Name: a plate competition assay as a quick preliminary assessment of disease suppression
Uploaded: 2018-10-28T15:00:00
Description: Watch this Scientific Journal Video about A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression at JoVE.com

佛蒙特州农业实验站竞争孵化计划 VT-HO1609 资助了这项研究。林恩在佛蒙特大学6的硕士论文中使用了这个方法。

1. 提前准备
<ol><li>主真菌培养 (试验有机体)<ol>
<li>从美国类型文化收集, 微生物学收集9, 由其 teleomorph Thanatephorus 螨(弗兰克) (ATCC 10154) 或妙4031。</li>
		<li>或者: 收集由作者完成的本地隔离。红萝卜苗 (萝卜) 作为诱饵厂工作良好;收集土壤从已知有阻尼关闭或根腐烂的历史记录由R. 病菌造成。播种种子在土壤和孤立从根病变时幼苗是3到4周大。<ol>
<li>从土壤中?...

<ol>
	<li>Gonzalez Garcia, G., Onco, M. A. P., Susan, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review.+Biology+and+systematics+of+the+form+genus+Rhizoctonia.">Review. Biology and systematics of the form genus Rhizoctonia.</a> Spanish Journal of Agricultural Research. 4 (1), 55-79 (2006).</li><li>Bonanomi, G., Antignani, V., Pane, C., Scala, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+soilborne+fungal+diseases+with+organic+amendments.">Suppression of soilborne fungal diseases with organic amendments.</a> Journal of Plant Pathology. 89, 311-324 (2007).</li><li>Noble, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risks+and+benefits+of+soil+amendment+with+composts+in+relation+to+plant+pathogens.">Risks and benefits of soil amendment with composts in relation to plant pathogens.</a> Australasian Plant Pathology. 40, 157-167 (2011).</li><li>Wichuk, K. M., McCartney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compost+stability+and+maturity+evaluation+-+a+literature+review.">Compost stability and maturity evaluation - a literature review.</a> Canadian Journal of Civil Engineering. 37, 1505-1523 (2010).</li><li>Alfano, G., Lustrato, G., Lima, G., Vitullo, D., Ranalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+composted+olive+mill+wastes+to+predict+potential+plant+disease+suppressiveness.">Characterization of composted olive mill wastes to predict potential plant disease suppressiveness.</a> Biological Control. 3, 199-207 (2011).</li><li>Fang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biological indicators of compost-mediated disease suppression against the soilborne plant pathogen Rhizoctonia solani. , (2015).</li><li>Bonanomi, G., Antignani, V., Capodilupo, M., Scala, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+characteristics+of+organic+soil+amendments+that+suppress+soilborne+plant+diseases.">Identifying the characteristics of organic soil amendments that suppress soilborne plant diseases.</a> Soil Biology and Biochemistry. 42, 136-144 (2010).</li><li>Neher, D. A., Fang, L., Weicht, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecoenzymes+as+indicators+of+compost+to+suppress+Rhizoctonia+solani.">Ecoenzymes as indicators of compost to suppress Rhizoctonia solani.</a> Compost Science and Utilization. 25 (4), 251-261 (2017).</li><li>. ATCC&#174; Mycology Culture Guide Available from: <a target="_blank" href="https://www.atcc.org/~/media/PDFs/Culture%20Guides/Mycology_Guide.ashx">https://www.atcc.org/~/media/PDFs/Culture%20Guides/Mycology_Guide.ashx</a> (2018)</li><li>Berns, A. E., Phillip, H., Narres, H. -. D., Burauel, P., Vereecken, H., Tappe, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+gamma-sterilization+and+autoclaving+on+soil+organic+matter+structure+as+studied+by+solid+state+NMR,+UV+and+fluorescence+spectroscopy.">Effect of gamma-sterilization and autoclaving on soil organic matter structure as studied by solid state NMR, UV and fluorescence spectroscopy.</a> European Journal of Soil Science. 59, (2008).</li></ol>

我们从以前的研究中知道, 某些堆肥是有效的抑制病菌和抑制作用是由于微生物生活在堆肥, 而不是非生物性质的堆肥6,8。使用高压灭菌作为一种手段, "杀死" 微生物群已被批评, 因为它影响碳化学的媒体10。我们通过 Whatman 1 号纸将高压灭菌与真空过滤进行了比较。既不过滤也不蒸压的治疗显示病菌生长的最大抑制<sup class=...

纹枯代表了真菌的一个广泛的复杂, 其中Thanatephorus 螨(弗兰克) 反主动 (无性型 =纹枯病菌库恩) 是导致根腐病和阻尼1。纹枯病菌是一种积极的病原体和腐生植物, 可以作为菌核在恶劣环境条件下存活1。因此, 它有一个全球性的分布, 并可能导致广泛的工厂主机的疾病, 包括茄科, 豆科, 菊科, 和十字花科造成严重的经济损失。
堆肥有能力为某些植物病原体提供2的生物防治剂。然而, 并非所有的堆肥都一样, 也不影响所有病原体同样3。木质碳的木质素比干草或稻草碳基堆肥的纤维素比例高。R. 病菌更喜欢在稻草中找到的容易获得的碳。相比之下, 生物控制真菌, 如木霉菌, 更有效时, 碳是不太容易得到。有益真菌和细菌进入堆肥可以通过竞争、对抗或调节植物生长3来抑制植物疾病。所建议的检测方法主要检测抗生素、ecoenzymes 或螯合剂的产生对病原体有害的拮抗作用。
植物生物鉴定是确定堆肥有利于或阻止植物生长4的黄金标准。然而, 植物生物鉴定是耗时的 (几周到几个月) 完成这可能是长于预期的, 需要更多的劳动力, 以提取植物根系, 以量化的严重程度的根系统。相对稳健, 但更快 (天) 的检测将是质量控制计划的理想选择。本文的目的是对堆肥的抑制电位进行比较快速、准确的试验。该方法阿尔法诺等。5除两种例外外, 堆肥提取物被稀释, 水琼脂用于代替马铃薯葡萄糖琼脂 (PDA)。R. 病菌在简单的水琼脂培养基上生长迅速, 而 PDA 促进细菌和其他真菌的生长, 污染了文化6。
此板块测定是一种抑制指标, 适用于土壤中存活的一系列植物病原体, 如腐生生物病菌7、链孢摧残、枯萎病、疫霉根腐、腐霉根腐病。板块竞争检测是有用的筛选一系列堆肥产品的包含微生物群落作为土壤病原体的生物控制剂。该检测是商业堆肥产品中最一致的疾病抑制指标之一6,8。产品在配方、成熟度和生产过程中的差异被选择。

<table>
	<tbody>
		<tr>
			<td>autoclavable narrow-neck glass conical flask</td>
			<td>Fisher Scientific</td>
			<td>10-040D</td>
			<td>125-mL</td>
		</tr>
		<tr>
			<td>dehydrated granulated agar</td>
			<td>Fisher Scientific</td>
			<td>DF0145-17-0</td>
			<td>500 g quantity</td>
		</tr>
		<tr>
			<td>heat resistant gloves</td>
			<td>Fisher Scientific</td>
			<td>MEMGG1314WL</td>
			<td>several brands available</td>
		</tr>
		<tr>
			<td>Parafilm (strips of 2-3 cm wide)</td>
			<td>Fisher Scientific</td>
			<td>PM992 13-374-16</td>
			<td>5 or 10 cm widths work</td>
		</tr>
		<tr>
			<td>disposable polystyrene petri dishes</td>
			<td>Fisher Scientific</td>
			<td>R80116</td>
			<td>comes in sleeves of 20/ea or cases of 500</td>
		</tr>
		<tr>
			<td>dehydrated potato dextrose agar</td>
			<td>Fisher Scientific</td>
			<td>DF0013-15-8</td>
			<td>comes in quantities of 100, 500 and 2000 grams</td>
		</tr>
		<tr>
			<td>benchtop reciprocal shaker</td>
			<td>Thomas Scientific</td>
			<td>1227Y31</td>
			<td>other models will work</td>
		</tr>
		<tr>
			<td>water bath</td>
			<td>ThermoScientific</td>
			<td>S37363</td>
			<td>5L general purpose</td>
		</tr>
		<tr>
			<td>clear ruler, flat, at least 10 cm</td>
			<td>Any</td>
			<td></td>
			<td>use metric rule</td>
		</tr>
		<tr>
			<td>ATCC culture</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 10154</td>
			<td>teleomorph Thanatephorus cucumeris (Frank) (ATCC 10154) or MYA 4031;</td>
		</tr>
		<tr>
			<td>lab tape</td>
			<td>Fisher Scientific</td>
			<td>15935</td>
			<td>autoclavable and removable, 1&#34; wide preferred</td>
		</tr>
		<tr>
			<td>water resistant marker</td>
			<td>office or scientific supply</td>
			<td>Sharpie fine tip</td>
			<td>write sample number on tape</td>
		</tr>
	</tbody>
</table>

a plate competition assay as a quick preliminary assessment of disease suppression

其目的是开发和优化一个简单, 负担得起和有效的生物测定, 以检测特定堆肥对土传真菌纹枯病菌的疾病抑制能力。R. 病菌是全球范围内各种植物宿主的病原体。真菌在土壤中存活为腐生植物, 在简单的水琼脂培养基上迅速生长。钢板法是一种快速的方法来比较堆肥的能力, 以减缓病菌的生长。该检测还与其他土传真菌病原体的抑制有很好的相关性, 如链孢早期摧残、枯萎病菌、霉菌根腐病和腐霉根腐病等土壤中腐生生物。

成品堆肥应该是稳定和成熟的, 两个术语, 经常互换使用, 所以它可以安全包装和运输, 并没有造成不良影响, 在其最终使用4。稳定性是一种抗分解性, 通常使用微生物活性指标来确定。微生物呼吸的一般措施可以测量堆肥的稳定性, 但不一定能抑制侵袭性病原体和腐生植物如R. 病菌7的疾病。本研究着眼于成熟度, 推断?...

Watch this Scientific Journal Video about A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression at JoVE.com

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

介绍了一种平板竞争试验的协议, 以确定特定堆肥是否可能含有抑制纹枯病菌生长的细菌和真菌。

一种快速的疾病抑制初步评估的板块竞争分析法

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

该方法有助于回答与土壤植物病原体生物控制有关的关键问题。测试堆肥等材料的黄金标准是进行植物生物测定，以测试它是否对植物有毒。然而，这需要三个星期。此方法可以在两到三天内完成。这种技术的主要优点是简单、经济、有效。这种检测是特定堆肥的快速筛选，以抑制Rhizoctonia solani的生长。然而，这种测定也可用于其他土壤传播的植物病原体，如富萨里姆、紫土和植物。此方法的视觉演示至关重要，因为它在避免污染的同时，注意隔离和维护真菌的纯培养物。证明隔离程序的将是泰勒·雷迪霍夫，一个研究生从我的实验室。艾玛·赖特，我实验室的本科生，将演示竞争检测。首先，在已知有R.solani病史的土壤中播种红萝卜种子。三到四周后，将幼苗从土壤中取出，然后用自来水冲洗。使用剃须刀刀片，切割具有棕色的低边根和根的一厘米段。然后，使用火焰消毒钳将部分浸入 10% 漂白剂溶液中一分钟。在此之后，用无菌水冲洗段。接下来，使用火焰消毒钳将段转移到纸巾上。将段子擦干，然后将它们与水加水一起转移到培养皿中。将培养皿放在带盖子的容器内，并在室温下孵育分段。要建立女儿文化，请将其中一个段转移到一盘新鲜土豆葡萄糖的水果的中心。在此之后，可 <3>5毫升试管含有10毫升水。将每个测试样品的两个 0.5 克样本添加到一对带 10 毫升无菌水的自 <3>开式试管中，并摇动试管过夜。24小时后，在一对圆锥烧瓶中加入1.5克普通加糖和90毫升蒸馏水。将烧瓶高压灭后，放入45摄氏度的水浴中30分钟。接下来，将自动处理参考样品和活样品倒入熔融的加糖中。将每个烧瓶中的混合物倒入五个培养皿中。为了保护本地微生物，重要的是不要添加堆肥，而阿加太热。因此，在添加堆肥之前，让阿加冷却至45摄氏度。使用无菌技术，将 R.solani 片段转移到每对样品板。然后，在室温下孵育板一至两天。最后，使用透明、扁平的尺子和立体显微镜测量每个板中米丝的半径到最接近的毫米。在该协议中，从活堆肥中提取的提取物抑制R.solani的生长明显超过自 <3>发样本，表明其作用是微生物的，而不是基于营养的。与自动 <3> <3>控制相比，抑制度被测量为增长的减少。生长不受堆肥成熟或固化期的影响。生长受到堆肥方法和配方成分的显著影响，特别是与紫杉木，风向道，和食谱与硬木树皮。开发后，该技术可成为用于疾病抑制特性的堆肥商业认证的候选产品。

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