Name: a gnotobiotic system for studying microbiome assembly in the phyllosphere and in vegetable fermentation
Uploaded: 2020-06-03T14:00:00
Description: Watch this Scientific Journal Video about A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation at JoVE.com

这项工作得到了美国农业部-NIFA赠款的支持：2017-67013-26520。特蕾西·德本波特和克莱尔·福根提供了技术支持，Ruby Ye和Casey Cosetta对这份手稿的早期版本提供了有益的评论。

1. 种植无细菌卷心菜
<ol><li>准备种植无菌卷心菜的设备<ol>
<li>清洁烧结粘土以去除细小的灰尘颗粒<ol>
<li>用自来水冲洗钙化粘土（材料表）至少3倍;排水。 注意：钙化粘土产生非常细的灰尘，建议在洗涤时戴上防护面罩（材料表）。</li>
			<li>将烧结粘土作为薄层（±4厘米）铺入自动包装托盘，并在干燥循环（121°C加热20分钟和20分钟干燥时间）上进行解洗，进行灭菌?...

<ol>
	<li>Grady, K. L., Sorensen, J. W., Stopnisek, N., Guittar, J., Shade, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+seasonality+of+core+phyllosphere+microbiota+on+perennial+biofuel+crops.">Assembly and seasonality of core phyllosphere microbiota on perennial biofuel crops.</a> Nature Communications. 10 (1), 4135 (2019).</li><li>Pii, Y., 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=Microbial+interactions+in+the+rhizosphere:+beneficial+influences+of+plant+growth-promoting+rhizobacteria+on+nutrient+acquisition+process.+A+review.">Microbial interactions in the rhizosphere: beneficial influences of plant growth-promoting rhizobacteria on nutrient acquisition process. A review.</a> Biology and Fertility of Soils. 51 (4), 403-415 (2015).</li><li>Berendsen, R. L., Pieterse, C. M. J., Bakker, P. A. H. 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+rhizosphere+microbiome+and+plant+health.">The rhizosphere microbiome and plant health.</a> Trends in Plant Science. 17 (8), 478-486 (2012).</li><li>Bai, Y., 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=Functional+overlap+of+the+Arabidopsis+leaf+and+root+microbiota.">Functional overlap of the Arabidopsis leaf and root microbiota.</a> Nature. 528 (7582), 364-369 (2015).</li><li>Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van Themaat, E., Schulze-Lefert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+functions+of+the+bacterial+microbiota+of+plants.">Structure and functions of the bacterial microbiota of plants.</a> Annual Review of Plant Biology. 64, 807-838 (2013).</li><li>Dinu, L. D., Bach, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+viable+but+nonculturable+Escherichia+coli+O157:H7+in+the+phyllosphere+of+lettuce:+a+food+safety+risk+factor.">Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor.</a> Applied and Environmental Microbiology. 77 (23), 8295-8302 (2011).</li><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> Journal of Applied Microbiology. 104 (3), 613-626 (2008).</li><li>Bringel, F., Cou&#233;e, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pivotal+roles+of+phyllosphere+microorganisms+at+the+interface+between+plant+functioning+and+atmospheric+trace+gas+dynamics.">Pivotal roles of phyllosphere microorganisms at the interface between plant functioning and atmospheric trace gas dynamics.</a> Frontiers in Microbiology. 6, 486 (2015).</li><li>Maignien, L., DeForce, E. A., Chafee, M. E., Eren, A. M., Simmons, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+succession+and+stochastic+variation+in+the+assembly+of+Arabidopsis+thaliana+phyllosphere+communities.">Ecological succession and stochastic variation in the assembly of Arabidopsis thaliana phyllosphere communities.</a> mBio. 5 (1), e00682 (2014).</li><li>Carlstr&#246;m, C. I., 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=Synthetic+microbiota+reveal+priority+effects+and+keystone+strains+in+the+Arabidopsis+phyllosphere.">Synthetic microbiota reveal priority effects and keystone strains in the Arabidopsis phyllosphere.</a> Nature Ecology &amp; Evolution. 3 (10), 1445-1454 (2019).</li><li>Meyer, K. M., Leveau, J. H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiology+of+the+phyllosphere:+a+playground+for+testing+ecological+concepts.">Microbiology of the phyllosphere: a playground for testing ecological concepts.</a> Oecologia. 168 (3), 621-629 (2012).</li><li>Humphrey, P. T., Nguyen, T. T., Villalobos, M. M., Whiteman, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+abundance+of+phyllosphere+bacteria+are+linked+to+insect+herbivory.">Diversity and abundance of phyllosphere bacteria are linked to insect herbivory.</a> Molecular Ecology. 23 (6), 1497-1515 (2014).</li><li>Williams, T. R., Marco, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phyllosphere+microbiota+composition+and+microbial+community+transplantation+on+lettuce+plants+grown+indoors.">Phyllosphere microbiota composition and microbial community transplantation on lettuce plants grown indoors.</a> mBio. 5 (4), e01564 (2014).</li><li>Di Cagno, R., Coda, R., De Angelis, M., Gobbetti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploitation+of+vegetables+and+fruits+through+lactic+acid+fermentation.">Exploitation of vegetables and fruits through lactic acid fermentation.</a> Food Microbiology. 33 (1), 1-10 (2013).</li><li>K&#246;berl, M., 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=Deciphering+the+microbiome+shift+during+fermentation+of+medicinal+plants.">Deciphering the microbiome shift during fermentation of medicinal plants.</a> Scientific Reports. 9 (1), 13461 (2019).</li><li>Yu, A. O., Leveau, J. H. J., Marco, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance,+diversity+and+plant-specific+adaptations+of+plant-associated+lactic+acid+bacteria.">Abundance, diversity and plant-specific adaptations of plant-associated lactic acid bacteria.</a> Environmental Microbiology Reports. 12 (1), 16-29 (2020).</li><li>Miller, E. R., 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=Establishment+limitation+constrains+the+abundance+of+lactic+acid+bacteria+in+the+Napa+cabbage+phyllosphere.">Establishment limitation constrains the abundance of lactic acid bacteria in the Napa cabbage phyllosphere.</a> Applied and Environmental Microbiology. 85 (13), e00269 (2019).</li><li>Stamer, J. R., Stoyla, B. O., Dunckel, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+rates+and+fermentation+patterns+of+lactic+acid+bacteria+associated+with+sauerkraut+fermentation.">Growth rates and fermentation patterns of lactic acid bacteria associated with sauerkraut fermentation.</a> Journal of Milk and Food Technology. 34 (11), 521-525 (1971).</li><li>Yildiz, F., Westhoff, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+growth+of+lactic+acid+bacteria+in+cabbage+juice.">Associative growth of lactic acid bacteria in cabbage juice.</a> Journal of Food Science. 46 (3), 962-963 (1981).</li><li>Zabat, M. A., Sano, W. H., Wurster, J. I., Cabral, D. J., Belenky, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+community+analysis+of+sauerkraut+fermentation+reveals+a+stable+and+rapidly+established+community.">Microbial community analysis of sauerkraut fermentation reveals a stable and rapidly established community.</a> Foods. 7 (5), 77 (2018).</li><li>Lee, S. H., Jung, J. Y., Jeon, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Source+tracking+and+succession+of+kimchi+lactic+acid+bacteria+during+fermentation.">Source tracking and succession of kimchi lactic acid bacteria during fermentation.</a> Journal of Food Science. 80 (8), M1871 (2015).</li><li>Trivedi, P., Schenk, P. M., Wallenstein, M. D., Singh, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiny+Microbes,+Big+Yields:+enhancing+food+crop+production+with+biological+solutions.">Tiny Microbes, Big Yields: enhancing food crop production with biological solutions.</a> Microbial Biotechnology. 10 (5), 999-1003 (2017).</li><li>Knief, C., 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=Metaproteogenomic+analysis+of+microbial+communities+in+the+phyllosphere+and+rhizosphere+of+rice.">Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice.</a> The ISME Journal. 6 (7), 1378-1390 (2012).</li><li>Wuyts, S., 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=Carrot+Juice+Fermentations+as+Man-Made+Microbial+Ecosystems+Dominated+by+Lactic+Acid+Bacteria.">Carrot Juice Fermentations as Man-Made Microbial Ecosystems Dominated by Lactic Acid Bacteria.</a> Applied and Environmental Microbiology. 84 (12), AEM.00134 (2018).</li><li>Niu, B., Paulson, J. N., Zheng, X., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+and+representative+bacterial+community+of+maize+roots.">Simplified and representative bacterial community of maize roots.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (12), E2450-E2459 (2017).</li><li>Steinkraus, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lactic+acid+fermentation+in+the+production+of+foods+from+vegetables,+cereals+and+legumes.">Lactic acid fermentation in the production of foods from vegetables, cereals and legumes.</a> Antonie van Leeuwenhoek. 49 (3), 337-348 (1983).</li></ol>

无细菌的纳帕卷心菜植物已被用来研究纳帕卷心菜植物圈17中乳酸菌的分散性限制。无细菌的纳帕卷心菜也可以用来测试植物圈中的个体或成对生长（图1）。对三种不同品种的卷心菜进行了无菌蔬菜提取物的制作方法：红、绿、纳帕三种。每个 SVES 都充当可靠的增长介质;接种的微生物在不同的介质中持续生长。SVE的单株增长率（图2）</strong...

植物层的多样性在维持植物健康方面起着重要作用，也会影响植物承受环境压力的能力,1、2、3、4、5。2,3,4,5反过来，作物的健康直接影响到食品安全和质量。,7植物在生态系统功能中起着一定的作用，它们相关的微生物群落既影响植物开展这些活动的能力，又直接影响环境本身。虽然科学家已经开始破译植物圈的功能和组成，但影响植物圈微生物群落群的生态过程尚未完全了解。,10植物层微生物群是研究微生物群落生态学的优良实验系统。这些社区相对简单，许多社区成员可以在标准实验室媒体10，12，13,12,上成长。
发酵蔬菜是植物圈社区结构具有重要后果的一个系统。在酸菜和泡菜中，自然存在于蔬菜叶子上的微生物（布拉西卡物种 的 植物层）是发酵14，15的孵化。乳酸菌（LAB）被认为是植物微生物群落中无处不在的成员，然而它们在植物圈16中丰度低。发酵过程中强烈的非生物选择驱动微生物群落成分的变化，使乳酸菌的丰度增加。随着实验室的成长，他们生产乳酸，从而创造了发酵蔬菜产品的酸性环境。植物层和发酵之间的联系提供了一个机会，利用蔬菜作为模型，以了解微生物群系的结构。
我们已经开发出了种植无细菌的纳帕卷心菜的方法，并使用喷雾瓶用特定的微生物群落为它们接种。这是一种廉价而可靠的方法，用单个微生物或混合群落均匀地接种卷心菜。无菌蔬菜提取物（SVE）也从三种不同的卷心菜类型/品种开发：红色和绿色卷心菜（布拉西卡油菜菜）和纳帕卷心菜（B.拉帕）。向这些 SVEs 添加盐可复制发酵环境，并允许对发酵微生物群组装进行小规模和相对较高的实验性研究。这些方法可用于研究植物圈中的微生物群落，以及如何将植物圈中的微生物群落动力学与植物发酵的成功联系在一起。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>VWR</td>
			<td>20170-650</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>7-way tray tray</td>
			<td>Sigma Magenta</td>
			<td>T8654</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber Round Boston Glass Bottle</td>
			<td></td>
			<td>GPS 712OZSPPK12BR</td>
			<td>Ordered on Amazon.com from various suppliers</td>
		</tr>
		<tr>
			<td>Basket coffee filters</td>
			<td>If you care</td>
			<td></td>
			<td>(unbleached paper) Purchased from Wholefoods</td>
		</tr>
		<tr>
			<td>Bleach (mercury-free)</td>
			<td>Austin&#39;s</td>
			<td>50-010-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass tubes</td>
			<td>VWR</td>
			<td>47729-586</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcined clay</td>
			<td>Turface</td>
			<td>MVP</td>
			<td>Ordered on Amazon.com from Root Naturally 6 Quart Bags. Particle size approximately 3-5 mm</td>
		</tr>
		<tr>
			<td>Cuisinart blender</td>
			<td>Cuisinart</td>
			<td></td>
			<td>Cuisinart Mini-Prep Plus Food Processor, 3-Cup</td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>7-389-A</td>
			<td>American Educational Products</td>
			<td>Ordered on Amazon.com</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>89125-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Aven</td>
			<td>18434</td>
			<td>Ordered on Amazon.com</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr>
			<td>KleenGuard M10</td>
			<td>Kimberley-Clark</td>
			<td>64240</td>
			<td></td>
		</tr>
		<tr>
			<td>Large plastic container</td>
			<td>Rubbermaid</td>
			<td></td>
			<td>Ordered on Amazon.com</td>
		</tr>
		<tr>
			<td>Light racks</td>
			<td>Gardner&#39;s Supply</td>
			<td>39-357</td>
			<td>full-spectrum T5 fluorescent bulbs</td>
		</tr>
		<tr>
			<td>Magenta tm 2-way caps</td>
			<td>Millipore Sigma</td>
			<td>C1934</td>
			<td></td>
		</tr>
		<tr>
			<td>Man, Rogosa, and Sharpe</td>
			<td>Fisher Scientific</td>
			<td>DF0881-17-5</td>
			<td>This media is for broth and 15 g of agar is added to make plates</td>
		</tr>
		<tr>
			<td>Micro pH probe</td>
			<td>Thermo Scientific</td>
			<td>8220BNWP</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropestle</td>
			<td>Carolina</td>
			<td>215828</td>
			<td>Also called Pellet Pestle</td>
		</tr>
		<tr>
			<td>MS nutrient broth</td>
			<td>Millipore Sigma</td>
			<td>M5519</td>
			<td>Murashige and Skoog Basal Medium</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Napa cabbage seeds</td>
			<td>Johnny&#39;s Select Seeds</td>
			<td>2814G</td>
			<td>B. rapa var pekinensis (Bilko)</td>
		</tr>
		<tr>
			<td>Petri dish 100 mm x 15 mm</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>Used to make agar plates</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline</td>
			<td>Fisher Scientific</td>
			<td>50-842-941</td>
			<td>Teknova</td>
		</tr>
		<tr>
			<td>Plant tissue culture box</td>
			<td>Sigma</td>
			<td>Magenta GA-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Serologial pipettes</td>
			<td>VWR</td>
			<td>89130-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile dowel</td>
			<td>Puritan</td>
			<td>10805-018</td>
			<td>Autoclave before use to sterilize</td>
		</tr>
		<tr>
			<td>Sterilizing 0.2 &#181;m filter</td>
			<td>Nalgene</td>
			<td>974103</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptic soy agar</td>
			<td>Fisher Scientific</td>
			<td>DF0370-17-3</td>
			<td>This media is for broth and 15 g of agar is added to make plates</td>
		</tr>
		<tr>
			<td>Wide orifice pipette tips</td>
			<td>Rainin</td>
			<td>17007102</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast, peptone and dextrose</td>
			<td>Fisher Scientific</td>
			<td>DF0428-17-5</td>
			<td>This media is suitable but media can also be made using yeast, peptone and dextrose, add 15 g of agar when making plates</td>
		</tr>
	</tbody>
</table>

a gnotobiotic system for studying microbiome assembly in the phyllosphere and in vegetable fermentation

植物层是植物的地上部分，可以由微生物殖民，是一个有用的模型系统，用于识别微生物群落的组装过程。该协议概述了一个系统，用于研究纳帕卷心菜植物植物圈中的微生物群落动力学。它描述了如何在试管中用钙化粘土和养分汤基质种植无细菌植物。接种具有特定微生物培养物的无细菌植物为测量植物圈中的微生物生长和社区动力学提供了机会。也可以评估，通过使用从卷心菜中产生的无菌蔬菜提取物在发酵过程中发生的微生物群落中发生的变化。该系统在实验室中设置相对简单且价格低廉，可用于解决微生物群落中的关键生态问题。它还提供了了解植物圈群落组成如何影响蔬菜发酵的微生物多样性和质量的机会。这种发展侏儒大白菜植物群落的方法可以应用于其他野生和农业植物物种。

纳帕卷心菜的增长率 种子灭菌方法测试了几个不同的纳帕卷心菜（B. 拉帕瓦尔佩基尼斯; 补充图1）来自多个不同供应商，且增长率一直持续。然而，用不同种类的布拉西 卡测试 方法（B. 拉帕： 萝卜紫顶; B. 奥莱拉西亚： 开罗杂交， 热带巨人杂交; B. 露营者： 朴崔玩具崔混合; B. juncea： 芥末红巨人） 给...

Watch this Scientific Journal Video about A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation at JoVE.com

A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation

一种种植无细菌的纳帕卷心菜的方法已经开发出来，使研究人员能够评估单一微生物物种或多物种微生物群落在卷心菜叶表面是如何相互作用的。还提出了一种无菌蔬菜提取物，可用于测量蔬菜发酵过程中社区成分的变化。

植物圈和蔬菜发酵微生物群研究的侏儒生物系统

The phyllosphere, the above ground portion of the plant that can be colonized by microbes, is a useful model system to identify processes of microbial ...

使用此协议，我们可以解决有关微生物如何在植物层中聚集和相互作用的关键问题。利用无菌蔬菜提取物，将植物球群落的结构与植物发酵过程中的功能联系起来，这具有额外的好处。这些技术使科学家能够测试微生物在植物球中是如何相互作用的，然后跟踪这些植物球群落如何影响发酵。要表面消毒卷心菜种子，首先将100个种子放入1.5毫升微型离心管中。将一毫升70%乙醇加入管子，然后旋涡5分钟。然后用移液器丢弃乙醇。加入一毫升50%漂白剂，在管子中旋转5分钟，然后丢弃漂白剂。冲洗种子四次，加入一毫升的自洗脱水，使管子旋转，然后丢弃水。最后一次冲洗后，将种子浸泡在消毒的去维化水中两到八小时，在种植前软化种子涂层。重量10克干净的钙粘土到15由2.5厘米玻璃管。在每个管中加入大约九毫升的已准备好的 MS Broth，以覆盖钙化粘土。用 22 毫米的双向试管盖松散地盖住玻璃管。在121摄氏度下将管材在121摄氏度下自动处理60分钟。从自动管卸下管子时，将盖推到玻璃管上以密封，然后在使用前将管冷却至室温。当管子冷却后，使用超长的钳子轻轻地将一个无菌卷心菜种子放入每个管子的中心。将管子放在一个七路托盘中，将托盘放在灯架下，在 24 摄氏度下循环 16 小时。种子将在五天后发展出他们的第一个真叶，这是第一个血管叶后，科蒂龙已经形成。它有一个皱纹的边缘，并覆盖在三梳。要测试无菌卷心菜的不育性，请用消毒钳轻轻握住植物的底座，然后将其拉出。在将卷心菜从管子中完全取出之前，用消毒解剖剪刀小心切开根部。然后，将卷心菜叶压缩成1.5毫升微离心管。在每个微型离心管中加入400微升PBS，并使用无菌微虫使卷心菜均质。刀片 100 微升的卷心菜均质到阿加板上，确保使用宽孔移液器尖端。要使甘油储存的接种菌株，轻轻地条纹出个别菌落到两个或三个新的板的同一介质。将菌落排行两到五天，然后将它们从盘子刮入15毫升锥形管中，用15毫升的15%甘油。将管子彻底搅拌以混合。将一毫升的混合甘油储存放入微型离心管中，并将其储存80摄氏度，直到与剩余的14毫升甘油库存一起准备使用。使用前一周，将一毫升等分液解冻在冰上，并放在几个不同的稀释液中，以确定接种液的浓度。当准备给卷心菜接种时，将甘油在冰上解冻，并稀释到所需的浓度。将稀释库存的 10 毫升添加到无菌泵瓶中，并将 5 种喷雾泵入大型废物收集烧杯中。从卷心菜管上取下盖子，将卷心菜向喷雾瓶倾斜。喷洒每个卷心菜的三个泵的接种溶液，然后收获一部分卷心菜，以评估实际输入接种浓度。要收获卷心菜，请用消毒钳将其从管子中取出。用无菌解剖剪刀切断根部，小心地放在预称无菌的微型离心管中。记录卷心菜的重量以进行将来的计算。将400微升PBS加入每个管与卷心菜，并研磨30次与微虫，然后板卷心菜均质使用宽孔移液器提示。取出并丢弃卷心菜的最外层叶子，并切碎所有剩余的卷心菜，放入搅拌机中。混合成细浆，称量卷心菜均质。每克卷心菜加入两毫升蒸馏水。然后通过两层篮子咖啡过滤器过滤卷心菜浆。将浆料分配到离心管中，并在20000次G下离心20分钟，或直到大颗粒从溶液中沉淀出来。拆下上一液，注意不要打扰颗粒卷心菜碎屑。要用标准盐浓度重新创建发酵条件，在提取物中加入2%的重量。将蔬菜提取物与0.2微米过滤器连接到真空，将其放入无菌管中，并在零下80摄氏度下冷冻。种子灭菌方法测试了几个纳帕卷心菜，他们都表现出类似的增长率。然而，其他品种的黄铜给予有限的成功，因为植物要么在消毒后发芽率低，要么茎迅速拉长，使旋转不健康的植物。微生物分离物作为单株分离物接种，或与其他分离物结合接种。每次治疗共测试了15个无菌卷心菜，并在接种后4天或10天内立即收获。分离表明，在纳帕卷心菜植物圈的快速生长。两个酵母和三种细菌被接种到三种不同类型的无菌蔬菜提取物或SVE由红色，绿色和纳帕卷心菜。通过将每次治疗的5微升电镀到 MRS 或 YPD 的加糖板上，记录了 14 天内接种量的增长。在整个发酵过程中对pH值的测量表明，乳酸菌能够酸化SVE至pH4以下水平，表明发酵是安全的，食用。这些方法特别适用于卷心菜微生物群，但在其他植物系统中可能有用。保持系统无菌可能具有挑战性，我们的大纲协议非常详细，但我们发现每个步骤都很重要。

Research

JoVE Journal

Environment

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

Methods for Performing Crosses in Setaria viridis, a New Model System for the Grasses

方法中进行杂交<em&gt;狗尾草</em&gt;，新模型体系的草

Setaria viridis is an emerging model system for C4 grasses. It is closely related to the bioenergy feed stock switchgrass and the grain crop foxtail ...

科研

环境科学

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

技术树内组织学习节肢动物和微生物群落

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

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

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

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

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

Methodology for Developing Life Tables for Sessile Insects in the Field Using the Whitefly, Bemisia tabaci, in Cotton As a Model System

用虱、烟粉虱在棉花中开发无梗昆虫生命表的方法作为模型系统

Life tables provide a means of measuring the schedules of birth and death from populations over time. They also can be used to quantify the sources and ...

Techniques for Investigating the Anatomy of the Ant Visual System

蚂蚁视觉系统解剖学研究方法

This article outlines a suite of techniques in light microscopy (LM) and electron microscopy (EM) which can be used to study the internal and external eye ...

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

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 ...

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

In Vitro Rearing of Solitary Bees: A Tool for Assessing Larval Risk Factors

体外孤蜂饲养: 一种评估幼虫危险因素的工具

Although solitary bees provide crucial pollination services for wild and managed crops, this species-rich group has been largely overlooked in pesticide ...