Name: establishing fungal entomopathogens as endophytes: towards endophytic biological control
Uploaded: 2013-04-11T13:00:00
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生产和实验工作提出的雷纳尔多·帕雷哈本文反映的虔诚和热情帮助。资助哥伦比亚的行政主管部门的科学，技术和创新（COLCIENCIAS）和通过“探索大挑战”主动比尔和梅林达·盖茨基金会的资助。

1。植物<ol><li>表面消毒豆种子（栽培品种Calima）浸渍两分钟在0.5％的次氯酸钠和两分钟，在70％的乙醇。在无菌蒸馏水冲洗种子三次。 </li><li>评估您的灭菌100微升的最后的漂洗水镀上的马铃薯dextroxe琼脂（PDA）媒体，和在25温育10天的板的成功℃。终止并重​​新启动这个实验，如果有任何增长被认为是上盘。 </li><li>三个一组的植物的种子，在花盆中含有无菌土壤和沙子的混合物以2:1的比率。盆转移至在25℃下的生长室中，约。 50％RH和12小时光照。一个星期后发芽，消除至少有两个壮苗。水，每2-3天用无菌蒸馏水和6 g / L的水溶液，NPK 15-15-15肥料种植，施肥后10至20天。 </li></ol> 2。菌<ol><li>获取市售的CIAL制定的球孢白僵菌菌株GHA（SE Mycotrol，Laverlam，卡利，哥伦比亚）。 </li><li>要生成一个单孢子培养物，暂停约。一个充满分生孢子接种环在1毫升的0.1％的Triton-X 100的水溶液和涡流，持续10秒。然后，板100μL的悬浮2.5％高尚的琼脂孵育24小时，在25°C。分生孢子到100毫米的板，包含PDA传输单一的发芽和成长，直到它涵盖了整个板块（约3-4周）。 </li><li>在无菌条件下，刮从介质表面的真菌的生长，并在10ml无菌的0.1％的Triton-X 100暂停。涡旋1分钟。然后，暂停通过无菌纱布过滤除去菌丝体和获得股票停牌。 </li><li>使用血球计数仪，估计孢子浓度股票停牌。为了方便孢子的数量，准备了10000倍稀释的股票，每次转移100微升Øf 900微升0.1％的Triton X-100的分生孢子悬浮液中，并涡旋10秒之前，下一个稀释。 </li><li>要生成的接种物，调节的纸浆悬浮液的最终浓度为10 8个分生孢子/毫升，使用下面的公式： <img src="/files/ftp_upload/50360/50360eq1.jpg" alt="式（1）" fo:src="/files/ftp_upload/50360/50360eq1highres.jpg"/></li><li>分生孢子的可行性评估，板100微升2.5％高尚的琼脂孵育24小时的10000倍稀释，在25°C。然后，检查三个随机组100分生孢子萌发率估计。考虑时发芽分生孢子长于可见芽管从它的分生孢子项目的直径的一半。仅使用暂停时的平均萌发率超过90％。 </li></ol> 3。接种<ol><li>接种到植物，当他们到达的第一真叶期（约播种后14天）。用无菌水植物对土壤的能力提炼编辑水前24小时接种。 </li><li>对于叶面喷洒的方法，使用的一本手册雾化器应用的孢子悬浮液（处理）或0.1％的Triton-X 100（对照）的近轴（上）表面的叶子，直到它们达到饱和。用铝箔盖顶的锅，以避免孢子径流的土壤。喷药后，用塑料袋24小时保持较高的湿度水平，促进霉菌侵袭的地被植物。 </li><li>对于土壤浸润法，使用量筒申请10毫升的分生孢子悬浮液（处理）或0.1％的Triton X-100（对照）在植物底部的土壤表面。 </li><li>接种后，返回植物生长室，安排他们在一个完全随机区组设计。不低于4个附加的实验块应安装允许评价植物的生长，除了内生细菌定植的评价，在同一实验中。 </li></ol> 4。评估uations <ol><li>评估实验中，一个块的时间，选择的块以随机顺序。这是特别重要的，不能评价的单日为大型实验。 </li><li>处理植物之前，测量并记录其高度从基座到顶端分生组织。然后，铲除和自来水彻底清洗。 </li><li>从每一个工厂，样品两个瓣叶，根和两片，两片干。选择单张随机抽样，从第一片真叶的植物。然后，获取两个干样品，3厘米长的每一个，从中间的植物，从附近的土壤表面。最后，获得两个主根样品，也3厘米长的每一个，从中间的根和从落后1厘米根尖。将样品在三个不同的文件袋和标签适当。 </li><li>洗涤后，在一个块中的所有的植物和采样，从通过处理叶子，然后根，和最后的茎。 </li><li>表面消毒吨问题在无菌层流罩在1.1以上。三次冲洗每个样品浸泡在无菌蒸馏水中，并让它干燥，无菌巾纸。然后，解剖和丢弃的外边缘，内生真菌可能已被淘汰，由于消毒剂接触。 </li><li>切分为六个部分，剪裁后的样品平均6x6毫米的叶和6毫米长的茎和根。板60毫米培养皿用PDA培养基中于2 mg / L的每个与抗生素四环素，链霉素和青霉素的六个部分。用封口膜密封该板，并在黑暗中孵育在25℃下每一种植物产生六大板块，每两个工厂的一部分。 </li><li>改变一个给定的植物部分的每个块的处理后的冲洗水。在丢弃用过的漂洗水，板100微升样品PDA培养基上，并培育10天，在25℃下，以评估灭菌成功。如果随之而来的真菌生长，不考虑相应的样品进行分析。 </li><li>检查板，每2-3天，20天，观察并记录真菌的生长。新鲜的PDA平板消费税和转让植物部分参展内生真菌的存在。这将避免在原板的相邻的工厂部分的污染。 </li><li>记录B.可以识别的特征白森森的菌丝体成为边缘浅黄色，奶油色到工厂部分。 球孢白僵菌孢白僵菌增长。当有疑问时，安装试样中的一滴水，并在显微镜下检查，寻找的球形分生孢子和锯齿形分生孢子梗，特征的品种。 </li><li>使用额外的实验块的处理对植物生物量的影响进行评估。首先，测量它们的高度从底部到顶部的顶端分生组织。然后，小心地根除和洗植物在自来水中，并让它们在45℃干燥三天，以确定其干重。 </li></ol>

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(15), e683 (2008).</li><li>Parsa, S., Sotelo, G., Cardona, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+herbivore+resistance+mechanisms:+spittlebugs+on+Brachiaria+spp.+as+an+example.">Characterizing herbivore resistance mechanisms: spittlebugs on Brachiaria spp. as an example.</a> J. Vis. Exp. (52), e3047 (2011).</li><li>Atamian, H., Roberts, P., Kaloshian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+and+low+throughput+screens+with+root-knot+nematodes+Meloidogyne+spp.">High and low throughput screens with root-knot nematodes Meloidogyne spp.</a> J. Vis. Exp. (61), e3629 (2012).</li></ol>

作者宣称，他们没有竞争的金融利益。

很多因素可以影响的实验，建立了内生菌的真菌昆虫病原微生物的具体结果。我们的结果表明，接种方法是其中之一。实验生物因素包括作物种类和品种选择和真菌昆虫病原微生物物种应变或隔离使用。等因素来考虑操纵包括接种物的浓度，植物在接种的年龄，和植物的生长条件。 这将是理想的接种的方法导致全身厂殖民由真菌昆虫病原微生物14,17,18,21。相反，它似乎接种方法往往是一个特定的模式有利于当地的殖民统治。例如，在咖啡，叶面喷洒青睐叶定植，而土壤浸液青睐根定植17。我们发现了同样的模式在菜豆。最终，接种方法的选择应该是指导工厂内的内生真菌的预定位置，大概匹配的目标草食动物或植物病原真菌的利基。 虽然常用，内生菌的检测和定量分析的基础上的媒体文化可以是昂贵的，困难和容易出错的。例如在一个实验中，共10800工厂部分（镀1800培养皿上）进行了评价，以优化B。白僵菌接种香蕉16。在这些被殖民统治，4496节的假定B.球孢白僵菌 ，确定主要是通过菌落形态。显然，一个微小的验证各殖民地的物种将是一个可取的，但买不起的步骤。另一方面，1176的部分被由其他真菌定植和被丢弃，视为缺少数据16的 。存在的概率，但是，B。白僵菌是一个贫穷的竞争对手或更慢的增长，并可能最终摆脱那些部分，如果llowed足够的时间。因此，根据媒体文化的内生真菌检测方法假阳性和假阴性。因此，发展的更可靠的检测和定量的方法，例如那些基于聚合酶链反应（PCR）检测20,21,24，以及合理的。 接种实验的最终目标应该是发展对草食动物和/或疾病的有效治疗方法，可提供持久的全身抵抗力。一个可行的，但目前还未经检验的假设是内生殖民的程度与内生真菌介导的耐药程度呈正相关。因此，一个自然的下一步改进接种方法后，可以检查此相关。一些视频协议可以帮助研究人员设计一个合适的电阻检测的靶标害虫和病原体29日至31日 。最终，正是这种分析将决定成功的接种TION方法，以及相关的潜在的内生的生物控制。

，真菌entomopathogens的重要调节昆虫种群具有相当的潜力mycopesticides 1。然而，只是在最近，有真菌entomopathogens被证实发生作为内生真菌，自然和响应于各种接种方法2。生态功能内生真菌entomopathogens的仍然是未知，但一些研究已经牵连他们在草食动物电阻5-8，3,4，植物生长和抗病9,10。这里介绍的方法的总体目标是介绍一种真菌的内生真菌，昆虫病原微生物的内生生物控制做好后续评估的准备工作。 球孢白僵菌 （尔萨莫）Vullemin“（子囊菌Hypocreales）是最好的学习内生真菌昆虫病原微生物5-9,11-19，和它是作为一个商业mycopesticide提供的。接种方法测试，以建立<eM&gt; B。作为一个内生真菌球孢白僵菌包括的土壤淋14,17，种子包衣18和浸泡14，13,15胚根敷料，根及根茎浸泡11,16,18，叶面喷洒干注射17，14,17,20和花喷雾剂19。使用这些方法，研究人员已经推出B.球孢白僵菌 11日 ，入香蕉，咖啡，可可豆7 13 17 7，玉米，棉7，椰枣12 21，黄麻，罂粟20 7，南瓜，辐射松18，高粱14 7，番茄和小麦7。最近的证据表明，内生的B.孢白僵菌具有的潜力，以保护植物不仅从节肢动物害虫5-7,22-27，但也从某些植物病原体9。 菜豆（ 四季豆 ）跻身最容易受到病虫害的作物和疾病。它可能会受到400多名害虫和200个病原体，其攻击被认为是最重要的限制豆生产要素跨区域的28。因此，菜豆可能是一个很好的模式作物，全方位的内生生物控制B.检查球孢白僵菌 。作为第一步，在这个方向，本文介绍了叶面喷施和土壤淋接种方法介绍B.bassiana作为一个内生真菌在菜豆。

<table border="1">
<tbody>
	<tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 	<td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td></td>
 </tr>
 <tr>
 	<td>Mycotrol SE	</td>
 <td>Laverlam</td>
 <td>4167</td>
 <td></td>
 </tr>
 <tr>
 	<td>Noble agar</td>
 <td>Sigma</td>
 <td>A5431-250G</td>
 <td></td>
 </tr>
 <tr>
 	<td>Penicillin G sodium salt</td>
 <td>Sigma</td>
 <td>P3032-25MU</td>
 <td></td>
 </tr>
 <tr>
 	<td>Petri dish (100 x 15 mm)</td>
 <td>Fisher</td>
 <td>08-757-12</td>
 <td></td>
 </tr>
 <tr>
 	<td>Petri dish (60 x 15 mm)</td>
 <td>Fisher</td>
 <td>08-757-13A</td>
 <td></td>
 </tr>
 <tr>
 	<td>Potato dextrose agar</td>
 <td>Difco</td>
 <td>213400</td>
 <td></td>
 </tr>
 <tr>
 	<td>Regular bleach (NaOCl)</td>
 <td>CLOROX</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 	<td>Streptomycin sulfate salt</td>
 <td>Sigma</td>
 <td>S6501-25G</td>
 <td></td>
 </tr>
 <tr>
 	<td>Tetracycline</td>
 <td>Sigma</td>
 <td>T3258-25G</td>
 <td></td>
 </tr>
 <tr>
 	<td>Triple quince (NPK)</td>
 <td>ABOCOL</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 	<td>Triton X-100</td>
 <td>Sigma</td>
 <td>X-100</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 	<td>Biological safety cabinet</td>
 <td>NuAire</td>
 <td>NU-425-600</td>
 <td></td>
 </tr>
 <tr>
 	<td>Hemocytometer</td>
 <td>Fisher</td>
 <td>02-671-10 </td>
 <td></td>
 </tr>
 <tr>
 	<td>Leica DM LB microscope</td>
 <td>Leica</td>
 <td>N/A</td>
 <td></td>
 </tr>
 </tbody>
</table>

establishing fungal entomopathogens as endophytes: towards endophytic biological control

白僵菌是一种真菌昆虫病原微生物的能力拓殖植物endophytically。作为一个内生菌，B.白僵菌可能发挥的作用，在保护植物，草食动物和疾病。该协议演示了两种接种方法，建立B.白僵菌 endophytically菜豆（ 四季豆 ），内生生物控制做好后续评估的准备工作。两个星期前收到B.从表面消毒的种子植物生长球孢白僵菌 10 8个分生孢子/毫升（或水）的治疗应用，无论是作为叶面喷施或土壤浸润。两个星期后，采收和取样，以评估他们的叶，茎和根内生真菌。对于这一点，将样品分别进行表面灭菌，切成多个部分，并在马铃薯葡萄糖琼脂培养基上培养20天。媒体检查，每2-3天观察真菌的生长，工厂部分相关记录发生的B.白僵菌估计其内生殖民化的程度。接种成功的比较分析发生B.球孢白僵菌在一个给定的植物部分（ 即叶，茎或根）在治疗和控制。除了接种方法，具体的实验结果可取决于目标作物物种或品种，真菌昆虫病原微生物物种应变或隔离使用，和植物的生长条件。

球孢白僵菌能endophytically殖民P.寻常响应于证明接种处理（ 图1）。叶面喷施和土壤淋导致内生的殖民统治B.孢白僵菌在超过80％的处理过的植物中（ 图2）。然而，定植程度取决于评价的植物部分和所使用的接种方法。叶最好的喷雾接种。的根，在另一方面，回应只淋接种。最后，茎回应同样都接种方法。孢白僵菌的对照植物的任何部分中未检测出。 独立的治疗方法，内生以外B.白僵菌增长15％的工厂部分的评估，但他们从介质板进行解剖，才可以侵入邻近的区域和影响的结果。 治疗组和对照植株明显区分两个星期后接种。无显着差异在其干重，并在它们的高度进行检测。 <img src="/files/ftp_upload/50360/50360fig1.jpg" alt="图1"/> 图1。代表豆类植物（ 菜豆品种。Calima） 白僵菌内生殖民接种处理结果。左上：控板没有增长。右上图：从工厂部分内生真菌污染整个板块。左下：B.球孢白僵菌生长的两个工厂的部分。右下图：内生B.球孢白僵菌孢子和分生孢子在显微镜下看到。 <img src="/files/ftp_upload/50360/50360fig2.jpg" alt="图2" fo:content-width="4.5in" fo:src="/files/ftp_upload/50360/50360fig2highres.jpg"/> 图2。东亚银行的内生殖民的影响接种处理N植物（ 菜豆品种。Calima）。定殖率与应变GHA接种球孢白僵菌 ，两个星期后的殖民工厂部分除以培养的部分由数量。

Watch this Scientific Journal Video about Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control at JoVE.com

Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control

该协议说明了两个的接种方法引入真菌昆虫病原微生物<em&gt;球孢白僵菌</em作为内生真菌在菜豆（<em&gt;菜豆</em&gt;），内生生物控制的后续​​评估的准备工作。

建立真菌Entomopathogens作为内生菌：建立内生生物控制

 Beauveria bassiana is a fungal entomopathogen with the ability to colonize plants endophytically. As an endophyte, B. bassiana may play a role in ...

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