Nov 2016



Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination

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Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black fungal spores. While it is possible to directly use this spore material to infect Brachypodium seeds, in many cases it is more useful to isolate individual strains of U. bromivora for a genetically homogenous population. This protocol describes how to collect and germinate the spores of U. bromivora on plate in order to obtain strains derived from a single cell.

Keywords: Brachypodium distachyon (二穗短柄草), Ustilago bromivora (雀麦黑粉菌), Biotrophic interaction (活体营养互作), Plant pathogen (植物病原体), Filamentous fungus (丝状真菌), Head smut (黑粉病)


Ustilago maydis infecting maize (Zea mays) has long been established as a model system for studying biotrophic pathogens (Brefort et al., 2009). This has led to many discoveries concerning the nature of biotrophic interactions but has limitations due to the practical difficulties of working with maize in the laboratory. The same is true for the model fungus Ustilago hordei infecting barley (Hordeum vulgare) (Laurie et al., 2012). In contrast to these crop plants, the model grass Brachypodium distachyon has a small genome, undemanding growth conditions and is amenable to genetic manipulation (Draper et al., 2001). B. distachyon has also been used to study non-host resistance to Puccinia striiformis f. sp. tritici due to the genetic complexity of its usual host, wheat (An et al., 2016). Recently, we have described Ustilago bromivora, a smut fungus related to U. maydis, which is able to infect Brachypodium sp. and proposed this as a new model system for studying biotrophic interactions (Rabe et al., 2016).

During infection of Brachypodium sp. by U. bromivora, no visible symptoms can be detected for most of the infection. The only visible symptom of infection occurs during flowering when the plant produces spikelets that are filled with black, fungal spores. These spores can be used to directly infect new seeds but contain genetically disparate fungal strains. For most purposes, a pure culture from a single cell is preferable as it can be cultured axenically, characterized and genetically manipulated before being used to infect further seeds. This protocol describes the process of germinating the U. bromivora spores in vitro. It bears similarities to spore germination protocols of other smut fungi, for example, U. maydis (Heinze, 2009; Nadal et al., 2016), but has been modified to account for differences in the host species and infected organs. Please be aware that U. bromivora shows a mating type bias leading to the survival of only one mating type (mat a) on plate after spore germination (Rabe et al., 2016). This means that it will require additional effort to generate the second mating type or the use of the strain which we have previously isolated.

Materials and Reagents

  1. Paper bag (HERA, catalog number: 716P50 )
  2. 1.5 ml microcentrifuge tubes (SARSTEDT, catalog number: 72.690.001 )
  3. Petri dish (SARSTEDT, catalog number: 82.1473.001 )
  4. Micro-homogenizer (Carl Roth, catalog number: K994.1 )
  5. Pipetman Diamond tips, D200 (Gilson, catalog number: F161931 )
  6. Pipetman Diamond tips, D1000 (Gilson, catalog number: F161671 )
  7. Glass beads (Sigma-Aldrich, catalog number: 18406 )
  8. Copper sulfate (CuSO4) (AppliChem, catalog number: 131270 )
  9. Ampicillin (Carl Roth, catalog number: K029.2 )
  10. Tetracycline (Duchefa Biochemie, catalog number: T0150 )
  11. Chloramphenicol (AppliChem, catalog number: A1806 )
  12. Potato dextrose broth (BD, catalog number: 254920 )
  13. Agar (BD, catalog number: 214040 )
  14. PDAmp, Tet, ChlA plates (see Recipes)


  1. Scissors
  2. Cooled incubator (ST) ST 1 (Pol-Eko Aparatura, catalog number: ST 1 )
  3. Pipetman P1000 (Gilson, catalog number: F123602 )
  4. Pipetman P200 (Gilson, catalog number: F123601 )
  5. Vortex
  6. HeraeusTM PicoTM 17 Microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 , catalog number: 75002410)


  1. Harvest the infected spikelets from the plant by cutting them off using a pair of scissors. Figure 1 shows the appearance of the infected spikelets. Spikelets should be stored in a paper bag for ~10 days at 28 °C to dry out. They can then be stored at room temperature until use.

    Figure 1. Spikelets of B. distachyon infected by U. bromivora. The black masses of fungal spores are clearly visible in both the hydrated (right) and dehydrated (left) spikelet. The scale bar represents approximately 1 cm. The image background of the spikelets was digitally removed.

  2. Carefully grind the spikelets filled with spore material with a micro-homogenizer in 1.5 ml microcentrifuge tubes to break up the spikelet. The spores are very robust to survive harsh environments and will not be damaged by the grinding.
  3. Add 500 µl ddH2O and continue to grind softly. It is best to use a rotating motion to grind the spikelet as pushing into the microcentrifuge tube will cause the liquid to splash out. The liquid will turn black as it is ground, indicating that the spores have been released and are properly suspended.
  4. Incubate the ground spores in ddH2O for 1 h at room temperature to allow faster-germinating, contaminating, fungal spores to germinate. This will leave them more susceptible to the sterilisation treatment and reduce overall contamination levels.
  5. Add 500 µl 3% CuSO4 and mix the solution either by pipetting up and down or by using a vortex. This will kill most, if not all, of the contaminating spores that have germinated but not the U. bromivora spores which can take up to 17 h to germinate.
    Note: CuSO4 is a heavy metal and should be handled and discarded according to its MSDS.
  6. Incubate the spore/CuSO4 mixture for 15 min at room temperature.
  7. Centrifuge the spore mixture at 1,200 x g for 5 min. This will cause the spores to pellet. Carefully pour out the liquid and re-suspend the spores in 1 ml ddH2O.
  8. Repeat step 7 three times but, on the third time, instead of re-suspending the spores in ddH2O, proceed to step 9.
  9. Re-suspend the spores in 300 µl ddH2O with antibiotics (ampicillin, tetracycline and chloramphenicol). These will kill any non-fungal cells that might have survived the CuSO4 treatment.
  10. Make a dilution series of the fungal spore suspension (100-10-4) in ddH2O with the three antibiotics.
  11. Plate 100 µl of each dilution on a PDAmp, Tet, ChlA plate (see Recipes), spread using glass beads and incubate at 21 °C for several days to obtain colonies.
  12. As U. bromivora spores contain tetrads, the original colonies should be singled out on PD plates (with or without antibiotics) to obtain colonies derived from a single cell.


While we have provided information on the specific equipment and reagents used, we have no reason to believe that it is essential to use them exactly. The equivalent equipment or reagents from other manufacturers should be just as suitable.


  1. PDAmp, Tet, ChlA plates
    2.4% (weight/volume) potato dextrose broth
    2.0% (weight/volume) agar
    Note: Measure out the appropriate amount of potato dextrose broth and agar for the intended volume. Dissolve them in ddH2O then autoclave the mixture at 121 °C for 15 min. Once it has cooled to approximately 50 °C, add ampicillin, tetracycline and chloramphenicol to their final concentrations. Pour the mixture into Petri dishes (20 ml per 9 cm Petri dish) and wait ~20 min for it to set.


This protocol was developed in the Djamei laboratory and has had aspects contributed and/or changed by multiple members of the Djamei group. Due to this, the people listed as authors are not the only ones who have been involved in developing the protocol (for this see Rabe et al., 2016) but are merely the ones who have prepared it for publication. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement No. [EUP0012 ‘Effectomics’], the Austrian Science Fund (FWF): [P27429-B22, P27818-B22, I 3033-B22], and the Austrian Academy of Science (OEAW).


  1. An, T., Cai, Y., Zhao, S., Zhou, J., Song, B., Bux, H. and Qi, X. (2016). Brachypodium distachyon T-DNA insertion lines: a model pathosystem to study nonhost resistance to wheat stripe rust. Sci Rep 6: 25510.
  2. Brefort, T., Doehlemann, G., Mendoza-Mendoza, A., Reissmann, S., Djamei, A. and Kahmann, R. (2009). Ustilago maydis as a pathogen. Annu Rev Phytopathol 47: 423-445.
  3. Draper, J., Mur, L. A., Jenkins, G., Ghosh-Biswas, G. C., Bablak, P., Hasterok, R. and Routledge, A. P. (2001). Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol 127(4): 1539-1555.
  4. Heinze, B. (2009). Comparative analysis of the maize smut fungi Ustilago maydis and Sporisorium reilianum. Philipps-Universitat Marburg.
  5. Laurie, J. D., Ali, S., Linning, R., Mannhaupt, G., Wong, P., Guldener, U., Munsterkotter, M., Moore, R., Kahmann, R., Bakkeren, G. and Schirawski, J. (2012). Genome comparison of barley and maize smut fungi reveals targeted loss of RNA silencing components and species-specific presence of transposable elements. Plant Cell 24(5): 1733-1745.
  6. Nadal, M., Takach, J., Andrews, D., and Gold, S. (2016). Mating and progeny isolation in the corn smut fungus Ustilago maydis. Bio Protoc: e1793.
  7. Rabe, F., Bosch, J., Stirnberg, A., Guse, T., Bauer, L., Seitner, D., Rabanal, F. A., Czedik-Eysenberg, A., Uhse, S., Bindics, J., Genenncher, B., Navarrete, F., Kellner, R., Ekker, H., Kumlehn, J., Vogel, J. P., Gordon, S. P., Marcel, T. C., Munsterkotter, M., Walter, M. C., Sieber, C. M., Mannhaupt, G., Guldener, U., Kahmann, R. and Djamei, A. (2016). A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem. Elife 5.


Ustilago bromivora 是感染Brachypodium sp的生物营养性真菌真菌。 它与大麦感染黑斑病大西洋黑格尔(Hstilago hordei)密切相关,并且与研究良好的胆汁诱导型模型病原体Ustilago maydis有关。 开花时,U的小穗 受感染的植物充满黑色真菌孢子。 虽然可以直接使用这种孢子材料感染种子,但是在许多情况下,分离个体U株更有用。 bromivora 用于遗传上均匀的群体。 该协议描述了如何收集和发芽的U孢子。 溴代,以获得源自单细胞的菌株。
【背景】长期以来,感染玉米的Ustilago maydis已经被确定为用于研究生物营养性病原体的模型系统(Brefort等人,2009)。这导致了关于生物相互作用的性质的许多发现,但由于在实验室中与玉米的实际困难而受到限制。对于感染大麦(Hordeum vulgare )(Laurie等人,2012)的模型真菌Ustilago hordei也是如此。与这些作物植物相反,模式草(Brachypodium distachyon)具有小的基因组,不成熟的生长条件,并且易于遗传操作(Draper等人,2001)。  B中。 distachyon 也被用于研究非对虾对小麦条锈菌的抗性。 SP。由于其通常的宿主,小麦(An 等人,2016)的遗传复杂性,小麦具有。最近,我们已经描述了Ustilago bromivora ,这是一种与 U有关的真菌。 maydis ,能够感染
 在sp。通过romivora ,大多数感染都无法检测到明显的症状。当植物产生充满黑色,真菌孢子的小穗时,唯一可见的感染症状发生在开花期间。这些孢子可用于直接感染新种子,但含有遗传上不同的真菌菌株。对于大多数目的,来自单细胞的纯培养物是优选的,因为它可以在被用于感染另外的种子之前被种植,表征和遗传操作。该协议描述了萌发U的过程。溴菌素体外孢子。它与其他真菌真菌的孢子萌发方案相似,例如 U。 mayis (Heinze,2009; Nadal等人,2016),但已被修改以解释宿主物种和感染器官的差异。请注意,溴孢菌素显示出一种交配型偏倚,导致孢子萌发后板上只有一种交配型(垫子a)的存活(Rabe等人,2016)。这意味着需要额外的努力才能产生第二种交配型或使用我们以前隔离的菌株。

关键字:二穗短柄草, 雀麦黑粉菌, 活体营养互作, 植物病原体, 丝状真菌, 黑粉病


  1. 纸袋(HERA,目录号:716P50)
  2. 1.5ml微量离心管(SARSTEDT,目录号:72.690.001)
  3. 培养皿(SARSTEDT,目录号:82.1473.001)
  4. 微均化器(Carl Roth,目录号:K994.1)
  5. Pipetman钻石提示,D200(Gilson,目录号:F161931)
  6. 皮皮特钻石提示,D1000(Gilson,目录号:F161671)
  7. 玻璃珠(Sigma-Aldrich,目录号:18406)
  8. 硫酸铜(CuSO 4)(AppliChem,目录号:131270)
  9. 氨苄青霉素(Carl Roth,目录号:K029.2)
  10. 四环素(Duchefa Biochemie,目录号:T0150)
  11. 氯霉素(AppliChem,目录号:A1806)
  12. 马铃薯葡萄糖肉汤(BD,目录号:254920)
  13. 琼脂(BD,目录号:214040)
  14. PD Amp,Tet,ChlA板(参见食谱)


  1. 剪刀
  2. 冷藏培养箱(ST)ST 1(Pol-Eko Aparatura,目录号:ST 1)
  3. Pipetman P1000(Gilson,目录号:F123602)
  4. Pipetman P200(Gilson,目录号:F123601)
  5. 涡流
  6. Heraeus TM Pico TM微量离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Pico TM 17,目录号:75002410)


  1. 使用一把剪刀将受感染的小穗从植物中收获。图1显示感染的小穗的外观。小穗应在28℃下储存在纸袋中约10天以便干燥。然后可以在室温下储存,直到使用。

    图1. 的小穗 B。 distachyon 感染 U。 bromivora 即可。 真菌孢子的黑色质量在水合(右)和脱水(左)小穗中均清晰可见。比例尺表示大约1厘米。数字地删除小穗的图像背景。

  2. 用微量匀浆器在1.5ml微量离心管中仔细研磨填充有孢子物质的小穗,以分解小穗。孢子非常坚固,可以在恶劣的环境中生存,不会被磨削损坏。
  3. 加入500μlddH 2 O,并继续轻轻研磨。最好使用旋转运动来研磨小穗,因为推入微量离心管会导致液体飞溅。液体会在接地时变黑,表明孢子已被释放并被正确悬浮
  4. 在室温下将ddH 2 O中的地面孢子孵育1小时,以使更快的发芽,污染,真菌孢子发芽。这将使他们更容易进行灭菌处理,并降低总体污染水平
  5. 加入500μl3%CuSO 4,并通过上下移动或使用涡流混合溶液。这将会杀死大部分(如果不是全部)已经发芽而不是U型的污染孢子。 bromivora 孢子,其可能需要17小时才能发芽。
    注意:CuSO 4 是一种重金属,应根据其MSDS进行处理和丢弃。
  6. 在室温下孵育孢子/ CuSO 4 混合物15分钟
  7. 将孢子混合物以1,200×g离心5分钟。这将导致孢子沉淀。仔细倒出液体,并将孢子重新悬浮在1ml ddH 2 O中。
  8. 重复步骤7三次,但是第三次,而不是在ddH <2> O中重新悬浮孢子,请继续执行步骤9.
  9. 用抗生素(氨苄青霉素,四环素和氯霉素)将孢子重悬于300μlddH 2 O中。这些将杀死可能在CuSO 4 治疗中存活的任何非真菌细胞。
  10. 使用三种抗生素稀释ddH 2 O中的真菌孢子悬浮液(10μg/ ml)。
  11. 将板100μl的PD Amp,Tet,ChlA板(参见食谱)上的每种稀释液用玻璃珠扩散,并在21℃下孵育数天以获得菌落。
  12. 由于孢子含有四分体,因此应在PD平板上挑选出原始菌落(含有或不含抗生素),以获得源于单个细胞的菌落。




  1. PD Amp,Tet,ChlA板
    注意:测量适量的马铃薯葡萄糖肉汤和琼脂用于预期体积。将它们溶解在ddH 2 中,然后将混合物在121℃高压15分钟。一旦冷却至约50°C,将氨苄青霉素,四环素和氯霉素加入其最终浓度。将混合物倒入培养皿(每9厘米培养皿中20毫升),等待约20分钟即可进行。


该协议是在Djamei实验室开发的,并且已经由Djamei组的多个成员贡献和/或改变了方面。因此,列为作者的人不是唯一参与制定协议的人(参见Rabe 等人,2016年),但只是为了准备出版物。导致这些结果的研究得到欧洲研究理事会根据欧盟第七框架计划(FP7 / 2007-2013)/ ERC拨款协议号[EUP0012“效应”],奥地利科学基金(FWF)的资助: P27429-B22,P27818-B22,I3033-B22]和奥地利科学院(OEAW)。


  1. An,T.,Cai,Y.,Zhao,S.,Zhou,J.,Song,B.,Bux,H. and Qi,X.(2016)。&nbsp; T型DNA插入线:研究非小麦耐药性的模型病理系统条纹锈迹。 Sci Rep 6:25510.
  2. Brefort,T.,Doehlemann,G.,Mendoza-Mendoza,A.,Reissmann,S.,Djamei,A.and Kahmann,R。(2009)。&nbsp; Ustilago maydis 作为病原体。 Annu Rev Phytopathol 47:423-445。
  3. Draper,J.,Mur,LA,Jenkins,G.,Ghosh-Biswas,GC,Bablak,P.,Hasterok,R。和Routledge,AP(2001)。&lt; a class =“ke-insertfile”href = “http://www.ncbi.nlm.nih.gov/pubmed/11743099”target =“_ blank”> Brachypodium distachyon 。草原功能基因组学的新模型系统。植物生理学127(4):1539-1555。
  4. Heinze,B。(2009)。&nbsp; 比较分析玉米真菌真菌Ustilago maydis 和 Sporisorium reilianum Philipps-Universitat Marburg 。
  5. Laurie,JD,Ali,S.,Linning,R.,Mannhaupt,G.,Wong,P.,Guldener,U.,Munsterkotter,M.,Moore,R.,Kahmann,R.,Bakkeren,G.and Schirawski ,J.(2012)。&nbsp; 大麦和玉米真菌真菌显示RNA沉默组分的靶向丢失和转座因子的物种特异性存在。植物细胞 24(5):1733-1745。
  6. Nadal,M.,Takach,J.,Andrews D.和Gold,S。(2016)。玉米屎肠杆菌中的交配和后代分离Ustilago maydis 生物原核生物: e1793。
  7. Rabe,F.,Bosch,J.,Stirnberg,A.,Guse,T.,Bauer,L.,Seitner,D.,Rabanal,FA,Czedik-Eysenberg,A.,Uhse,S.,Bindics, ,Genenncher,B.,Navarrete,F.,Kellner,R.,Ekker,H.,Kumlehn,J.,Vogel,JP,Gordon,SP,Marcel,TC,Munsterkotter,M.,Walter,MC,Sieber,CM ,Mannhaupt,G.,Guldener,U.,Kahmann,R.and Djamei,A。(2016)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih。 gov / pubmed / 27835569“target =”_ blank“>用于研究Ustilago bromivora 和 Brachypodium sp的完整工具集。作为真菌温带草病系统。 Elife 5.
  • English
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Copyright Bosch and Djamei. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Bosch, J. and Djamei, A. (2017). Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination. Bio-protocol 7(14): e2392. DOI: 10.21769/BioProtoc.2392.
  2. Rabe, F., Bosch, J., Stirnberg, A., Guse, T., Bauer, L., Seitner, D., Rabanal, F. A., Czedik-Eysenberg, A., Uhse, S., Bindics, J., Genenncher, B., Navarrete, F., Kellner, R., Ekker, H., Kumlehn, J., Vogel, J. P., Gordon, S. P., Marcel, T. C., Munsterkotter, M., Walter, M. C., Sieber, C. M., Mannhaupt, G., Guldener, U., Kahmann, R. and Djamei, A. (2016). A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem. Elife 5.

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如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。