Name: measuring volatile and non-volatile antifungal activity of biocontrol products
Uploaded: 2020-12-05T14:00:00
Description: Watch this Scientific Journal Video about Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products at JoVE.com

我们非常感谢弗兰克·耶茨的宝贵建议。这项工作得到了苏普生物技术公司的支持。

1. 伊诺库拉准备
<ol><li>在实验之前，铺设5μL的三叶草。SBT10-2018 孢子储存在马铃薯脱糖agar介质（PDA）的4°C，在30°C下孵育4天，定期照射光线，促进锥状形成42（图1，面板A）。 注： 特里乔德玛。SBT10-2018已被与木材分离，并作为本研究的模型，以快速生长和容易孢子回收。我们的实验室保存了这种菌株。</li>
	<li>恢复锥体（...

此处介绍的方法允许评估最小加工植物衍生产品的抗真菌特性。在此协议中，使用 2 mm 玻璃珠实现在 agar 表面上孢子的均质分布。这一步骤要求处理技能正确分配珠子并获得可重复的结果，最终允许比较真菌生长不同阶段的抗真菌效果。我们发现，5毫米玻璃珠或过度旋转，而在传播过程中均质可能导致可变的生长直径。因此，我们建议在实验前进行培训，以掌握孢子分布。此外，当必须测试植物...

全球水果和蔬菜损失可能高达产量的50%，主要由于田间或收获后储存2，3的真菌变质造成的食物腐烂，尽管自20世纪4世纪中叶以来大量使用合成杀菌剂。正在重新考虑使用这些物质，因为它对环境和健康构成严重危害。由于这些药物的有害后果正在整个生态系统中显现出来，而且潜在的健康影响的证据已经累积了5，6，新的替代旧的预防策略正在开发用于收获前和收获后治疗7，8，9。因此，我们面临的挑战是双重的。新的杀菌策略首先必须保持食品保护对植物病原体的功效水平，其次，有助于大幅减少农业做法的环境足迹。为了实现这一雄心勃勃的目标，在植物进化的自然防御的启发下，提出了战略，因为超过1000种植物因其抗菌特性而得到强调。例如，开发天然杀菌剂对抗植物病原体的植物是探索开发新的生物控制产品的新资源。精油是这类的旗舰分子。例如，Origanum精油保护番茄植物免受10号温室的灰霉菌的侵害，而Solideago甘蔗剂L.和卡西亚精油已被证明可以保护收获后的草莓免受灰霉菌的损害11，12。这些例子说明，生物控制，特别是植物衍生产品，是一种结合了生物功效和环境可持续性的解决方案。
因此，植物是作物保护行业潜在利益分子的重要资源。然而，只有少数植物产品被建议用作生物控制产品，即使它们被普遍认为是安全的，非植物毒性和环保的2。观察到从实验室到现场的转换存在一些困难，例如在体内2、9中应用后疗效降低。因此，提高实验室测试的能力以更好地预测现场效果变得非常重要。在这方面，植物衍生产品的抗真果测试方法对于评估其抗真果效果和确定其最佳使用条件都是必要的。具体来说，生物控制产品通常不如化学杀菌剂有效，因此更好地了解其作用模式对于提出适当的配方、确定在田间的应用模式以及确定病原体的哪个发育阶段容易受到候选生物制品的影响非常重要。
目前处理抗菌和抗真菌活动的方法包括扩散方法，如阿加盘扩散、稀释、生物电图和流动细胞测量13.这些技术中，更具体地说，标准的抗真菌易感性测试（agar-磁盘扩散和稀释检测）非常适合评估液体悬浮剂中细菌和真菌孢子上可溶性化合物的抗菌活性14.然而，这些方法通常不适合测试固体化合物，如干植物粉末或量化在霉菌生长期间的抗真菌活性，因为它们需要孢子稀释或孢子在阿加板上传播和/或稀释抗真菌化合物13.在食物中毒方法中，含有抗真菌剂的阿加板接种了直径为5-7毫米的磁盘，从7天老真菌培养物中采样，而不考虑启动菌的精确数量。孵化后，抗真菌活性被确定为径向生长抑制的百分比17,18,19.通过这种方法，我们可以评估我天体生长的反真菌活动。相比之下，进行阿加稀释法可以确定直接接种含有抗真菌化合物的阿加板表面孢子上的抗真菌活性13,20,21.这两种方法在抗真果活动方面具有互补性。然而，这是两种并行使用的独立技术，不能提供准确并排比较孢子和菌体上抗真菌化合物的相对功效17,20,22 由于启动真菌材料的数量在两种方法中有所不同。此外，植物衍生产品的抗真菌活性往往来自植物合成的抗真菌分子与面对病原体的组合。这些分子包括蛋白质、肽23,24代谢物具有广泛的化学多样性，属于不同类别的分子，如多酚、三苯、藻类25，葡萄糖素8，和有机硫化合物26.其中一些分子在病原体攻击期间挥发性或变挥发性27.这些制剂通常是水溶性差和高蒸汽压力化合物，必须通过水蒸馏作为精油回收，其中一些抗菌活动已经建立28.已开发出蒸汽相介导易感性检测，以测量蒸发和通过蒸汽相迁移后挥发性化合物的抗菌活性29.这些方法基于在距离微生物培养物较远的地方引入抗真菌化合物29,30,31,32,33.在常用的蒸汽相阿加检测中，精油沉积在纸盘上，并放置在培养皿盖的中心，距离细菌或真菌孢子悬架（在 agar 介质上传播）很远。然后以与 agar-磁盘扩散方法相同的方式测量生长抑制区的直径20,24.还开发了其他方法，以提供对精油的蒸汽相抗真菌性的定量测量，这种测量方法来自计算抑制性蒸汽相介导抗菌活性的肉汤稀释方法32，或从阿加磁盘扩散检测中提取31.这些方法通常特定于蒸汽相活动研究，不适合接触抑制检测。这排除了确定挥发性和非挥发性制剂对复杂生物活性混合物抗真菌活动的相对贡献。
我们开发的定量方法旨在测量干植物粉末对受控孢子数量的抗真菌作用，并生长沉积在阿加介质表面的菌株，以再现植物病变在感染植物15 期间的空中生长，以及相互连接的my天体网络16。该方法是基于阿加稀释和食物中毒方法的改进实验设置，在同一实验设置中，还允许并排量化挥发性和非挥发性抗真果代谢物的贡献。在这项研究中，该方法以三种特征良好的抗真真药制剂的活动为基准。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Autoclave-vacuclav 24B+</td>
			<td>Melag</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbendazim</td>
			<td>Sigma&#160;</td>
			<td>378674-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Sarstedt</td>
			<td>72.706</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>Falcons tubes</td>
			<td>Sarstedt</td>
			<td>547254</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Five millimeters diameter stainless steel tube</td>
			<td>retail store</td>
			<td></td>
			<td>/</td>
		</tr>
		<tr>
			<td>Food dehydrator</td>
			<td>Sancusto</td>
			<td></td>
			<td>six trays</td>
		</tr>
		<tr>
			<td>Garlic powder</td>
			<td>Organic shop</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>CLOUP</td>
			<td>65020</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/61798/61798eq1.png" /> 2 mm</td>
		</tr>
		<tr>
			<td>Hemocytometer counting cell</td>
			<td>Jeulin</td>
			<td>713442</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert</td>
			<td>&#160;UM400</td>
			<td>30 &#176;C</td>
		</tr>
		<tr>
			<td>Knife mill</td>
			<td>Bosch</td>
			<td>TSM6A013B</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual cell counter</td>
			<td>Labbox</td>
			<td>HCNT-001-001</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Measuring ruler</td>
			<td>retail store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological safety cabinets</td>
			<td>FASTER</td>
			<td></td>
			<td>FASTER BHA36, TYPE II, Cat 2</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Mettler-Toledo</td>
			<td>17014407</td>
			<td>100 - 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Mettler-Toledo</td>
			<td>17014411</td>
			<td>20 - 200 &#181;L</td>
		</tr>
		<tr>
			<td>Micropipette</td>
			<td>Mettler-Toledo</td>
			<td>17014412</td>
			<td>2 - 20 &#181;L</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82-1194500</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/61798/61798eq1.png" /> 55 mm</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82-1473&#160;</td>
			<td><img alt="Equation 1" src="/files/ftp_upload/61798/61798eq1.png" /> 90 mm</td>
		</tr>
		<tr>
			<td>Pipette Controllers-EASY 60</td>
			<td>Labbox</td>
			<td>EASY-P60-001</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Potato Dextrose Agar</td>
			<td>Sigma&#160;</td>
			<td>70139-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision scale-RADWAG</td>
			<td>Grosseron</td>
			<td>B126698</td>
			<td>AS220.R2-ML 220g/0.1mg&#160;</td>
		</tr>
		<tr>
			<td>Rake</td>
			<td>Sarstedt</td>
			<td>86-1569001</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Reverse microscope AE31E trinocular</td>
			<td>Grosseron</td>
			<td>M097917</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Sterile graduated pipette</td>
			<td>Sarstedt</td>
			<td>1254001</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Thymus essential oil</td>
			<td>Drugstore</td>
			<td></td>
			<td>Essential oil 100%</td>
		</tr>
		<tr>
			<td>Tips 1000 &#181;L&#160;</td>
			<td>Sarstedt</td>
			<td>70.762010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 20 &#181;L&#160;</td>
			<td>Sarstedt</td>
			<td>70.760012</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips 200 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.760002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tooth pick</td>
			<td>retail store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trichoderma spp strain</td>
			<td></td>
			<td></td>
			<td>Strain of LRPIA laboratory</td>
		</tr>
		<tr>
			<td>Tween-20&#160;</td>
			<td>Sigma&#160;</td>
			<td>P1379-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Sigma&#160;</td>
			<td>P1754-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Labbox</td>
			<td>FORS-001-002</td>
			<td>/</td>
		</tr>
	</tbody>
</table>

measuring volatile and non-volatile antifungal activity of biocontrol products

所述协议基于插头传输技术，可准确确定微生物数量及其发育阶段。特定数量的孢子分布在阿加板上。这种阿加板的孵育期为一定时期，使真菌达到预期的发展阶段，但不需要孵化的孢子除外。下一步，由孢子、催眠或菌斑覆盖的 Agar 插头将被提取并转移到含有抗真菌化合物的 agar 介质中，以测试放置在离真菌或接触的距离。此方法适用于测试液体提取物和固体样品（粉末）。它特别适合量化挥发性和非挥发性制剂在生物活性混合物中的相对贡献，并确定其影响，特别是对孢子、早期催眠和菌精的影响。
该方法与生物控制产品，特别是植物衍生产品抗真菌活性的定性具有高度相关性。事实上，对于植物处理，结果可用于指导应用模式的选择和建立触发阈值。

为了评估定量方法区分不同类型抗真果化合物作用模式的能力，我们比较了三种知名抗真果剂的疗效。卡本达齐姆是一种非挥发性合成杀菌剂，已广泛用于控制植物中广泛的真菌疾病39，40。提木斯粗俗精油主要被描述为其抗菌和抗真菌活性，并被用作天然食品防腐剂41。大蒜粉被选为植物衍生生物制品的模型。传统上，它一直?...

Watch this Scientific Journal Video about Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products at JoVE.com

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

我们描述了一种基于阿加的改进方法，旨在量化植物衍生产品的抗真果效应。可通过本议定书评估对抗真果活动的挥发性和非挥发性贡献。此外，在一项实验设置中，可在关键发育阶段测量对真菌的疗效。

测量生物控制产品的挥发性和非挥发性抗真菌活性

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

This protocol provides a method for making accurate side-by-side comparisons of the relative efficacy of volatile and non-volatile antifungal compounds at different fungal growth stages. This method is well-suited for evaluating the antifungal activity of plant-derived products used in dried or liquid forms that contain a wide diversity of molecules. This method can provide valuable information about the mode of application of plant-derived products and is particularly well-suited in the field of biocontrol.Demonstrating the procedure will be Valentina Gligorijevic, an engineer assistant from the Sup'Biotech laboratory. For conidia collection, layer three milliliters of 0.05%Tween 20 on a trichoderma mycelium culture and use a rake to release the conidia from the conidiophores taking care not to press down on the mycelium to prevent the hyphae from being torn away. When all of the conidia have been released, use a micropipette to quickly recover and add the conidia solution to a 15 milliliter tube for counting.To prepare fungal plates, deposit 100 microliters of spores at a three times 10 to the six spores per milliliter concentration onto a nine centimeter Petri dish containing PDA medium, and use a sterile spatula to add 10 grams of two millimeter diameter glass beads to the plate. Then gently shake the plate forward and backward and spin and rotate the plate in 90 degree segments until it has been fully rotated to evenly distribute the spores across the surface of the agar. Then incubate the plate at 30 degrees Celsius until growth inhibition analysis.To perform a contact-inhibition assay with garlic powder, use a sterile spatula to weigh the desired garlic powder quantity into individual 50 milliliter tubes. to obtain concentrations generally ranging from 0.25 to 16 milligrams per milliliter. Add 10 milliliters of approximately 45 degrees Celsius PDA to each tube and carefully invert each tube several times to evenly distribute the powder throughout the agar.Quickly, pour the homogenized suspensions into five centimeter diameter Petri dishes and allow the agar to solidify at room temperature. To perform a contact-inhibition assay, use a five millimeter diameter sterile stainless steel tube to plot a circle in the center of the agar in the control and antifungal compound-treated dishes, and use a sterile toothpick to dispose of the agar cylinders. Next, use a new five millimeter diameter stainless steel tube to plot 15 to 20 circles randomly into the fungal plates, and use sterile toothpicks to carefully transfer the spore, hyphae, or mycelium-covered cylinders into the empty regions of the PDA plates.Then return the plates to the 30 degree Celsius incubator. To prepare a plates for a vapor-phase antifungal inhibition assay, pour 10 milliliters of PDA medium into the lid of a five centimeter diameter Petri dish containing 10 milliliters of PDA medium containing antifungal compounds or PDA medium alone, and allow the agar to solidify at room temperature. Using a 50 milliliter centrifical tube as a calibration tool, make a circle of PDA in the center of the agar and use a sterile spatula to remove the agar around the circle.Use a five millimeter diameter sterile stainless steel tube to plot a circle in the center of the medium in the lid and use a sterile toothpick to discard the auger cylinder. Then use a five millimeter diameter sterile stainless steel tube to randomly make plugs in the prepared fungal plates, and use a sterile toothpick to carefully transfer spore, hyphae, or mycelium-coated plugs into the lids of the assay plates. In this analysis, trichoderma species SBT 10-2018 growth inhibition triggered by three antifungal compounds was evaluated using contact and vapor-phase inhibition assays as demonstrated for each fungal stage.A higher spore sensitivity to carbendazim was observed compared to early hyphae and mycelium networks when trichoderma and antifungal compounds were in contact. In contrast, carbendozim had no antifungal effect on trichoderma when the fungus was placed at a distance from the fungicide in accordance with the low volatility of this substance. When thymus vulgaris essential oil was used as an antifungal compound, a higher sensitivity was observed for spores and early hyphae as compared to mycelium growth inhibition at 0.025%essential oil.As expected, thymus vulgaris essential oil presented identical antifungal activity irrespective of the distance between the fungus and the oil. When garlic powder was used, a higher efficacy was observed against spore germination and early hyphae elongation than for mycellium growth. Particular attention must be paid during the fungal plate preparation to make sure that the spores are evenly distributed on the surface of the agar plate to ensure comparable results.

Research

JoVE Journal

Environment

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

科研

环境科学

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

Measuring Carbon-based Contaminant Mineralization Using Combined CO2 Flux and Radiocarbon Analyses

测量碳为基础的污染物矿化结合使用CO<sub&gt; 2</sub&gt;助焊剂和放射性碳分析

A method is described which uses the absence of radiocarbon in industrial chemicals and fuels made from petroleum feedstocks which frequently contaminate ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

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Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

百里香精油应用于肉类干燥过程中微生物负荷的影响

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

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

Measuring Stolons and Rhizomes of Turfgrasses Using a Digital Image Analysis System

利用数字图像分析系统测量草坪的茎和根状茎

Length and diameter of stolons or rhizomes are usually measured using simple rulers and calipers. This procedure is slow and laborious, so it is often ...

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope

木本植物的赛莱默水分布,用低温扫描电子显微镜可视化

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water ...