概念化、H.C. 和 z.q.f.;调查, h.c., J, 和 z.q. F;资源, z.q.f.;文字原件草稿, i.a.p.;写作-审阅 & 编辑, i.a.p., h.c., s.l.t. 和 z.q.f.;可视化, h.c.;监督、z.q.f.;资金购置、z.q. F 和 f.q.l。
南卡罗来纳州支持的项目提高教师素质高等教育国家补助金标题,通过参与植物科学研究提高中级理科教师对植物过程、结构和功能的认识 (植物科学)
美国教育部颁发的资金 (CFDA 号 84.367B)
作者要感谢佛罗里达大学的年王博士提供来自受感染的柑橘的瓦伦西亚的基因组 DNA 样本。

1. 使用基因组提取试剂盒分离植物组织中的基因组 DNA
<ol><li>用干净的剪刀从柑橘树上切下一整片新鲜的叶子, 将叶子放在干净的塑料三明治袋中, 获得柑橘叶组织。 注: 在收集后应尽快将含有叶组织的包放在4摄氏度冰箱或保温容器中的冰上, 以避免变质。</li>
	<li>加入少量液氮冷却砂浆和杵。当它们冷却时, 用剪刀剪下一小片叶子组织, 大小大约1平方英寸。将切割组织放在砂浆中立即将其冻结。如果需要, 添加更多的液氮。 注: 在处理液氮时, 应始终佩戴绝缘手套。</li>
	<li>用砂浆将植物组织快速研磨成细粉。继续研磨直至液氮完全蒸发, 并保持良好的绿色粉末。</li>
	<li>在 10-十五年代, 在液态氮气中短暂冷却金属铲, 然后用刮刀将砂浆内的地面组织舀入1.5 毫升的离心管中。</li>
	<li>添加600µL 核溶解溶液 (见材料表) 使用1000µL 吸管和涡流样品 1-3 s, 然后孵化在65°c 水浴15分钟。</li>
	<li>添加3µL RNase 溶液的裂解液使用10µL 吸管, 反转管 2-5 倍, 以混合, 并孵化37°c 在一个橱柜孵化器为15分钟。 注: 允许混合物冷却到室温, 然后再继续。</li>
	<li>添加200µL 的蛋白质沉淀溶液, 在二十年代高速涡旋, 然后离心机3分钟在 1.3万 x g 形成一个坚实的颗粒。当样品被离心, 添加600µL 室温异丙醇到一个新鲜的1.5 毫升离心管。</li>
	<li>使用1000µL 吸管, 小心地从离心样品中取出上清, 将其转移到含有异丙醇的新鲜1.5 毫升离心管。颗粒现在可能被丢弃。 注意: 避免用蛋白质污染上清液, 在颗粒上方留下少量的上清液。</li>
	<li>反转管直到 DNA 变为可见的一大类线程状股, 然后离心机在 1.3万 x g 1 分钟室温。</li>
	<li>醒酒上清液, 将70% 乙醇的600µL 添加到颗粒中, 多次反转管以清洗 DNA, 离心机在 1.3万 x g 处进行1分钟。</li>
	<li>小心地吸干乙醇, 留下松散的 DNA 颗粒, 然后打开并倒置管子, 搁在吸水性纸上。室温空气干燥15分钟。</li>
	<li>将100µL dna 补液溶液添加到干 dna 颗粒中, 然后在65摄氏度的水浴中孵化出1小时的 dna, 同时通过轻敲管进行混合。</li>
	<li>将1µL 的纯净水放在 microcuvette 上, 并将其插入分光光度计中用作空白。通过在 microcuvette 上放置1µL 样品, 并插入分光光度计, 计算出目前 DNA 的浓度。DNA 浓度 (ng/µL) 可以计算为 (260-320) x 稀释因子 x 50 ng/µL。 注意: 协议可以在这里暂停。样品可以储存在4摄氏度。</li>
</ol>2. 对基因组 DNA 样品进行 PCR 处理
<ol><li>结合10µL 2x PCR 大师组合 (参见材料表), 1 µL 前底漆 (下午10点/µL), 1 µL 反向底漆 (下午10点/µL) (见表 2为底漆序列), 100 ng 的基因组 DNA 提取的样本, 和纯净水 (最多20µL 最终体积) 在50µL PCR 管, 轻弹混合, 并简要离心机。</li>
	<li>将 PCR 管插入 thermocycler 并相应运行 (见表 1)。 注意: 协议可以在这里暂停。如有必要, 在反应完成后, 将样品贮存在4摄氏度。</li>
</ol>3. Electrophorese 琼脂糖凝胶中扩增的 DNA
<ol><li>重0.4 克琼脂糖粉, 并添加它连同1x 泰的缓冲器到一个干净的200毫升锥形瓶, 填补多达50毫升。</li>
	<li>微波在高为1.5 分钟, 摇晃每三十年代, 直到琼脂糖完全地被溶化。 注: 在处理液体琼脂糖时应佩戴绝缘手套以防止烫伤。</li>
	<li>添加2.5 µL 10 毫克/毫升溴溴化液琼脂糖和摇动混合, 然后倒入液琼脂糖凝胶成水平凝胶模具。快速插入标准的12牙凝胶梳后浇。</li>
	<li>允许凝胶在室温下冷却20分钟, 然后去除凝胶梳, 并将凝胶放入一个充满1x 泰缓冲器的水平凝胶盒中。</li>
	<li>使用微, 将总扩增的 DNA 样本加载到水井中。添加5µL 的 DNA 阶梯 (见材料表) 在一个单独的井从你的样品。</li>
	<li>Electrophorese 样品35分钟在恒定的 90 V, 然后去除胶凝体和地方在紫外 transilluminator。</li>
	<li>在302纳米紫外线透视的情况下, 对凝胶条纹图案进行照相和分析。</li>
</ol>

<ol>
	<li>Field Office, F. l. o. r. i. d. 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> Citrus October Forecast Maturity Test Results and Fruit Size. , (2016).</li><li>Singerman, A., Useche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+citrus+greening+on+citrus+operations+in+Florida.">Impact of citrus greening on citrus operations in Florida.</a> Food and Resource Economics Department, UF/IFAS Extension. , (2015).</li><li>Spreen, T. H., Hodges, 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> The economic impact of HLB on the florida citrus industry. , (2012).</li><li>UICEP. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathology. , (2013).</li><li>Jagoueix, S., Bove, J. M., Garnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phloem-limited+bacterium+of+greening+disease+of+citrus+is+a+member+of+the+alpha+subdivision+of+the+Proteobacteria.">The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria.</a> Int J Syst Bacteriol. 44 (3), 379-386 (1994).</li><li>Fujikawa, T., Iwanami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+and+robust+detection+of+citrus+greening+(huanglongbing)+bacterium+&amp;#34;Candidatus+Liberibacter+asiaticus&amp;#34;+by+DNA+amplification+with+new+16S+rDNA-specific+primers.">Sensitive and robust detection of citrus greening (huanglongbing) bacterium &amp;#34;Candidatus Liberibacter asiaticus&amp;#34; by DNA amplification with new 16S rDNA-specific primers.</a> Mol. Cell. Probes. 26 (5), 194-197 (2012).</li><li>Davis, M. J., Mondal, S. N., Chen, H., Rogers, M. E., Brlansky, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-cultivation+of+'Candidatus+Liberibacter+asiaticus'+with+actinobacteria+from+citrus+with+huanglongbing.">Co-cultivation of 'Candidatus Liberibacter asiaticus' with actinobacteria from citrus with huanglongbing.</a> Plant Dis. 92 (11), 1547-1550 (2008).</li><li>Department of Agriculture &amp; National Agriculture Statistics Service. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Citrus Fruits 2015 Summary (September 2015). , (2015).</li><li>Hocquellet, A., Toorawa, P., Bov&#233;, J. M., Garnier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+the+two+Candidatus+Liberobacter+species+associated+with+citrus+huanglongbing+by+PCR+amplification+of+ribosomal+protein+genes+of+the+&#946;+operon.">Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the &#946; operon.</a> Mol. Cell. Probes. 13 (5), 373-379 (1999).</li><li>Mafra, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+genes+for+accurate+transcript+normalization+in+citrus+genotypes+under+different+experimental+conditions.">Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.</a> PLoS One. 7 (2), e31263 (2012).</li><li>Li, W., Hartung, J. S., Levy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+real-time+PCR+for+detection+and+identification+of+Candidatus+Liberibacter+species+associated+with+citrus+huanglongbing.">Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing.</a> J. Microbiol. Meth. 66 (1), 104-115 (2006).</li></ol>

丰泉、伊恩？帕默、建安、明昌、斯蒂芬·汤普森、刘和郑庆府宣告没有利益冲突。

至关重要的是, 所有的步骤都是完全遵循, 以达到最佳的实验条件。全叶组织在本示范期间使用;然而, 该协议可以修改为理论上包括任何类型的植物组织。此前, 研究人员从叶静脉组织中提取基因组 DNA, 而不是全叶组织, 并取得了类似的结果11。如果与柑橘FBOX基因相对应的带不出现, 结果无效, 在操作过程中可能存在一个或多个技术错误。常见的错误包括: 使用受污染或不适?...

柑橘的绿化, 也被称为黄龙, 已经造成了大量的柑橘树的损失。一个典型的案例是佛罗里达州, 预计该疾病将导致佛罗里达州橙色盒的生产减少 70%, 从1997至1998季的2亿4400万箱, 7000万盒 2016 2017 赛季1。佛罗里达州90% 以上的柑橘类植物感染了2, 据估计, 由于柑橘类疾病, 佛罗里达柑橘业每年损失约10亿美元, 其中柑橘绿化在3起主要作用。柑橘的绿化主要由侵袭性的亚洲柑橘木虱Diaphorina 桔传播,但也可以通过嫁接感染的组织传播.该病引起静脉发黄, 黄叶斑驳不对称, 过早落叶, 枝复生, 根腐, 植物死亡。重要的是, 这种疾病使柑橘类植物生产出的季节开花, 很少产生水果。被感染的柑橘类植物生产的水果是不成熟的, 绿色的和苦涩的品尝4。
该方法的目的是准确和准确地识别Candidatus liberibacter 菌的运动细菌, 其致病剂为柑橘的绿化, 生活在被感染的柑桔树的韧皮部内5。基因组 DNA 从整个叶组织中提取, 其中含有活细菌。这种提取的基因组 DNA 被用来作为常规 PCR 的模板, 其中寡核苷酸补充细菌的 16S rDNA 序列被用来放大这个序列。寡核苷酸互补的柑橘FBOX基因作为内部放大控制。我们选择使用这个方法, 因为它已经被证明是成功的在以前的研究6。
这种方法有明显的优势, 简单, 相对便宜, 并能够在任何设备正常的生物化学实验室进行。此外, PCR 仍然是最准确和最精确的方法, 以检测这种病原体, 由于难以培养这个病原体7, 和病原体生存和繁殖的能力在无症状的主机8年。许多不同的特异性 PCR 检测方法已经成功地发现了这种病原体;然而, 常规 PCR 仍然是最简单的检测, 特别是在无症状宿主8。

<table><tbody><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120</td><td>Source of Nuclei Lysis, RNase, Protein Precipitation, and DNA Rehydration solutions</td></tr><tr><td>Liquid nitrogen</td><td>Air Products</td><td>N/A</td><td></td></tr><tr><td>Mastercycler pro with control panel</td><td>Eppendorf</td><td>6321000019</td><td></td></tr><tr><td>1250 W Microwave</td><td>Panasonic</td><td>N/A</td><td></td></tr><tr><td>5424 table top centrifuge</td><td>Eppendorf</td><td>05-403-93</td><td></td></tr><tr><td>Isotemp 215 digital water bath</td><td>Fisher scientific</td><td>15-462-15Q</td><td></td></tr><tr><td>Metal spatula</td><td>Sigma-Aldrich</td><td>Z283274</td><td></td></tr><tr><td>Mortar and pestle</td><td>Sigma-Aldrich</td><td>Z247464</td><td></td></tr><tr><td>LSE Vortex Mixer</td><td>Corning</td><td>6775</td><td></td></tr><tr><td>2-propanol</td><td>Sigma-Aldrich</td><td>I9516</td><td></td></tr><tr><td>Sterile 10 &amp;mu;L tips</td><td>TipOne</td><td>1161-3730</td><td></td></tr><tr><td>Sterile 200 &amp;mu;L tips</td><td>TipOne</td><td>1163-1730</td><td></td></tr><tr><td>Sterile 1250 &amp;mu;L tips</td><td>TipOne</td><td>1161-1750</td><td></td></tr><tr><td>Variable Volume Pipettor Kit</td><td>VWR</td><td>75788-460</td><td></td></tr><tr><td>Ethidium bromide</td><td>Sigma-Aldrich</td><td>E1510</td><td></td></tr><tr><td>Agarose</td><td>IBI Scientific</td><td>IB70041</td><td></td></tr><tr><td>EDTA</td><td>Thermo Fisher Scientific</td><td>17892</td><td></td></tr><tr><td>Acetic acid</td><td>Fisher Chemical</td><td>A38-212</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>10708976001</td><td></td></tr><tr><td>&amp;mu;cuvette</td><td>Eppendorf</td><td>6138000018</td><td></td></tr><tr><td>Stackable casting tray and combs</td><td>Carolina</td><td>213655</td><td></td></tr><tr><td>PowerPac Basic Power Supply</td><td>Bio-Rad</td><td>1645050</td><td></td></tr><tr><td>Mini-Sub Cell GT System</td><td>Bio-Rad</td><td>1704487EDU</td><td></td></tr><tr><td>Biospectrometer basic</td><td>Eppendorf</td><td>6135000009</td><td></td></tr><tr><td>0.2 mL PCR tubes</td><td>Fisher scientific</td><td>AB-0620</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Thermo Fisher Scientific</td><td>3439</td><td></td></tr><tr><td>2X Taq Green PCR Master Mix</td><td>Promega</td><td>M7122</td><td></td></tr><tr><td>Forward Primer</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Reverse Primer</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Water, PCR grade</td><td>Sigma-Aldrich</td><td>3315953001</td><td></td></tr><tr><td>Gel Doc XR+</td><td>Bio-Rad</td><td>1708195</td><td></td></tr><tr><td>1 KB+ DNA ladder</td><td>Thermo Fisher Scientific</td><td>10787018</td><td></td></tr></tbody></table>

specific and accurate detection of the citrus greening pathogen candidatus liberibacter spp. using conventional pcr on citrus leaf tissue samples

柑橘类绿化, 又称黄龙, 是一种破坏性的柑橘病, 在全球范围内肆虐柑橘养殖场。这种疾病导致不对称的黄叶斑驳, 静脉变黄, 落叶, 根腐, 最终死亡的柑橘植株。在感染时, 柑橘类植物生长发育迟缓, 并在季节内产生花朵。这些花很少产生果实, 而那些产生小的, 苦涩的, 不规则形状的柑橘类水果是不可取的。这种疾病是由亚洲柑橘木虱、 Diaphorina 桔和被感染的柑橘组织嫁接而传播的。病原体在柑橘植株内有长而多变的潜伏期--有时在症状出现前数年。在体外培养这种病菌的尝试是不成功的, 可能是由于感染的柑橘组织中的病原体浓度低和不均匀, 或者因为很难复制有利于生长的环境条件。病原体。由于其潜伏期长, 研究人员无法培养病原体, 很难在病毒传播前发现疾病。因此, 这种疾病只在突然破坏柑橘农民的全部产量之后才变得明显。本文介绍了一种准确、特异地检测柑桔绿化病原体、 Candidatus liberibacter 的方法. 使用基因组 DNA 提取试剂盒和 PCR 技术。该方法简单、高效、成本效益高, 适用于定量分析。该方法可用于任何柑橘组织;然而, 它有可能受组织中的病原体数量的限制。然而, 这种方法将使柑橘养殖者能够及早识别受感染的柑橘类植物, 并遏制这种破坏性疾病的蔓延, 然后才能进一步扩散。

一个正面结果产生一个不同的波段对应于 500 bp 为Candidatus Liberibacter 胭脂Las606/Lss6和/或一个独特的带对应于 700 bp 为Laa2/Laj5,插入在16S 核糖体β操纵9。内部放大控制带也必须出现在 400 bp。这个波段对应于柑橘FBOX基因10的放大, 并且必须出现一个结果被认为是有效的 (图 1</s...

Watch this Scientific Journal Video about Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples at JoVE.com

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

柑橘类绿化是影响全球柑橘作物的一种特殊破坏性疾病。本文介绍了一种利用 PCR 和基因组 DNA 提取柑桔叶组织的简便方法, 对柑桔绿化病原体、 Candidatus liberibacter 进行准确、准确的鉴定。

利用常规 PCR 技术对柑桔叶片组织样品Candidatus liberibacter的特异和准确检测

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

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Universidad San Sebastian

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JoVE Journal

Environment

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

7.6K 次观看.  University of South Carolina. 柑橘类绿化是影响全球柑橘作物的一种特殊破坏性疾病。本文介绍了一种利用 PCR 和基因组 DNA 提取柑桔叶组织的简便方法, 对柑桔绿化病原体、 Candidatus liberibacter 进行准确、准确的鉴定。

视频: Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

用便携式实时 PCR 系统进行土壤载体 Phytopathogens 的现场分子检测

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management ...

科研

环境科学

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 ...

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

使用 循环介质等温扩 增检测灌溉水中的植物浮肿

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

细菌性叶渗透试验使用的植物防御反应精细表征<em&gt;拟南芥，丁香假单胞菌</em&gt;病害系统

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

免疫与感染

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

Genotyping of Plant and Animal Samples without Prior DNA Purification

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors.
PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. 
Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. 
Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size.

The Direct PCR approach presented here facilitates PCR amplification directly from small amounts of unpurified plant and animal tissue.

基因分型的植物和动物样品，恕不另行DNA的纯化

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and ...

生物学

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while &#34;Candidatus Liberibacter asiaticus&#34; has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California&#39;s citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step.
Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/&#181;L) that was comparable with the CCPP&#39;s hand-chopping protocol (77.84 ng/&#181;L). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

We engineered, fabricated, and validated an instrument that rapidly processes phloem-rich bark citrus budwood tissues. Compared to current methods, the budwood tissue extractor (BTE) has increased sample throughput and decreased the required labor and equipment costs.

通过仪器工程自动化柑橘接穗加工，用于下游病原体检测

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious ...

生物工程

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A simple method for rapid and portable Huanglongbing (HLB) diagnosis is presented here that combines recombinase polymerase amplification and a fluorescent reporter utilizing the nuclease activity of the clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a (CRISPR-Cas12a) system. The sensitivity of this technique is much higher than PCR. Furthermore, this method showed similar results to qPCR when leaf samples were used. Compared with conventional CLas detection methods, the detection method presented here can be completed in 90 min and works in an isothermal condition that does not require the use of PCR machines. In addition, the results can be visualized through a handheld fluorescent detection device in the field.

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

In this work, a rapid, sensitive, and portable detection method for Candidatus Liberibacter asiaticus based on recombinase polymerase amplification combined with CRISPR-Cas12a was developed.

使用重组酶聚合酶扩增结合CRISPR-Cas12a进行现场部署的亚洲 自由念珠 菌检测

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A ...

使用直接输注装置在植物中递送分子

Direct Infusion Device for Molecule Delivery in Plants

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed.
To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant&#39;s trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed.
Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for&#160;testing and facilitate research and screening purposes.

作者聚焦：用于植物分子递送的直接输注装置  

This manuscript describes a novel direct plant infusion device for screening the effectiveness of molecules against the bacteria (Candidatus Liberibacter asiaticus) or its insect vector (Diaphorina citri, Kuwayama) that, in combination, are associated with Huanglongbing citrus disease.

用于植物分子输送的直接输液装置

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but ...

利用常规 PCR 技术对柑桔叶片组织样品Candidatus liberibacter的特异和准确检测

摘要

探索更多视频

此视频中的章节

用便携式实时 PCR 系统进行土壤载体 Phytopathogens 的现场分子检测

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

使用循环介质等温扩增检测灌溉水中的植物浮肿

细菌性叶渗透试验使用的植物防御反应精细表征

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

基因分型的植物和动物样品，恕不另行DNA的纯化

通过仪器工程自动化柑橘接穗加工，用于下游病原体检测

使用重组酶聚合酶扩增结合CRISPR-Cas12a进行现场部署的亚洲自由念珠菌检测

用于植物分子输送的直接输液装置

用于植物分子输送的直接输液装置

利用常规 PCR 技术对柑桔叶片组织样品Candidatus liberibacter的特异和准确检测

摘要

探索更多视频

此视频中的章节

用便携式实时 PCR 系统进行土壤载体 Phytopathogens 的现场分子检测

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

使用 循环介质等温扩 增检测灌溉水中的植物浮肿

细菌性叶渗透试验使用的植物防御反应精细表征

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

基因分型的植物和动物样品，恕不另行DNA的纯化

通过仪器工程自动化柑橘接穗加工，用于下游病原体检测

使用重组酶聚合酶扩增结合CRISPR-Cas12a进行现场部署的亚洲 自由念珠 菌检测

用于植物分子输送的直接输液装置

用于植物分子输送的直接输液装置

使用循环介质等温扩增检测灌溉水中的植物浮肿

使用重组酶聚合酶扩增结合CRISPR-Cas12a进行现场部署的亚洲自由念珠菌检测