステージ）昆虫宿主に引き付けられ、それに寄生されることを前提としている選択的な手順である。この技術はまた、真菌、および細菌8,10のような他の昆虫の病原体の単離のために考慮することができる。 我々はまた、感染した昆虫のホスト7,14から線虫の子孫を検索するために使用される修正されたホワイトトラップ技術を実証する。これが満たさ簡単です劣化昆虫死体からの不要な物を「クリーンな」線虫の子孫を取得する利点を提供していますHOD。 最後に、この記事で説明して示した技術が考案および/または、私たちの研究室だけでなく、様々な同僚や協力者によって記載されているもので行われるものに対応する。

<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)

昆虫病原性線虫（別名EPN）は昆虫の広い範囲に寄生する土壌に生息する線虫の基を表す。 Steine​​rnematidaeとHeterorhabditidae：これらの線虫は二つの家族に属しています。今まで、70以上の種がSteine​​rnematidaeに記載されており、Heterorhabditidae約20種が存在する。線虫は、腸内細菌と共生のパートナーシップを持っており、彼らは一緒に昆虫種の広い範囲を殺す強力な殺虫複合体として機能します。 ここに、私たちは土壌から、EPNを収集するために考慮される最も一般的な技術に焦点を当てています。このプレゼンテーションの後半では、昆虫苦しませる技術、土壌サンプルからEPNを単離するために広く使用されているアプローチ、および感染した昆虫から、これらの線虫の回復のために使用されている修正されたホワイトトラップ技術に焦点を当てています。これらの方法および技術は、EPNカルトの成功確立のための重要なステップであるURES研究室でも、他の生物学の分野での研究のためのモデル生物として、これらの線虫を考え、他のバイオアッセイのための基礎を形成する。このプレゼンテーションに示す手法が実施され、/またはSPストック研究室のメンバーだけでなく、様々な著者が記載されているものが設計したものに相当する。

土壌サンプルの採取 EPNの3段目の感染少年は、土壌（ 図1）に存在するこれらの線虫のライフサイクルの中で唯一の自由生活のステージです。したがって、土壌サンプルを採取することは、これらの線虫から回復するための非常に効率的な方法である。 図4は、サンプルを採取する位置の深さを示す土壌断面を示している。サンプルの水分を維持?...

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

昆虫病原性線虫、昆虫の広い範囲に寄生する土壌に生息する回虫です。それぞれ、土壌サンプル、感染昆虫死体から線虫の回復のために、昆虫苦しませると変更されたホワイトトラップ：我々は2つ​​の技術を使用した土壌からこれらの線虫を分離するために使用されるサンプリング方法を示しています。

土壌のサンプリングと昆虫病原線虫の分離（Steine​​rnematidae、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 ビュー.  University of Arizona. 昆虫病原性線虫、昆虫の広い範囲に寄生する土壌に生息する回虫です。それぞれ、土壌サンプル、感染昆虫死体から線虫の回復のために、昆虫苦しませると変更されたホワイトトラップ：我々は2つ​​の技術を使用した土壌からこれらの線虫を分離するために使用されるサンプリング方法を示しています。 

ビデオ: 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.

連続の設計および動作<sup&gt; 13</sup&gt; Cと<sup&gt; 15</sup均一または差動、代謝および構造·&gt; N標識化会議所、工場同位体ラベル化

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

環境学

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.

統合フィールドLysimetryと間隙水土壌中の化学物質の移動性の評価のためのサンプリングと設立植生

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.

の単離のための最適化された濃縮技術<em&gt;アルスロ</em&gt;バクテリオファージ種土壌サンプルの分離株から

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.

で捕食摂食行動を分析します<em&gt; Pristionchus</em&gt;およびその他の線虫

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.

マチ付きThermogradient表は、植物の成長を評価し、土壌プロセスの監視のための土壌の温度を制御します

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.

測定と土壌侵食と堆積土炭酸濃度農業経営に関連のパターンのマッピング

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.

土壌透水排水灌漑への影響: 結合フィールド サンプリングと飽和透水係数の実験室測定

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.

実験で多年生の草の根、根、土壌微生物の分離と解析

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.

主成分の土壌の再分配と土壌の有機炭素をマップに地形モデルをスケール アップ用

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法による植物バイオマスの結晶セルロース含有量の推定

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 ...

土壌のサンプリングと昆虫病原線虫の分離（Steine​​rnematidae、Heterorhabditidae）

土壌のサンプリングと昆虫病原線虫の分離（Steinernematidae、Heterorhabditidae）