植物圈和蔬菜发酵微生物群研究的侏儒生物系统
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 种植无细菌卷心菜 准备种植无菌卷心菜的设备 清洁烧结粘土以去除细小的灰尘颗粒 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）。 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?…
Representative Results
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…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1.5 mL microcentrifuge tubes | VWR | 20170-650 | |
| 15 mL conical tubes | Falcon | 352096 | |
| 7-way tray tray | Sigma Magenta | T8654 | |
| Amber Round Boston Glass Bottle | GPS 712OZSPPK12BR | Ordered on Amazon.com from various suppliers | |
| Basket coffee filters | If you care | (unbleached paper) Purchased from Wholefoods | |
| Bleach (mercury-free) | Austin's | 50-010-45 | |
| Borosilicate Glass tubes | VWR | 47729-586 | |
| Calcined clay | Turface | MVP | Ordered on Amazon.com from Root Naturally 6 Quart Bags. Particle size approximately 3-5 mm |
| Cuisinart blender | Cuisinart | Cuisinart Mini-Prep Plus Food Processor, 3-Cup | |
| Dissection scissors | 7-389-A | American Educational Products | Ordered on Amazon.com |
| Ethanol | VWR | 89125-172 | |
| Forceps | Aven | 18434 | Ordered on Amazon.com |
| Glycerol | Fisher Scientific | 56-81-5 | |
| KleenGuard M10 | Kimberley-Clark | 64240 | |
| Large plastic container | Rubbermaid | Ordered on Amazon.com | |
| Light racks | Gardner's Supply | 39-357 | full-spectrum T5 fluorescent bulbs |
| Magenta tm 2-way caps | Millipore Sigma | C1934 | |
| Man, Rogosa, and Sharpe | Fisher Scientific | DF0881-17-5 | This media is for broth and 15 g of agar is added to make plates |
| Micro pH probe | Thermo Scientific | 8220BNWP | |
| Micropestle | Carolina | 215828 | Also called Pellet Pestle |
| MS nutrient broth | Millipore Sigma | M5519 | Murashige and Skoog Basal Medium |
| NaCl | Sigma Aldrich | S9888 | |
| Napa cabbage seeds | Johnny's Select Seeds | 2814G | B. rapa var pekinensis (Bilko) |
| Petri dish 100 mm x 15 mm | Fisher | FB0875712 | Used to make agar plates |
| Phosphate buffer saline | Fisher Scientific | 50-842-941 | Teknova |
| Plant tissue culture box | Sigma | Magenta GA-7 | |
| Serologial pipettes | VWR | 89130-900 | |
| Sterile dowel | Puritan | 10805-018 | Autoclave before use to sterilize |
| Sterilizing 0.2 µm filter | Nalgene | 974103 | |
| Tryptic soy agar | Fisher Scientific | DF0370-17-3 | This media is for broth and 15 g of agar is added to make plates |
| Wide orifice pipette tips | Rainin | 17007102 | |
| Yeast, peptone and dextrose | Fisher Scientific | DF0428-17-5 | This media is suitable but media can also be made using yeast, peptone and dextrose, add 15 g of agar when making plates |
References
- Grady, K. L., Sorensen, J. W., Stopnisek, N., Guittar, J., Shade, A. Assembly and seasonality of core phyllosphere microbiota on perennial biofuel crops. Nature Communications. 10 (1), 4135 (2019).
- Pii, Y., et al. Microbial interactions in the rhizosphere: beneficial influences of plant growth-promoting rhizobacteria on nutrient acquisition process. A review. Biology and Fertility of Soils. 51 (4), 403-415 (2015).
- Berendsen, R. L., Pieterse, C. M. J., Bakker, P. A. H. M. The rhizosphere microbiome and plant health. Trends in Plant Science. 17 (8), 478-486 (2012).
- Bai, Y., et al. Functional overlap of the Arabidopsis leaf and root microbiota. Nature. 528 (7582), 364-369 (2015).
- Bulgarelli, D., Schlaeppi, K., Spaepen, S., Ver Loren van Themaat, E., Schulze-Lefert, P. Structure and functions of the bacterial microbiota of plants. Annual Review of Plant Biology. 64, 807-838 (2013).
- Dinu, L. D., Bach, S. Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor. Applied and Environmental Microbiology. 77 (23), 8295-8302 (2011).
- Heaton, J. C., Jones, K. Microbial contamination of fruit and vegetables and the behaviour of enteropathogens in the phyllosphere: a review. Journal of Applied Microbiology. 104 (3), 613-626 (2008).
- Bringel, F., Couée, I. Pivotal roles of phyllosphere microorganisms at the interface between plant functioning and atmospheric trace gas dynamics. Frontiers in Microbiology. 6, 486 (2015).
- Maignien, L., DeForce, E. A., Chafee, M. E., Eren, A. M., Simmons, S. L. Ecological succession and stochastic variation in the assembly of Arabidopsis thaliana phyllosphere communities. mBio. 5 (1), e00682 (2014).
- Carlström, C. I., et al. Synthetic microbiota reveal priority effects and keystone strains in the Arabidopsis phyllosphere. Nature Ecology & Evolution. 3 (10), 1445-1454 (2019).
- Meyer, K. M., Leveau, J. H. J. Microbiology of the phyllosphere: a playground for testing ecological concepts. Oecologia. 168 (3), 621-629 (2012).
- Humphrey, P. T., Nguyen, T. T., Villalobos, M. M., Whiteman, N. K. Diversity and abundance of phyllosphere bacteria are linked to insect herbivory. Molecular Ecology. 23 (6), 1497-1515 (2014).
- Williams, T. R., Marco, M. L. Phyllosphere microbiota composition and microbial community transplantation on lettuce plants grown indoors. mBio. 5 (4), e01564 (2014).
- Di Cagno, R., Coda, R., De Angelis, M., Gobbetti, M. Exploitation of vegetables and fruits through lactic acid fermentation. Food Microbiology. 33 (1), 1-10 (2013).
- Köberl, M., et al. Deciphering the microbiome shift during fermentation of medicinal plants. Scientific Reports. 9 (1), 13461 (2019).
- Yu, A. O., Leveau, J. H. J., Marco, M. L. Abundance, diversity and plant-specific adaptations of plant-associated lactic acid bacteria. Environmental Microbiology Reports. 12 (1), 16-29 (2020).
- Miller, E. R., et al. Establishment limitation constrains the abundance of lactic acid bacteria in the Napa cabbage phyllosphere. Applied and Environmental Microbiology. 85 (13), e00269 (2019).
- Stamer, J. R., Stoyla, B. O., Dunckel, B. A. Growth rates and fermentation patterns of lactic acid bacteria associated with sauerkraut fermentation. Journal of Milk and Food Technology. 34 (11), 521-525 (1971).
- Yildiz, F., Westhoff, D. Associative growth of lactic acid bacteria in cabbage juice. Journal of Food Science. 46 (3), 962-963 (1981).
- Zabat, M. A., Sano, W. H., Wurster, J. I., Cabral, D. J., Belenky, P. Microbial community analysis of sauerkraut fermentation reveals a stable and rapidly established community. Foods. 7 (5), 77 (2018).
- Lee, S. H., Jung, J. Y., Jeon, C. O. Source tracking and succession of kimchi lactic acid bacteria during fermentation. Journal of Food Science. 80 (8), M1871 (2015).
- Trivedi, P., Schenk, P. M., Wallenstein, M. D., Singh, B. K. Tiny Microbes, Big Yields: enhancing food crop production with biological solutions. Microbial Biotechnology. 10 (5), 999-1003 (2017).
- Knief, C., et al. Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice. The ISME Journal. 6 (7), 1378-1390 (2012).
- Wuyts, S., et al. Carrot Juice Fermentations as Man-Made Microbial Ecosystems Dominated by Lactic Acid Bacteria. Applied and Environmental Microbiology. 84 (12), AEM.00134 (2018).
- Niu, B., Paulson, J. N., Zheng, X., Kolter, R. Simplified and representative bacterial community of maize roots. Proceedings of the National Academy of Sciences of the United States of America. 114 (12), E2450-E2459 (2017).
- Steinkraus, K. H. Lactic acid fermentation in the production of foods from vegetables, cereals and legumes. Antonie van Leeuwenhoek. 49 (3), 337-348 (1983).
Cite This Article
Miller, E. R., O’Mara Schwartz, J., Cox, G., Wolfe, B. E. A Gnotobiotic System for Studying Microbiome Assembly in the Phyllosphere and in Vegetable Fermentation. J. Vis. Exp. (160), e61149, doi:10.3791/61149 (2020).