作者感谢苏珊·威菲尔, 准备的数字和卡蜜尔马丁和希拉里 Samagaio 帮助一些实验。

<ol>
	<li>Heaton, J. C., Jones, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+contamination+of+fruit+and+vegetables+and+the+behaviour+of+enteropathogens+in+the+phyllosphere:+a+review.">Microbial contamination of fruit and vegetables and the behaviour of enteropathogens in the phyllosphere: a review.</a> J. Appl. Microbiol. 104, 613-626 (2008).</li><li>Beuchat, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+factors+influencing+survival+and+growth+of+human+pathogens+on+raw+fruit+and+vegetables.">Ecological factors influencing survival and growth of human pathogens on raw fruit and vegetables.</a> Microbes Infect. 4, 413-423 (2002).</li><li>Berger, C. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharides+cellulose,+poly-beta-1,6-n-acetyl-D-glucosamine,+and+colanic+acid+are+required+for+optimal+binding+of+Escherichia+coli+O157:H7+strains+to+alfalfa+sprouts+and+K-12+strains+to+plastic+but+not+for+binding+to+epithelial+cells.">Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells.</a> Appl. Environ. Microbiol. 74, 2384-2390 (2008).</li><li>Matthysse, A. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+medium+promotes+the+attachment+of+Agrobacterium+tumefaciens.strain+NT1+to+carrot+cells.">Conditioned medium promotes the attachment of Agrobacterium tumefaciens.strain NT1 to carrot cells.</a> Protoplasma. 183, 131-136 (1994).</li><li>Matthysse, A. G., McMahan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+Agrobacterium+tumefaciens.+attR+mutation+on+attachment+and+root+colonization+differs+between+legumes+and+other+dicots.">The effect of the Agrobacterium tumefaciens. attR mutation on attachment and root colonization differs between legumes and other dicots.</a> Appl. Environ. Microbiol. 67, 1070-1075 (2001).</li><li>Jeter, C., Matthysse, A. 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R., Fuqua, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+analyses+of+Agrobacterium.">Phenotypic analyses of Agrobacterium.</a> Curr. Protoc. Microbiol. , (2012).</li><li>Brandl, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+lesions+promote+the+rapid+multiplication+of+Escherichia+coli+O157:H7+on+postharvest+lettuce.">Plant lesions promote the rapid multiplication of Escherichia coli O157:H7 on postharvest lettuce.</a> Appl. Environ. 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撰文人宣称她没有竞争的经济利益。

重要的是要知道所有的表面, 细菌可以坚持在实验中。因此, 如果能用玻璃管和吸管来完成可行的细胞计数, 就可以低估能够与玻璃结合的细菌。如果植物生长在琼脂或土壤中, 一些琼脂或土壤留在植物上, 那么细菌就会粘附在附着物上, 而不是植物。另一方面, 洗涤植物表面, 特别是在根的情况下, 可能去除天然表面涂层如粘液, 从而改变粘附试验的结果。
重要的是要确定, 添加到孵化混合物中的细菌在实验中仍然活着。因而可生存细胞计数自由并且附有的细菌应该通常被做。一些治疗或细菌突变可能会降低细菌的生长速度, 或者实际上导致部分细菌数量的死亡。除非使用特殊污渍, 否则活体和死细菌在显微镜下可能无法区分。有一个有用的染色试剂盒的活/死细菌, 这取决于排除染料的活菌。然而, 如果有混合的细菌种类的人口存在, 那么可行的细胞计数的兴趣物种可能是最简单的方法来确定孵化是否导致细菌死亡。
培养基组成会影响细菌的生存和生长。根渗出物和从伤口和切割部位释放的物质将提供基质以支持适度的细菌生长。在这些条件下, 磷酸盐、氮和铁往往会受到限制。价阳离子如钙和镁可能影响黏附力。在某些情况下, 碳源会影响粘附。pH 值也很重要。一般来说, 根际的 pH 值介于5.5 和6.5 之间。
必须小心使用有抗生素耐药性的细菌。最常用的抗生素是利福平和萘啶酮酸。对这些抗生素的抗药性通常是由于染色体基因 (RNA 聚合酶和回旋酶) 的突变, 因此在孵化过程中不易转移到另一株。这种耐药性也不会导致抗生素的降解或改性。除非质粒不能转移到任何其他细菌, 否则不推荐用质粒传播的基因标记标记细菌。抗生素耐药性不应归因于抗生素的降解或化学改性, 因为抗生素敏感的细菌如果靠近耐药性细菌, 就能在抗生素板上生长。
本文所描述的方法对于小样本大小和/或实验中需要包含样本 (例如, 涉及人类病原体的实验) 非常有用。对于大样本大小 (超过100克的材料或超过50种植物), 其他方法或对这些方法的剧烈修改将需要10,19,32,19,33,34,35,36. 除了正在研究的物种以外, 存在大量的微生物也会造成重大问题。可能的解决办法包括使用标记有荧光蛋白或耐药性的细菌, 如步骤6.1.2 和7.3 所述。然而, 当有兴趣的细菌是稀有的个体在大量的其他微生物中, 这些标记可能不足以允许对所研究的细菌数量进行明确的评估。
这里描述的所有方法都是基于实验室的方法。温室研究需要稍加修改。对于那些原生动物、昆虫和其他牲畜捕食和气候变化使实验条件的提供复杂化的实地研究, 可能需要进行更多的重大修改。在未来, 这些方法可以扩展到包括两个或更多的微生物在植物表面的相互作用。

在三种不同的情况下, 对植物表面的细菌结合的测量变得非常重要。第一种情况是检查人类病原体在植物表面的传播1,2,3。这里的目标是防止细菌结合, 或清除或杀死绑定细菌, 从而减少疾病传播的植物材料。第二种情况是检查植物病原体对植物表面的约束力4。再次, 这里的目标是防止捆绑或删除或杀死绑定细菌, 从而减少疾病。第三种情况是对共生或植物生长促进细菌的结合的检查5,6。这里的目标是促进细菌的结合, 从而提高植物的健康和作物产量。
本文所描述的对植物表面细菌结合的测量技术是廉价的, 相对容易进行。唯一的要求是显微镜和材料一般发现在细菌学实验室。对于一些技术, 浴 sonicator 是有用的。所描述的技术是为使用相对较小的样本尺寸进行的结合实验设计的。对装订的测量是在实验室中进行的, 尽管可能可以修改其中的一些技术用于温室或田间使用。
这些技术被用来测量细菌结合到根, 芽, 切叶, 切水果, 和完整的樱桃西红柿在实验室7,8,9,10,11, 12,13,14,15。它们也被用来测量实验室16中生长在土壤或沙子中的植物的根系定植。该技术已应用于多种细菌种类, 包括农杆菌、根瘤菌苜蓿、大肠杆菌、沙门氏菌 enterica和铜绿假单胞菌荧光。在莫顿和福库 (2012)17中, 可以找到一个有用的描述方法来评估一个.在所有情况下, 所涉及的样本大小是小的, 一般少于 25-50 植物。所描述的技术适用于人类病原体, 需要在实验中加以保存。

<table border="0" cellpadding="0" cellspacing="0" style="width:731px;" width="731">
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	</colgroup>
	<tbody>
		<tr height="56">
			<td height="56" style="height:56px;width:143px;">light microscope</td>
			<td style="width:164px;">any</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;">phase contrast or Nomarski optics are helpful, for studies using fluorescent markers a fluorescence microscope is required.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">seeds</td>
			<td style="width:164px;">any&#160;</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;">make a note of the seed lot number and the cultivar</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">bleach</td>
			<td style="width:164px;">any</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">bath sonicator</td>
			<td style="width:164px;">any scientific supply company</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">Triton X-100</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">T9284</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">nutrient agar&#160;</td>
			<td style="width:164px;">Difco&#160;</td>
			<td style="width:125px;">001-01-8</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">soytone</td>
			<td style="width:164px;">Difco&#160;</td>
			<td style="width:125px;">24360</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">Sand</td>
			<td style="width:164px;">sea sand Fisher Scientific</td>
			<td style="width:125px;">S25</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">sand</td>
			<td style="width:164px;">&#160;Sigma-Aldrich</td>
			<td style="width:125px;">S9887</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">conetainers</td>
			<td style="width:164px;">Stuewe &#38; Sons, Inc.</td>
			<td style="width:125px;">Ray-Leach cone-tainers</td>
			<td style="width:299px;">many different sizes are available to suit the type of plant you wish to grow</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">parafilm</td>
			<td style="width:164px;">any scientific supply company</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">MS salts</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">M5524</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">parrafin</td>
			<td style="width:164px;">any</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">centrigfuge</td>
			<td style="width:164px;">any scientific company</td>
			<td style="width:125px;">N/A</td>
			<td style="width:299px;">to pellet most bacteri only a small centrifuge with a max force of 10,000 XG is needed</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">vortex mixer</td>
			<td style="width:164px;">any scientific company</td>
			<td style="width:125px;"></td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">micrometer</td>
			<td style="width:164px;">ACCU-SCOPE Accessories</td>
			<td style="width:125px;">A3145</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">Sedgwick-rafter counting cell</td>
			<td style="width:164px;">Hauser Scientific</td>
			<td style="width:125px;">HS3800</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">probe-clip press-seal incubatin chamber</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">Z359483</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">rifampicin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">R3501</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">naladixic acid</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:125px;">n8878</td>
			<td style="width:299px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:143px;">Live/dead stain</td>
			<td style="width:164px;">In Vitrogen Molecular Bioprobes</td>
			<td style="width:125px;">L7007</td>
			<td style="width:299px;"></td>
		</tr>
	</tbody>
</table>

adherence of bacteria to plant surfaces measured in the laboratory

本手稿描述了一种方法, 以测量细菌结合无菌植物表面在光显微镜和通过使用可行的细胞计数。所用的植物材料包括根、芽、叶和切果。所描述的方法便宜, 容易, 适合小样本大小。在实验室中测量装订, 并可使用各种孵化介质和条件。可以确定抑制剂的作用。还可以评估促进和抑制绑定的情况。在某些情况下, 有可能区分各种条件是否会因其对植物或细菌的影响而改变约束力。

殖民根表面。为了确定纤维素的细菌生产是否在根系定植中起着重要作用, 研究了防止纤维素合成的细菌突变对16的影响。使用了步骤1.3 和7.1 中描述的技术。番茄种子在无菌水中进行表面杀菌和发芽。当根约2厘米长, 他们被浸泡在悬浮 105细菌每毫升和种植在巴氏杀菌土中的容器。植物生长了14天在25°c 在 12 h light/12 h 黑暗的周期。在被表明的时间以后植物从容器被去除了。根被洗涤和微气泡在浴 sonicator 去除束缚的细菌。细菌数量是用可行的细胞计数来确定的。图 2显示了两种不同的纤维素-负突变对细菌对番茄根部的殖民能力的影响。虽然一些测量的标准偏差高达0.9 日志10 (这类测量的一个共同问题), 但纤维素-负突变体的束缚的减少显然是明显的, 我们可以得出结论, 细菌生产的纤维素在番茄根系的定植中有助于细菌的生长。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56599/56599fig2.jpg"> 图 2: 由野生类型和纤维素减去突变体的根癌.日志10以野生a 型为基础的 C58 和纤维素-减去突变体 C58:1 和 C58:A60, 从番茄根中回收的每 cm 根长度细菌总数。显示的数字是从最少的四个单独实验的手段。条形表示方法的标准偏差。根接种了浸泡在 105细菌每毫升一分钟的悬浮。这些植物是在容器中生长的, 而松散附着的细菌则是通过在玻璃瓶中洗涤缓冲液中的根部来去除的。紧密贴附的细菌被清除使用浴 sonicator 和由此产生的悬浮镀, 以确定可行的细胞计数16。此数字已从 Matthysse 和威廉·达拉戈·麦默恩修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
研究了多糖在大肠杆菌和其他细菌对苜蓿芽中的结合作用。由于大肠杆菌O157:H7, 一些腹泻病的暴发被追踪到受污染的苜蓿芽。用步骤1.1、5.1 和7.2 中描述的方法测量了无法使各种多糖的野生型细菌和突变体的结合。苜蓿芽在不育的水中以25摄氏度在黑暗中被消毒并发芽一天。四芽与附着的种子大衣被放置在无菌的塑料菜肴含有5毫升的水。在 Luria 汤中生长的细菌被添加到最后浓度约 5 x 103每毫升。接种芽在25摄氏度在黑暗中孵化了3天。用马达驱动的聚四氟乙烯玻璃均质机在洗涤缓冲器中进行剧烈倒置和匀质, 在5毫升的无菌水中洗净豆芽两次。以前的实验中使用的细菌标记的质粒携带 GFP 基因显示没有内化细菌, 虽然表面细菌很容易观察到。结果见表 1。检查了两株大肠杆菌O157:H7。在这两种菌株中, poly-β-1,6-glucuronic 酸 (PGA) 的产生对致病性大肠杆菌对植物表面的束缚作用最大。结肠酸在装订中也起着重要作用。虽然纤维素-负突变体中的结合量的减少是显著的, 但它并不像其他两种多糖那么大。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="4">多糖生产基因突变对大肠杆菌 O157:H7 与芽苗和开放种衣结合的影响</td>
		</tr>
<tr>
<td rowspan="2">菌株</td>
			<td rowspan="2">突变或基因型 (相关表型)</td>
			<td colspan="2">记录10每芽或种衣的细菌数量</td>
		</tr>
<tr>
<td>苜蓿芽b
</td>
			<td>打开种子大衣</td>
		</tr>
<tr>
<td>86-24</td>
			<td>无 (野生类型)</td>
			<td>4.7 @ 0。6</td>
			<td>5.6 @ 0。2</td>
		</tr>
<tr>
<td>8624N</td>
			<td>
yhjN(纤维素-减去)</td>
			<td>2.9 @ 0.7c
</td>
			<td>3.5 @ 0.6c
</td>
		</tr>
<tr>
<td>8624C</td>
			<td>
wcaD(结肠酸-减)</td>
			<td>1.8 @ 0.7c
</td>
			<td>2.4 @ 0.5c
</td>
		</tr>
<tr>
<td>8624P</td>
			<td>
pgaC(PGA-减去)</td>
			<td>< 1.0c
</td>
			<td>1.0 @ 1.0c
</td>
		</tr>
<tr>
<td>DEC4A</td>
			<td>无 (野生类型)</td>
			<td>5.6 @ 0。2</td>
			<td>6.1 @ 0。3</td>
		</tr>
<tr>
<td>DEC4AN</td>
			<td>
yhjN(纤维素-减去)</td>
			<td>4.8 ~ 0.8d
</td>
			<td>4.1 ~ 0.8d
</td>
		</tr>
<tr>
<td>DEC4AC</td>
			<td>
wcaD(结肠酸-减)</td>
			<td>3.9 @ 0.5c
</td>
			<td>4.8 ~ 0.8d
</td>
		</tr>
<tr>
<td>DEC4AP</td>
			<td>
pgaC(PGA-减去)</td>
			<td>< 1.0c
</td>
			<td>1.2 @ 0.7c
</td>
		</tr>
<tr>
<td colspan="4">一个平均的标准偏差至少三测量的数量 (日志10) 的细菌绑定3天后。</td>
		</tr>
<tr>
<td colspan="4">b 芽在测量前洗净。</td>
		</tr>
<tr>
<td colspan="4">c 显着不同于野生类型: P < 0.01。</td>
		</tr>
<tr>
<td colspan="4">d 显着不同于野生类型: P < 0.05。</td>
		</tr>
<tr>
<td colspan="4">此表已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。</td>
		</tr>
</tbody></table>表 1: 多糖生产基因突变对其结合性的影响大肠杆菌 O157:H7 芽。为了确定各种多糖和脂多糖对苜蓿芽苗的致病性大肠杆菌O157:H7 菌株的作用, 对一组突变体对芽苗和开皮种衣的约束力进行了研究, 采用了方法步骤6中所述。结果表明, poly-ß-1, 6n-乙酰氨基葡萄糖 (PGA) 似乎是必不可少的绑定到芽, 并认为纤维素和 colanic 酸是必需的最大结合大肠杆菌O157。此表已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。
为了确定 pga 的生产是否足以引起细菌对植物表面的结合, 采用克隆操纵编码的质粒 (pMM11) 将 pga 生产所需的基因引入到两种不同的细菌中, 这将不会通常可以绑定到西红柿根部10。农杆菌A1045 是野生型菌株 C58 的突变体, 不能使 cyclic-β-1,2 葡聚糖, 也不能与植物表面29结合。在苜蓿上形成根结节的根瘤菌苜蓿1021 未与非豆科植物 (包括西红柿12) 结合。步骤1.1、5.1、6.3 和7.1 中描述的技术用于确定是否能够使 PGA 通常增加细菌对根表面的结合。番茄种子在无菌水中进行表面杀菌和发芽。根被切成1厘米长的段, 放在不育的水中, 细菌接种。由于这两种细菌以不同的速率生长, 所以在不同的时间对结合进行了测定, 以允许大致相等的细菌生长量。质粒 pMM11 的存在导致了两种物种的束缚细菌数量的相似显著增加 (表 2)10。在光镜中也看到了捆绑的显著增加, 但这两种物种的结合是非常不同的 (图 3)。 A1045 作为单个细菌被绑定到根表面。苜蓿在大团簇中, 只有少数细菌直接附着在根部, 大多数细菌附着在其他细菌上。这个例子表明, 简单的分析细菌的数量, 而不包括显微观察, 可以给一个误导的印象, 实验结果。虽然两种方法 (可行细胞计数和显微观察) 表明, pMM11 增加了对番茄根系的细菌结合, 但由 PGA 生产所造成的结合类型不同的两个细菌10。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td colspan="3">质粒 pMM11 对番茄根系细菌结合的影响</td>
		</tr>
<tr>
<td>菌株</td>
			<td>质 粒</td>
			<td>每毫米根长度的细菌数量</td>
		</tr>
<tr>
<td>A1045 农杆菌
</td>
			<td>没有</td>
			<td>0.25 x 103 @ 0.25 x 103
</td>
		</tr>
<tr>
<td></td>
			<td>pBBR1mcs (矢量)</td>
			<td>0.25 x 103 @ 0.25 x 103
</td>
		</tr>
<tr>
<td></td>
			<td>pMM11 (PGA 合成)</td>
			<td>10 x 103 @ 0.25 x 103
</td>
		</tr>
<tr>
<td>S. 苜蓿 1021b
</td>
			<td>没有</td>
			<td>未检测到</td>
		</tr>
<tr>
<td></td>
			<td>pBBR1mcs (矢量)</td>
			<td>未检测到</td>
		</tr>
<tr>
<td></td>
			<td>pMM11 (PGA 合成)</td>
			<td>50 x 103 @ 5 x 103
</td>
		</tr>
<tr>
<td colspan="3">2小时后测定细菌结合量</td>
		</tr>
<tr>
<td colspan="3">b. 18 小时后细菌结合量的测定</td>
		</tr>
<tr>
<td colspan="3">此表已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。</td>
		</tr>
</tbody></table>表 2: 质粒携带基因对 PGA 合成的影响 A1045 和苜蓿 1021 到西红柿根段。为了研究 poly-ß-1, 6n-乙酰氨基葡萄糖 (PGA) 的能力, 以促进细菌对植物根系的结合, 一个质粒的影响, 使 PGA (pMM11) 的能力, 对两株植物相关细菌的结合, 以西红柿根被检查。在缺乏质粒或质粒存在的情况下, 两种细菌对番茄根部均无明显的约束力, 而不存在 PGA 合成 (pBBR1mcs) 的基因编码。加入 PGA 合成基因的质粒增加了两种细菌的结合。因为. ................. .........苜蓿后2小时的苜蓿结合量。所使用的技术是步骤7中描述的方法。此表已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56599/56599fig3.jpg"> 图 3: 质粒 pMM11 携带 PGA 生物合成基因对 A1045 和苜蓿1021 至番茄根毛的结合作用。对西红柿根毛的约束力) a.农杆菌 A1045, B) A1045 pMM11, C) s. 苜蓿1021 和 D) s. 苜蓿1021 pMM11。虽然两种细菌的结合度的增加大致相似, 但在光镜中看到的结合的细节却是截然不同的。所使用的技术是步骤6中描述的方法。此数字已从 Matthysse、迪欧、Mishra 和托雷斯10进行了修改。<a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
有时可以使用绑定到非生物表面, 以帮助区分细菌和植物在特定的相互作用的贡献。单极多糖 (UPP) 已被证明能够调解细菌结合到各种生物和非生物表面30。钙被观察, 以抑制约束的一个. 农杆菌的植物表面介导的 UPP28。为了确定细菌结合对植物表面的钙离子的抑制是由于对细菌或在植物表面的作用, 细菌的捆绑对尼龙螺纹被审查了。使用了步骤8.2 中描述的技术。在步骤1中描述的番茄种子表面被杀菌, 在水中发芽。细菌生长在最小的培养基与蔗糖, 并添加到番茄根或尼龙线的最终浓度约 105/毫升, 如步骤5.1 所述。显微镜观察了添加 CaCl2对番茄根系和尼龙丝细菌结合的影响。图 4显示了类似的抑制钙结合使用任何表面表明, 钙的影响主要是在细菌10。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56599/56599fig4.jpg"> 图 4: 钙对农杆菌结合对番茄根毛和尼龙丝的影响.在1:10 稀释 MS 盐和1:20 稀释 AB 最小培养基的情况下, 用西红柿根 (a 和 B) 或尼龙线 (C 和 D) 在24小时内培育了农杆菌; 0.4% 蔗糖 (A 和 C) 或1:10 稀释的 MS 盐和1:20 稀释的 AB 最小培养基, 0.4% 蔗糖含有60毫米 CaCl2 (B 和 D)31。添加的 CaCl2抑制细菌结合的根和尼龙线表明, 抑制主要是由于对细菌的影响, 而不是在植物表面。<a href="https://cloudflare.jove.com/files/ftp_upload/56599/56599fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Adherence of Bacteria to Plant Surfaces Measured in the Laboratory at JoVE.com

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

本文介绍了一种简单的方法来测量和表征细菌对植物的黏附力, 特别是根和芽。

细菌附着在实验室测量的植物表面

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

adherence-of-bacteria-to-plant-surfaces-measured-in-the-laboratory

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12.5K 次观看.  University of North Carolina at Chapel Hill. 本文介绍了一种简单的方法来测量和表征细菌对植物的黏附力, 特别是根和芽。

视频: Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

A New Application of the Electrical Penetration Graph (EPG) for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of electrical transmission between distant cells in an organism requires in vivo, artifact-free recordings of cells embedded within their native environment. The transmission of electrical signals from wounded to unwounded areas in a plant has since long piqued the interest of botanists. The phloem, the living part of the plant vasculature that is spread throughout the plant, has been postulated as a major tissue in electrical transmission in plants. The lack of suitable electrophysiological methods poses many challenges for the study of the electrical properties of the phloem cells in vivo. Here we present a novel approach for intracellular electrophysiology of sieve elements (SEs) that uses living aphids, or other phloem-feeding hemipteran insects, integrated in the electrical penetration graph (EPG) circuit. The versatility, robustness, and accuracy of this method made it possible to record and study in detail the wound-induced electrical signals in SEs of central veins of the model plant Arabidopsis thaliana1. Here we show that EPG-electrodes can be easily implemented for intracellular electrophysiological recordings of SEs in marginal veins, as well as to study the capacity of SEs to respond with electrical signals to several external stimuli. The EPG approach applied to intracellular electrophysiology of SEs can be implemented to a wide variety of plant species, in a large number of plant/insect combinations, and for many research aims.

A New Application of the Electrical Penetration Graph EPG for Acquiring and Measuring Electrical Signals in Phloem Sieve Elements

Electrical Penetration Graph (EPG) is a well-established technique for studying the feeding behavior of stylet-bearing insects. Here we show a new application of EPG as a non-invasive tool for the acquisition of intracellular electrophysiology recordings of sieve elements (SEs), the cells that form the phloem vasculature in plants.

在电气贯穿图（EPG）的韧皮部筛分子获取和测量电信号中的新应用

Electrophysiological properties of cells are often studied in vitro, after dissociating them from their native environments. However, the study of ...

科研

环境科学

Laboratory Simulation of an Iron(II)-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling from hydrothermal sources in the Precambrian ocean was oxidized by molecular oxygen [O2] produced by cyanobacteria. The oldest BIFs, deposited prior to the Great Oxidation Event (GOE) at about 2.4 billion years (Gy) ago, could have formed by direct oxidation of Fe(II) by anoxygenic photoferrotrophs under anoxic conditions. As a method for testing the geochemical and mineralogical patterns that develop under different biological scenarios, we designed a 40 cm long vertical flow-through column to simulate an anoxic Fe(II)-rich marine upwelling system representative of an ancient ocean on a lab scale. The cylinder was packed with a porous glass bead matrix to stabilize the geochemical gradients, and liquid samples for iron quantification could be taken throughout the water column. Dissolved oxygen was detected non-invasively via optodes from the outside. Results from biotic experiments that involved upwelling fluxes of Fe(II) from the bottom, a distinct light gradient from top, and cyanobacteria present in the water column, show clear evidence for the formation of Fe(III) mineral precipitates and development of a chemocline between Fe(II) and O2. This column allows us to test hypotheses for the formation of the BIFs by culturing cyanobacteria (and in the future photoferrotrophs) under simulated marine Precambrian conditions. Furthermore we hypothesize that our column concept allows for the simulation of various chemical and physical environments &#8212; including shallow marine or lacustrine sediments.

Laboratory Simulation of an IronII-rich Precambrian Marine Upwelling System to Explore the Growth of Photosynthetic Bacteria

We simulated a Precambrian ferruginous marine upwelling system in a lab-scale vertical flow-through column. The goal was to understand how geochemical profiles of O2 and Fe(II) evolve as cyanobacteria produce O2. The results show the establishment of a chemocline due to Fe(II) oxidation by photosynthetically produced O2.

一个铁（II）的实验室模拟的前寒武纪富海洋上升流系统探索光合细菌生长

A conventional concept for the deposition of some Precambrian Banded Iron Formations (BIF) proceeds on the assumption that ferrous iron [Fe(II)] upwelling ...

Procedures of Laboratory Fumigation for Pest Control with Nitric Oxide Gas

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on fresh products and procedures for residue analysis and product quality evaluation. An airtight fumigation chamber containing fresh fruit and vegetables is first flushed with nitrogen (N2) to establish an ultralow oxygen (ULO) environment followed by injection of NO. The fumigation chamber is then kept at a low temperature of 2 - 5 &#176;C for a specified time period necessary to kill a target pest to complete a fumigation treatment. At the end of a fumigation treatment, the fumigation chamber is flushed with N2 to dilute NO prior to opening the chamber to ambient air to prevent the reaction between NO and O2, which produces NO2 and may damage delicate fresh products. At different times after NO fumigation, NO2 in headspace and nitrate and nitrite in liquid samples were measured as residues. Product quality was evaluated after 2 weeks of post-treatment cold storage to determine effects of NO fumigation on product quality. Keeping&#160;O2 from reacting with NO is critical to NO fumigation and is an important part of the protocols. Measuring NO levels is challenging and a practical solution is provided. Possible protocol modifications are also suggested for measuring NO levels in the fumigation chambers as well as residues. NO fumigation has the potential to be a practical alternative to methyl bromide fumigation for postharvest pest control on fresh and stored products. This publication is intended to assist other researchers in conducting NO fumigation research for postharvest pest control and accelerating the development of NO fumigation for practical applications.

This paper describes nitric oxide (NO) fumigation protocols for postharvest pest control. Fumigation chambers are flushed with nitrogen (N2) to establish ultralow oxygen conditions before NO is injected. At the end, chambers are flushed with N2 to dilute NO before exposing products to ambient air to prevent exposure to NO2.

一氧化氮气体防治害虫实验室熏蒸程序

Nitric oxide (NO) is a newly discovered fumigant for postharvest pest control. This paper provides detailed protocols for conducting NO fumigation on ...

Experimental Methods of Dust Charging and Mobilization on Surfaces with Exposure to Ultraviolet Radiation or Plasmas

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using three recently developed experiments in which dust particles are exposed to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. The UV light source has a narrow bandwidth in wavelength centered at 172 nm. The beam electrons with the energy of 120 eV are created with a negatively biased hot filament. When the vacuum chamber is filled with the argon gas, a thermal plasma is created in addition to the electron beam. Insulating dust particles of a few tens of microns in diameter are used in the experiments. Dust particles are recorded to be lofted to a height up to a few centimeters with a launch speed up to 1 m/s. These experiments demonstrate that photo and/or secondary electron emission from a dusty surface changes the charging mechanism of dust particles. According to the recently developed &#34;patched charge model&#34;, the emitted electrons can be re-absorbed inside microcavities between neighboring dust particles below the surface, causing the accumulation of enhanced negative charges on the surrounding dust particles. The repulsive forces between these negatively charged particles may be large enough to mobilize and lift them off the surface. These experiments present the advanced understanding of dust charging and transport on dusty surfaces, and laid a foundation for future investigations of its role in the surface evolution of airless planetary bodies.

Dust charging and mobilization is demonstrated in three experiments with exposure to thermal plasma with beam electrons, beam electrons only, or ultraviolet (UV) radiation only. These experiments present the advanced understanding of electrostatic dust transport and its role in shaping the surfaces of airless planetary bodies.

紫外辐射或等离子体照射表面的粉尘充电和动员实验方法

Electrostatic dust transport has been hypothesized to explain a number of observations of unusual planetary phenomena. Here, it is demonstrated using ...

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.

Ecosystem Fabrication EcoFAB Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions

This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.

用于研究植物-微生物相互作用的实验室生态系统制造 (EcoFAB) 协议

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be critical for microbial energy production and persistence in many subsurface environments, especially those lacking soluble terminal electron acceptors. While EET-capable microbes have been successfully isolated from various environments, the diversity of bacteria capable of EET is still poorly understood, especially in difficult-to-sample, low energy or extreme environments, such as many subsurface ecosystems. Here, we describe an on-site electrochemical system to enrich EET-capable bacteria using an anode as a respiratory terminal electron acceptor. This anode is connected&#160;to a cathode capable of&#160;catalyzing abiotic oxygen reduction. Comparing this approach with electrocultivation methods that use a potentiostat for poising the electrode potential, the two-electrode system does not require an external power source. We present an example of our on-site enrichment utilized in an alkaline pond at the Cedars, a terrestrial serpentinization site in Northern California. Prior attempts to cultivate mineral reducing bacteria were unsuccessful, which is likely due to the low-biomass nature of this site and/or the low relative abundance of metal reducing microbes. Prior to implementing our two-electrode enrichment, we measured the vertical profile of dissolved oxygen concentration. This allowed us to place the carbon felt anode and platinum-electroplated carbon felt cathode at depths that would support anaerobic and aerobic processes, respectively. Following on-site incubation, we further enriched the anodic electrode in the laboratory and confirmed&#160;a distinct microbial community compared to the surface-attached or biofilm communities normally observed at the Cedars. This enrichment subsequently led to the isolation of the first electrogenic microbe from the Cedars. This method of on-site microbial enrichment has the potential to greatly enhance the isolation of EET-capable bacteria from low biomass or difficult to sample habitats.

On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET).

自立电化学建立富集阳极 respiring 菌现场

Anaerobic respiration coupled with electron transport to insoluble minerals (referred to as extracellular electron transport [EET]) is thought to be ...

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

植物感染试验: 以植物病原菌为载体的喷雾和伤口介导接种

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

一种灵活的低成本水培系统在无菌条件下评价植物对小分子的响应

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...