使用内核生物测定真菌孢子发生和霉菌毒素生产的定量
Summary
谷类作物种子感染真菌的破坏，促使大量的研究工作，以便更好地了解植物病原体相互作用。研究种子真菌的相互作用，在实验室环境中，我们开发了一个强大的真菌繁殖，生物质能和使用内核生物测定霉菌毒素污染的量化方法。
Abstract
由种子腐烂粮食感染真菌对全球谷物产量最大的经济挑战之一，更不用说对人类和动物健康的严重风险。其中谷物产量中，玉米无疑是受影响最严重的作物，由于粮食的完整性和霉菌毒素的种子污染的病原体引起的损失。玉米种植者和食品和饲料加工两个最普遍的问题霉菌毒素是黄曲霉毒素和伏马毒素， 黄曲霉菌和镰刀菌产生，分别。
分子植物-病原体相互作用的最近的研究已经证明，在了解相关的具体机制与植物的真菌感染和真菌毒素污染1,2,3,4,5,6的承诺。因为许多实验室正在使用的内核分析，研究植物病原体相互作用，有需要为不同的生物参数量化标准的方法，所以来自不同实验室的结果可以交叉进行解释。一个强大的和可重复性的种子的定量分析手段，我们已经制定了在实验室内核检测和随后的方法，以量化霉菌的生长，生物量和霉菌毒素污染。四消毒玉米籽粒真菌悬液（10 6）接种在玻璃小瓶和培养一个预定的期限内。样品瓶，然后选择由血球，麦角甾醇的生物分析，高效液相色谱（HPLC），黄曲霉毒素定量使用AflaTest荧光法，高效液相色谱法和伏马菌素量化为分生孢子枚举。
Protocol
1。玉米内核生物活性的两个星期前，文化马铃薯葡萄糖琼脂（PDA）的病原真菌在28°C。 选择具有类似的形状和大小的内核，最好是扁平的，所以他们打下水平的生物活性瓶的底部，并在50毫升猎鹰管。内核选择必须已同时在相同的环境，以确保类似种子的年龄和代谢产物的组成。 晃动管在室温下用70％乙醇，用无菌水1分钟，10分钟，5分钟与6％次氯酸钠表面消毒内核。冲洗，…
Discussion
<p class="jove_content"这里描述的方法已被广泛的测试和产生真菌孢子形成和霉菌毒素生产的量化结果被证明是强大的。此外，这些方法应该适用于从其他mycotoxigenic真菌（如花生，小麦，棉花，开心果等），很容易受到污染的植物物种的种子。主管植物病原菌互作分析，这是必须活着保存的种子。内核的胚胎方面的一个小伤口有利于感染，同时保持种子的活力。</p><p class="jove_content"有效运行的内核检测已被证明是来?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢他们的技术援助布兰登哈塞特和卡洛斯·奥尔蒂斯。这项工作是由国家科学基金会资助的IOB-0544428，内部监督办公室-0951272，的IOS-0925561博士迈克尔Kolomiets的支持，由美国农业部国家食品和农业研究所（粮农），AFRI植物育种和教育补助金＃2010-85117 -20539到博士。赛斯美利，托马斯Isakeit，和迈克尔Kolomiets的。
Materials
| Name of the reagent | Company | Catalog # |
| Potato Dextrose Agar | Fisher Scientifc | S71659A |
| Tween-20 | Fisher Scientifc | BP337-100 |
| Plastic incubation container | Sterilite | 1713LAB06 |
| Blender | Vicam | 20200 |
| 24 cm Fluted Filter Papers | Vicam | 31240 |
| 1.5 μm glass microfibre | Vicam | 31955 |
| Afla Test column | Vicam | G1024 |
| Afrla Test Developer | Vicam | 32010 |
| Methanol | Vicam | 35016 |
| Acetonitrile | Fisher Scientifc | AC14952-0025 |
| Ethanol | Fisher Scientifc | AC39769-0025 |
| C-18 solid phase extraction column (Prep SEP SPE C18 Column) | Fisher Scientifc | 60108-304 |
| O-phthalaldehyde (OPA) | Sigma Chemical Co | 79760-5g |
| Boric acid | Fisher Scientifc | BP168-500 |
| Sodium borate | Fisher Scientifc | RDCS0330500 |
| Mercaptoethanol | Fisher Scientifc | 45-000-231 |
| Shimadzu HPLC LC-20AT (Pump) | Shimadzu Scientific Instruments, Inc. | LC-20AT |
| Zorbax ODS column (4.6x150mm) | Agilent Technologies | 443905-902 |
| Shimatzu RF-10Axl fluorescence detector | Shimadzu Scientific Instruments, Inc. | RF-10AXL |
| Sodium phosphate | Fisher Scientifc | AC38987-0010 |
| FB1 standards | Sigma Chemical Co. | F1147-1mg |
| Chloroform | VWR | MK444410 |
| 13 mm syringe filter with 0.45 um nylon membrane (HPLC) | Pall Life Science | 4426 |
| Ergosterol | Sigma-Aldrich | 45480-50G-F |
| Scintillation vials | VWR | 66021-602 |
| Sodium Chloride | Vicam | G1124 |
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Tags
QuantificationFungal ColonizationSporogenesisMycotoxinsKernel BioassaysSeed-infecting FungiCereal ProductionMaizePathogen-induced LossesGrain IntegrityMycotoxin Seed ContaminationAflatoxinFumonisinAspergillus FlavusFusarium VerticillioidesMolecular Plant-pathogen InteractionsStandardized MethodQuantifying Biological ParametersCross-interpretationKernel AssaysQuantitative AnalysesFungal GrowthBiomassMycotoxin Contamination
Cite This Article
Christensen, S., Borrego, E., Shim, W., Isakeit, T., Kolomiets, M. Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays. J. Vis. Exp. (62), e3727, doi:10.3791/3727 (2012).