搜索

Fusarium graminearum Maize Stalk Infection Assay and Associated Microscopic Observation Protocol
玉米秸秆禾谷镰刀菌感染分析和显微镜观察   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae) causes Gibberella stalk rot in maize (Zea mays) and results in lodging and serious yield reduction. To develop methods to assess the fungal growth and symptom development in maize stalks, we present here a protocol of maize stalk inoculation with conidiospores of fluorescent protein-tagged F. graminearumand microscopic observation of the stalk infection process. The inoculation protocol provides repeatable results in stalk rot symptom development, and allows tracking of fungal hyphal growth inside maize stalks at cellular scale.

Keywords: Fusarium graminearum(禾谷镰刀菌), Maize stalk rot(玉米茎腐病), Intercellular invasion(细胞间侵袭), Plant fungal pathogen(植物真菌病原体), Inoculation(接种)

Background

Maize (Zea mays) is one of the most important crops across the world. The stalk is the main stem of a maize plant. It is composed of nodes and internodes (Figure 1). The internodes comprise rind and pith, and vascular tissues are scattered in the pith. Figure 1 shows the microscopic images of maize stalk sections under bright field or GFP fluorescence channel. This provides context for maize stalk inoculation and observation.

The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae) causes Gibberella stalk rot in maize (Zea mays) and results in lodging and yield loss (Jackson et al., 2009; Santiago et al., 2007). F. graminearumcan also cause seedling blight and ear rot of maize, and Fusarium head blight of wheat and barley (Jackson et al., 2009). In the field, ascospores of F. graminearumoverwinter on infected crop residues such as maize stalks and wheat straw, and may infect other plants through wounds (e.g., caused by hail or pests), or may enter through young roots, and start a new infection cycle (Jackson et al., 2009). Although the entering routes and disease development time course may differ, the final symptom for maize Gibberella stalk rot is in the stalk of adult maize plant, which directly leads to lodging and yield loss.


Figure 1. Anatomy of maize stalk. Uninfected internodes of maize stalk at V11 stage shown as reference for understanding F. graminearumprogression. V: vascular bundle. P: parenchyma cells. Green bar = 5 cm; White bars = 100 μm.

Compared to the inoculation method for assessing wheat head blight development (Pritsch et al., 2000 and 2001; Proctor et al., 1995), the inoculation method for maize stalk rot is less well established. To assess plant resistance or fungal virulence regarding maize stalk rot, two major types of inoculation methods have been reported. One is inoculation of young roots (Yang et al., 2010), which mimics the route of one type of natural infection, but has great variance in the time before stalk symptoms develop among individual maize plants, which makes follow-up microscopic observations difficult. The other is inoculation of mature stalk by wounding, usually at the internode immediately below the tassel (Zhou et al., 2010; Zheng et al., 2012) or at the internode immediately above aerial root node (Reid et al., 1996; Santiago et al., 2007) and then assessing lesions after 14 days. Recently we reported a method of wounding inoculation of maize stalks at lower internodes in combination with fluorescent protein-expressing fungi, which provided more synchronic symptom development for microscopic tracking of the infection process at the cellular scale (Zhang et al., 2016).

Materials and Reagents

  1. Sterile gauzes (regular cotton yarn 21s, 100% absorbent cotton, for medical use, many brands will work, we used 500 g pack from Shanghai HongLong Medical Material Company)
  2. Sterile 250 ml-centrifuge bottles (Beckman Coulter, catalog number: 356011 )
  3. 1.5 ml sterile centrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
  4. 500 ml flasks
  5. Single-side razor blades (CLOUD, YIZUN BRAND, catalog number: DD75 )
  6. Microscope slides (Grale Scientific, Sail Brand, catalog number: 7103 )
  7. Microscope cover slides (CITOTEST LABWARE MANUFACTURING, catalog number: 80330-2810 )
  8. Fungal strains: The F. graminearum strain PH-1 expressing fluorescent protein AmCyan under the promoter of VM3 from Neurospora crassa (AmCyanPH-1) (Yuan et al., 2008; Zhang et al., 2012).
  9. Plant material: Maize (Zea mays ssp. mays L.) cultivar B73 plants (Schnable et al., 2009) were cultivated in a phytotron at 22-26 °C with 65% relative humidity and a 14 h photoperiod for 8 weeks until the tenth leaf appeared.
  10. Glycerol (Sinopharm Chemical Reagent, catalog number: 10010618 )
  11. V8 vegetable juice (Campbell Soup Company, catalog number: V8® ORIGINAL )
  12. CaCO3
  13. 1.5% agar powder
  14. Mung bean
  15. V8 juice agar medium (see Recipes)
  16. Mung bean liquid medium (see Recipes)

Equipment

  1. Growth chamber (Jiangnan, model: RXZ-1000 )
  2. Incubator (Yiheng, model: MJ-150I )
  3. Biological safety cabinet (ESCO Micro, model: FHC1200A )
  4. Sterile tweezers (Stainless Steel Tweezers)
  5. Constant temperature shaker (Taicang, model: DHZ-DA )
  6. Haemocytometer (0.10 mm, 1/400 mm2 ) (QIUJING, model: XB-K-25 )
  7. Fluorescent microscope (Olympus, model: BX51 )
  8. Confocal microscope (Olympus, models: Fv10i and Fluoview FV1000 )
  9. Centrifuge (Beckman Coulter, model: Avanti J-E )

Software

  1. ImageJ software (http://rsbweb.nih.gov/ij/index.html)

Procedure

  1. Preparation of F. graminearum conidiospores
    Note: Operations in Procedure A should be carried out in a biological safety cabinet that has been sterilized under UV-light for at least 20 min.
    1. Pipet 10 μl of the conidia suspension of F. graminearum in 20%-30% glycerol from -80 °C freezer onto a V8 juice agar plate, and incubate in a 25 °C growth chamber for 3-5 days.
    2. Chop the V8 agar, now full of aerial hyphae, into small pieces with a sterilized tweezer, and transfer these pieces into 100 ml sterilized mung bean liquid medium, then incubate in a constant temperature shaker at 25 °C and 150 rpm for 3-5 days (Figure 2).


      Figure 2. Preparation for the inoculum of F. graminearum. A. Transfer the medium with hyphae of F. graminearum to mung bean liquid medium. B. Cultivate the liquid medium with hyphae of F. graminearum in a shaker at 25 °C and 150 rpm.

    3. Filter the mung bean liquid medium containing F. graminearum conidia through sterile gauze to remove any remaining agar, then collect the filtered liquid medium into a sterile 250 ml-centrifuge bottle. Centrifuge at 7,500 x g for 10 min at room temperature.
    4. Discard the supernatant and re-suspend the conidia pellet in 1 ml sterile water by pipetting, then transfer the conidia suspension into the 1.5 ml sterile centrifuge tube.
    5. Wash the pellet using sterile water three times and centrifuge at 7,500 x g for 1 min until all residual red medium is removed.
    6. Resuspend the conidia pellet in about 0.5 ml sterile water (the volume of water can be adjusted according to the amount of conidia), pipet 20 μl of conidia suspension onto the haemocytometer and adjust the concentration of the conidia into 105-106/ml (Figure 3A).
      Note: Fresh-made conidial suspension stored at room temperature should be used within 48 h.

  2. Maize stalk inoculation with F. graminearum conidia.
    1. Choose maize plants at V10-V11 stage (10th or 11th fully expanded leaf with the leaf collar) before anthesis for stalk infection (Figure 3B).
      Note: For B73, it usually takes about 5 weeks to grow to this stage.
    2. Keep two maize plants as untreated controls, and inoculate the others for experiments.
    3. Punch a small hole (about 10 mm in depth) in the stalk at the third or fourth internode above the soil using sterile tweezers (Figure 3C). Inject 20 μl of the conidia suspension (106/ml) into the hole.
      Note: Multiple maize plants should be inoculated in one experiment to allow at least three individual plants to be sampled per time point for observation and measurements. At least three independent experiments should be performed for statistical analysis of symptoms.
    4. As negative control, 20 μl sterile water was injected into the hole, called mock-inoculated.
    5. Wrap up the inoculated stalk with sterile gauze (Figure 3D) to retain moisture, and put it in a growth chamber (25 °C, 60% humidity, a 10 h light/day photoperiod).
    Note: The whole maize plant is used for stalk infections, not detached stems.


    Figure 3. Procedure for inoculating maize stalks with F. graminearum. A. The conidia suspension of F. graminearum for inoculation. B. The whole maize plant in V10 stage. C-D. The main steps for inoculation. Red arrow points to infect site. Yellow bar = 50 μm, Green bar = 5 cm.

  3. Microscopic observation of maize stalk infection by F. graminearum
    Conidia of F. graminearum germinate about 12 h after inoculation (hai): most hyphae grow intercellularly before 72 hai in stalks and colonize parenchymal cells around the infected site from 72 hai to 144 hai (Zhang et al., 2016).
    1. At given time-point after inoculation, maize plants were harvested, and the infected internode was split longitudinally for visual observation (Figure 4).


      Figure 4. Process for symptom observation. Black scale bar = 1 cm.

    2. Split internodes were photographed as to record lesion development (Figure 5).


      Figure 5. Representative images of the internodes inoculated by water (A) or F. graminearum (B). hai: hours after inoculation. Green scale bar = 5 cm.

    3. Split the infected maize stalk along the hole, chop half of the split internode longitudinally or transversely into small slice 5-10 mm in length and width, and place it on the glass slide immersed in sterile water covered by the coverslip.
    4. Settings for microscopic observation
      A BX51 (Olympus) taken, and then fluorescence images using a filter with excitation wavelength of 450-480 nm and emission wavelength of 515 nm. Set the three basic colors ratio as red:blue:green = 1.5:1:1. The bright field and fluorescence images were merged (Figure 6).


      Figure 6. Compound microscopic observation of F. graminearum hyphae growing in maize stalk pith tissues. H: F. graminearum AmCyan PH-1 hyphae, exhibiting green fluorescence. Hai: hours after inoculation. Scale bars = 100 μm.

    5. Confocal microscopic observation
      Choose the two channels: channel 1 with 405 nm excitation wavelength and 460-500 nm emission wavelength is for AmCyan fluorescence of F. graminearum hyphae, and channel 2 with 405 nm excitation wavelength and 570-670 nm emission wavelength is for cell wall autofluorescence. The final images are merged by the two channels.
      Note: Because maize stalk tissues also show autofluorescence, recognition of fungal fluorescence requires understanding of background maize signals as shown in Figure 1. For example, see Figure 7.


      Figure 7. Representative longitudinal section images of infected internodes at indicated time point. V: vascular bundle; P: parenchyma cells; hai: hours after inoculation. Note the autofluorescence signals from plant cell walls are stronger than in un-infected stalks. Scale bars = 100 μm.

Data analysis

The longitudinal length of brown infected areas was measured as the lesion size at the indicated time using ImageJ software (http://rsbweb.nih.gov/ij/index.html). The average distance from the wounding line for both the top and bottom fronts of the brown area was measured as half lesion size (illustrated in Figure 8). Hyphal advancing distance was measured using a fluorescence microscope with GFP filter (Figure 8).


Figure 8. Measurements of lesion size, half lesion size and distance of hyphal advance

Notes

This protocol uses horizontal wounding, instead of a 45 °C wounding angle. The horizontal protocol provides more uniform fungal growth.

Recipes

  1. V8 juice agar medium
    168 ml V8 vegetable juice
    1 g CaCO3
    Add up to 1 L with sterile double distilled water
    Divide it into small aliquots, and add 15 g agar powder
    Autoclave at 121 °C for 20 min
  2. Mung bean liquid medium
    40 g mung bean (Vigna radiata) (dried, available at Grocery stores or supermarket such as Carrefour, Figure 9)
    1 L sterile double distilled water
    Boil for about 10 min and let it cool to room temperature (usually need 2 h)
    Filter through gauze and discard the bean residue
    Autoclave at 121 °C for 20 min, Store at room temperature before use


    Figure 9. Mung beans used in preparing medium

Acknowledgments

This protocol was modified from previous inoculation method of Colletotrichum graminicola (Tang et al., 2006). We thank Dr. Sheila McCormick for editing the protocol. The research in the Tang lab was supported by the Ministry of Science and Technology of China (Grant 2016YFD0100600), the Natural Science Foundation of China (Grant 31570318) and the Ministry of Agriculture of China (Grant 2016ZX08009-003).

References

  1. Jackson, T. A., Rees, J. M. and Harveson, R. M. (2009). Common stalk rot diseases of corn. The Board of Regents of the University of Nebraska.
  2. Pritsch, C., Muehlbauer, G. J., Bushnell, W. R., Somers, D. A. and Vance, C. P. (2000). Fungal development and induction of defense response genes during early infection of wheat spikes by Fusarium graminearum. Mol Plant-Microbe In 13(2): 159-169.
  3. Pritsch, C., Vance, C. P., Bushnell, W. R., Somers, D. A., Hohn, T. M. and Muehlbauer, G. J. (2001). Systemic expression of defense response genes in wheat spikes as a response to Fusarium graminearum infection. Physiol Mol Plant P 58(1): 1-12.
  4. Proctor, R. H., Hohn, T. M. and McCormick, S. P. (1995). Reduced virulence of Gibberella zeae caused by disruption of a trichthecine toxin biosynthetic gene. MPMI 8(4): 593-601.
  5. Reid, L. M., Hamilton, R. I. and Mather, D. E. (1996). Screening maize for resistance to Gibberella ear rot. Agriculture and Agri-Food Canada: Ontario Technical Bulletin: 1996-5E.
  6. Santiago, R., Reid, L. M., Arnason, J. T., Zhu, X., Martinez, N. and Malvar, R. A. (2007). Phenolics in maize genotypes differing in susceptibility to Gibberella stalk rot (Fusarium graminearum Schwabe). J Agric Food Chem 55: 5186-5193.
  7. Schnable, P. S., Ware, D., Fulton, R. S., Stein, J. C., Wei, F., Pasternak, S., Liang, C., Zhang, J., Fulton, L., Graves, T. A., Minx, P., Reily, A. D., Courtney, L., Kruchowski, S. S., Tomlinson, C., Strong, C., Delehaunty, K., Fronick, C., Courtney, B., Rock, S. M., Belter, E., Du, F., Kim, K., Abbott, R. M., Cotton, M., Levy, A., Marchetto, P., Ochoa, K., Jackson, S. M., Gillam, B., Chen, W., Yan, L., Higginbotham, J., Cardenas, M., Waligorski, J., Applebaum, E., Phelps, L., Falcone, J., Kanchi, K., Thane, T., Scimone, A., Thane, N., Henke, J., Wang, T., Ruppert, J., Shah, N., Rotter, K., Hodges, J., Ingenthron, E., Cordes, M., Kohlberg, S., Sgro, J., Delgado, B., Mead, K., Chinwalla, A., Leonard, S., Crouse, K., Collura, K., Kudrna, D., Currie, J., He, R., Angelova, A., Rajasekar, S., Mueller, T., Lomeli, R., Scara, G., Ko, A., Delaney, K., Wissotski, M., Lopez, G., Campos, D., Braidotti, M., Ashley, E., Golser, W., Kim, H., Lee, S., Lin, J., Dujmic, Z., Kim, W., Talag, J., Zuccolo, A., Fan, C., Sebastian, A., Kramer, M., Spiegel, L., Nascimento, L., Zutavern, T., Miller, B., Ambroise, C., Muller, S., Spooner, W., Narechania, A., Ren, L., Wei, S., Kumari, S., Faga, B., Levy, M. J., McMahan, L., Van Buren, P., Vaughn, M. W., Ying, K., Yeh, C. T., Emrich, S. J., Jia, Y., Kalyanaraman, A., Hsia, A. P., Barbazuk, W. B., Baucom, R. S., Brutnell, T. P., Carpita, N. C., Chaparro, C., Chia, J. M., Deragon, J. M., Estill, J. C., Fu, Y., Jeddeloh, J. A., Han, Y., Lee, H., Li, P., Lisch, D. R., Liu, S., Liu, Z., Nagel, D. H., McCann, M. C., SanMiguel, P., Myers, A. M., Nettleton, D., Nguyen, J., Penning, B. W., Ponnala, L., Schneider, K. L., Schwartz, D. C., Sharma, A., Soderlund, C., Springer, N. M., Sun, Q., Wang, H., Waterman, M., Westerman, R., Wolfgruber, T. K., Yang, L., Yu, Y., Zhang, L., Zhou, S., Zhu, Q., Bennetzen, J. L., Dawe, R. K., Jiang, J., Jiang, N., Presting, G. G., Wessler, S. R., Aluru, S., Martienssen, R. A., Clifton, S. W., McCombie, W. R., Wing, R. A. and Wilson, R. K. (2009). The B73 maize genome: complexity, diversity, and dynamics. Science 326(5956): 1112-1115.
  8. Tang, W., Coughlan, S., Crane, E., Beatty, M. and Duvick, J. (2006). The application of laser microdissection to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum graminicola. MPMI 19(11): 1240-1250.
  9. Yang, Q., Yin, G., Guo, Y., Zhang, D., Chen, S. and Xu, M. (2010). A major QTL for resistance to Gibberella stalk rot in maize. Theor appl genet 121(4): 673-687.
  10. Yuan, T. L., Zhang, Y., Yu, X. J., Cao, X. Y. and Zhang, D. (2008). Optimization of transformation system of Fusarium graminearum. Plant Physiol Commun 44:251-256.
  11. Zhang, X. W., Jia, L. J., Zhang, Y., Jiang, G., Li, X., Zhang, D. and Tang, W. H. (2012). In planta stage-specific fungal gene profiling elucidates the molecular strategies of Fusarium graminearum growing inside wheat coleoptiles. Plant Cell 24(12): 5159-5176.
  12. Zhang, Y., He, J., Jia, L. J., Yuan, T. L., Zhang, D., Guo, Y., Wang, Y. and Tang, W. H. (2016). Cellular tracking and gene profiling of Fusarium graminearum during maize stalk rot disease development elucidates its strategies in confronting phosphorus limitation in the host apoplast. PLoS Pathog 12(3): e1005485.
  13. Zheng, D., Zhang, S., Zhou, X., Wang, C., Xiang, P., Zheng, Q. and Xu, J. R. (2012). The FgHOG1 pathway regulates hyphal growth, stress responses, and plant infection in Fusarium graminearum. PloS one 7(11): e49495.
  14. Zhou, X., Heyer, C., Choi, Y. E., Mehrabi, R. and Xu, J. R. (2010). The CID1 cyclin C-like gene is important for plant infection in Fusarium graminearum. Fungal Genet Biol 47(2): 143-151.

简介

子囊菌真菌禾本科禾谷镰菌(以前也称为玉蜀黍赤霉)导致玉米( Zea mays )中的赤霉菌并导致倒伏和严重的产量降低。为了开发方法来评估玉米茎的真菌生长和症状发展,我们在这里提出玉米茎接种与荧光蛋白标记的F的分生孢子的协议。禾谷镰刀菌和茎显影观察茎感染过程。该接种方案在茎腐病症发展中提供可重复的结果,并且允许跟踪在细胞规模的玉米茎中的真菌菌丝生长。
关键字: 镰孢镰孢茎侵蚀,植物真菌病原体,接种。

[背景] 玉米( Zea mays )是整个土壤中最重要的作物之一世界。茎是玉米植物的主茎。它由节点和节点组成(图1)。节间包括皮和髓,并且血管组织分散在髓中。图1显示了在明场或GFP荧光通道下的玉米茎部分的显微图像。这为玉米秆接种和观察提供了背景。  子囊菌真菌禾本科镰刀菌(以前也称为玉蜀黍赤霉)会导致玉米中的赤霉菌腐烂( Zea mays >),并导致倒伏和产量损失(Jackson等人,2009; Santiago等人,2007)。 F。禾谷镰孢也可以引起小麦和大麦的玉米的幼苗枯萎和耳腐病以及小麦和大麦的镰刀菌枯叶病(Jackson等人,2009)。在该领域中,子孢子的F。禾谷镰刀菌对受感染的作物残余物如玉米秆和小麦秆过冬,并且可能通过伤口(例如由冰雹或害虫引起的)感染其他植物,或者可能通过幼根进入,并开始新的感染周期(Jackson等人,2009)。尽管进入途径和疾病发展时间过程可能不同,但是玉米赤霉菌茎腐病的最终症状在成年玉米植物的茎中,这直接导致倒伏和产量损失。


图1。 玉米秆在V11阶段的未感染节间显示为了解F的参考。 gr胺进展。 V:维管束。 P:薄壁组织细胞。绿色条= 5厘米;白色条=100μm。

 与用于评估小麦枯萎病发展的接种方法相比(Pritsch等人,2000和2001; Proctor等人,1995),玉米的接种方法茎腐病不太成熟。为了评估关于玉米秆腐病的植物抗性或真菌毒力,已经报道了两种主要类型的接种方法。一个是年轻根的接种(Yang等人,2010),其模拟一种类型的自然感染的途径,但在个体玉米植物中茎症状发展之前的时间有很大差异使得后续的显微镜观察困难。另一种是通过伤口接种成熟茎,通常在紧靠穗下的节间处(Zhou等人,2010; Zheng等人,2012)或在节间直接在气根根节点上方(Reid等人,1996; Santiago等人,2007),然后在14天后评估损伤。最近我们报道了一种在较低节间接种玉米茎的方法,其与荧光蛋白表达真菌组合,其提供更多的同步症状发展用于细胞级的感染过程的微观追踪(Zhang等人。,2016)。

关键字:禾谷镰刀菌, 玉米茎腐病, 细胞间侵袭, 植物真菌病原体, 接种

材料和试剂

  1. 无菌纱布(普通棉纱21s,100%脱脂棉,用于医疗用途,许多品牌都会使用,我们使用上海虹龙医疗材料公司的500g包装)
  2. 无菌250ml离心瓶(Beckman Coulter,目录号:356011)
  3. 1.5ml无菌离心管(Corning,Axygen ,目录号:MCT-150-C)
  4. 500 ml烧瓶
  5. 单面剃刀刀片(CLOUD,YIZUN BRAND,目录号:DD75)
  6. 显微镜载玻片(Grale Scientific,Sail Brand,目录号:7103)
  7. 显微镜盖玻片(CITOTEST LABWARE MANUFACTURING,目录号:80330-2810)
  8. 真菌菌株: (AmCyanPH-1)的VM3启动子下的禾谷镰孢菌株PH-1表达荧光蛋白AmCyan(Yuan等人,2008; Zhang等人, em>等,2012)。
  9. 植物材料:玉米(Zea mays ssp。mays)L.栽培品种B73植物(Schnable等人,2009)在植物营养在22-26℃,65%相对湿度和14小时光周期8周,直到第十叶出现
  10. 甘油(Sinopharm chemical Reagent,目录号:10010618)
  11. V8蔬菜汁(Campbell Soup Company,目录号:V8 ORIGINAL)
  12. CaCO 3
  13. 1.5%琼脂粉
  14. 绿豆
  15. V8汁琼脂培养基(见配方)
  16. 绿豆液体培养基(见配方)

设备

  1. 生长室(Jiangnan,型号:RXZ-1000)
  2. 孵化器(Yiheng,型号:MJ-150I)
  3. 生物安全柜(ESCO Micro,型号:FHC1200A)
  4. 不锈钢镊子(不锈钢镊子)
  5. 恒温振荡器(太仓,型号:DHZ-DA)
  6. 血球计(0.10mm,1/400mm )(QIJUJING,型号:XB-K-25)
  7. 荧光显微镜(Olympus,型号:BX51)
  8. 共聚焦显微镜(奥林巴斯,型号:Fv10i和Fluoview FV1000)
  9. 离心机(Beckman Coulter,型号:Avanti J-E)

软件

  1. ImageJ软件( http://rsbweb.nih.gov/ij/index .html

程序

  1. 制备F。禾本科分生孢子
    注意:程序A中的操作应在已经在紫外线下消毒至少20分钟的生物安全柜中进行。
    1. 吸取10微升的F的分生孢子悬浮液。禾谷镰孢在20%-30%甘油中的溶液从-80℃冰箱冷冻到V8汁琼脂平板上,并在25℃生长室中孵育3-5天。
    2. 使用灭菌的镊子将现在充满气生菌丝的V8琼脂切成小块,并将这些碎片转移到100ml无菌绿豆液体培养基中,然后在25℃和150rpm的恒温摇床中温育3-5天天(图2)。


      图2.F的接种物的制备。禾本科 。A.用 F菌丝转移培养基。禾谷镰菌转移到绿豆液体培养基中。 B.用em菌丝培养液体培养基。禾谷镰刀菌在振荡器中在25℃和150rpm下培养
    3. 过滤含有F的绿豆液体培养基。禾谷镰孢分生孢子通过无菌纱布去除任何残留的琼脂,然后将过滤的液体培养基收集到无菌的250ml离心瓶中。在室温下以7,500×g离心10分钟
    4. 弃去上清液,通过移液将分生孢子沉淀重悬在1ml无菌水中,然后将分生孢子悬浮液转移到1.5ml无菌离心管中。
    5. 使用无菌水洗涤沉淀三次,并以7,500×g离心1分钟,直到除去所有残留的红色培养基。
    6. 在约0.5ml无菌水(可以根据分生孢子的量调节水的体积)中重悬分生孢子沉淀,将20μl分生孢子悬浮液吸取到血细胞计数器上,并将分生孢子的浓度调节到10 5/sup> -10 /ml(图3A)。
      注意:在室温下储存的新鲜分生孢子悬浮液应在48小时内使用。

  2. 用玉米秆接种玉米秆。禾谷镰菌分生孢子
    1. 在茎感染开花前(图3B),选择V10-V11阶段的玉米植物(具有叶缘的10个或11个完全扩展的叶)。
      注意:对于B73,通常需要大约5周才能成长到这个阶段。
    2. 保持两个玉米植物作为未处理的对照,并接种其他的实验。
    3. 使用无菌镊子在土壤上方的第三或第四节间处在茎中打出小孔(深度约10mm)(图3C)。将20μl分生孢子悬浮液(10 6/ml)注入孔中。
      注意:在一个实验中应接种多个玉米植物,以允许每个时间点对至少三个单独的植物进行取样用于观察和测量。应进行至少三次独立实验用于症状的统计分析。
    4. 作为阴性对照,将20μl无菌水注入孔中,称为模拟接种
    5. 用无菌纱布包裹接种的茎(图3D)以保持水分,并将其放置在生长室(25℃,60%湿度,10小时光/天光周期)中。
    注意:整个玉米植物用于茎感染,而不是分离的茎。


    图3.用F接种玉米茎的程序。禾本科 。 禾谷镰菌接种。 B.整个玉米植株在V10期。光盘。接种的主要步骤。红色箭头指向感染网站。黄色条=50μm,绿色条= 5cm
  3. 玉米茎感染的显微镜观察。禾本科
    分生孢子。禾谷镰孢在接种后约12小时发芽(hai):大多数菌丝在茎中72小时之前在细胞间生长,并且在感染位点周围从72小时到144小时定居在实质细胞中(Zhang等人。 ,2016)。
    1. 在接种后的给定时间点,收获玉米植物,并且将受感染的节间纵向分裂用于目视观察(图4)。


      图4.症状观察的过程。黑色比例尺= 1厘米。

    2. 拍摄分裂节间以记录病变发展(图5)。


      图5.由水(A)或em接种的节间的代表性图像。禾本科(B)。 hai:接种后数小时。绿色刻度条= 5厘米。

    3. 沿着孔分割感染的玉米秆,将分裂节间的一半纵向或横向切成长度和宽度为5-10mm的小切片,并将其放置在浸没在由盖玻片覆盖的无菌水中的载玻片上。
    4. 显微镜观察设置
      使用BX51(Olympus)拍摄,然后使用具有450-480nm的激发波长和515nm的发射波长的滤光器的荧光图像。将三个基本颜色比率设置为红色:蓝色:绿色= 1.5:1:1。将明场和荧光图像合并(图6)。


      图6.化合物显微镜观察。禾谷镰孢菌丝生长在玉米茎秆组织中。 H:禾谷镰菌AmCyan PH-1菌丝,显示绿色荧光。海:接种后数小时。比例尺=100μm。

    5. 共焦显微镜观察
      选择两个通道:具有405nm激发波长的通道1和460-500nm发射波长是用于F的AmCyan荧光。禾谷镰菌菌丝,具有405nm激发波长和570-670nm发射波长的通道2用于细胞壁自体荧光。最终图像由两个通道合并。
      注意:由于玉米茎组织也显示自发荧光,真菌荧光的识别需要理解背景玉米信号,如图1所示。例如,见图7.


      图7.在指定时间点感染的节间的代表性纵断面图像。 V:维管束; P:实质细胞;海:接种后数小时。注意来自植物细胞壁的自体荧光信号比未感染的茎更强。比例尺=100μm。

数据分析

在指定时间使用ImageJ软件( http://rsbweb.nih.gov/ij/index.html )。测量棕色区域的顶部和底部正面的从伤口线的平均距离作为半损伤尺寸(如图8所示)。使用具有GFP滤光片的荧光显微镜测量菌丝前进距离(图8)

图8.损伤尺寸,半损伤尺寸和菌丝前进距离的测量

笔记

该协议使用水平创伤,而不是45°C的创伤角度。水平协议提供更均匀的真菌生长。

食谱

  1. V8汁琼脂培养基
    168毫升V8蔬菜汁
    1克CaCO 3
    用无菌双蒸水
    加入1 L 将其分成小份,加入15克琼脂粉
    在121℃高压灭菌20分钟
  2. 绿豆液体培养基
    40克绿豆( Vigna radiata )(干燥,可在杂货店或超市如家乐福,图9)
    1升无菌双蒸水
    煮沸约10分钟,让其冷却至室温(通常需要2小时) 通过纱布过滤并丢弃豆渣
    在121°C高压灭菌20分钟,使用前在室温下保存


    图9.用于制备中等 的绿豆

致谢

该方案从以前的禾本科植物Colletotrichum graminicola的接种方法改进(Tang等人,2006)。我们感谢Sheila McCormick博士编辑协议。唐实验室的研究由中国科学技术部(授予2016YFD0100600),中国自然科学基金(拨款31570318)和中国农业部(拨款2016ZX08009-003)支持。

参考文献

  1. Jackson,TA,Rees,JM和Harveson,RM(2009)。  玉米的常见茎腐病。内布拉斯加大学董事会。
  2. Pritsch,C.,Muehlbauer,GJ,Bushnell,WR,Somers,DA和Vance,CP(2000)。  真菌发育和通过禾谷镰孢早期感染小麦穗期间的防御反应基因的诱导。 -Microbe In 13(2):159-169。
  3. Pritsch,C.,Vance,CP,Bushnell,WR,Somers,DA,Hohn,TM和Muehlbauer,GJ(2001)。< a class ="ke-insertfile"href ="http://www.sciencedirect。作为对禾谷镰孢感染的应答,小麦中的防御反应基因的系统表达。 P 58(1):1-12。
  4. Proctor,RH,Hohn,TM和McCormick,SP(1995)。  由于trichthecine毒素生物合成基因的破坏引起的玉蜀黍赤霉菌的降低的毒力。 MPMI 8(4): 593-601。
  5. Reid,LM,Hamilton,RI和Mather,DE(1996)。 
  6. Santiago,R.,Reid,LM,Arnason,JT,Zhu,X.,Martinez,N.and Malvar,RA(2007)。< a class ="ke-insertfile"href ="http://pubs。 acs.org/doi/abs/10.1021/jf070641e"target ="_ blank">对玉米赤霉病的敏感性不同的玉米基因型中的酚类化合物(禾谷镰刀菌Schwabe)。 J Agric Food Chem 55:5186-5193
  7. Schnable,PS,Ware,D.,Fulton,RS,Stein,JC,Wei,F.,Pasternak,S.,Liang,C.,Zhang,J.,Fulton,L.,Graves,TA,Minx, ,Reily,AD,Courtney,L.,Kruchowski,SS,Tomlinson,C.,Strong,C.,Delehaunty,K.,Fronick,C.,Courtney,B.,Rock,SM,Belter, F.,Kim,K.,Abbott,RM,Cotton,M.,Levy,A.,Marchetto,P.,Ochoa,K.,Jackson,SM,Gillam,B.,Chen,W.,Yan, ,Higginbotham,J.,Cardenas,M.,Waligorski,J.,Applebaum,E.,Phelps,L.,Falcone,J.,Kanchi,K.,Thane,T.,Scimone,A.,Thane, ,Henke,J.,Wang,T.,Ruppert,J.,Shah,N.,Rotter,K.,Hodges,J.,Ingenthron,E.,Cordes,M.,Kohlberg,S.,Sgro, ,Delgado,B.,Mead,K.,Chinwalla,A.,Leonard,S.,Crouse,K.,Collura,K.,Kudrna,D.,Currie,J.,He,R.,Angelova, ,Rajasekar,S.,Mueller,T.,Lomeli,R.,Scara,G.,Ko,A.,Delaney,K.,Wissotski,M.,Lopez,G.,Campos,D.,Braidotti, ,Ashley,E.,Golser,W.,Kim,H.,Lee,S.,Lin,J.,Dujmic,Z.,Kim,W.,Talag,J.,Zuccolo, ,Sebastian,A.,Kramer,M.,Spiegel,L.,Nascimento,L.,Zutavern,T.,Miller,B.,Ambroise,C.,Muller,S.,Spooner,W.,Narechania, ,Ren,L.,Wei,S.,Kumari,S.,Faga,B.,Levy,MJ,McMahan,L.,Van Buren,P.,Vaughn,MW,Ying,K.,Yeh,CT,Emrich ,SJ,Jia,Y.,Kalyanaraman,A.,Hsia,AP,Barbazuk,WB,Baucom,RS,Brutnell,TP,Carpita,NC,Chaparro,C.,Chia,JM,Deragon,JM,Estill,JC, P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. ,Myers,AM,Nettleton,D.,Nguyen,J.,Penning,BW,Ponnala,L.,Schneider,KL,Schwartz,DC,Sharma,A.,Soderlund,C.,Springer,NM,Sun, ,Wang,H.,Waterman,M.,Westerman,R.,Wolfgruber,TK,Yang,L.,Yu,Y.,Zhang,L.,Zhou,S.,Zhu,Q.,Bennetzen,JL,Dawe ,RK,Jiang,J.,Jiang,N.,Presting,GG,Wessler,SR,Aluru,S.,Martienssen,RA,Clifton,SW,McCombie,WR,Wing,RAand Wilson,RK(2009) ; B73玉米基因组:复杂性,多样性和动态。/a> Science 326(5956):1112-1115。
  8. Tang,W.,Coughlan,S.,Crane,E.,Beatty,M.and Duvick,J。(2006)。  玉米中抗赤霉病的主要QTL为玉米。 > Theor appl genet 121(4):673-687。
  9. (2008)。  优化禾谷镰刀菌的转化系统。

    植物生理学通讯44:251-256。
  10. Zhang,XW,Jia,LJ,Zhang,Y.,Jiang,G.,Li,X.,Zhang,D.and Tang,WH(2012)。  在植物阶段特异性真菌基因分析中阐明了小麦胚芽鞘内生长的禾本科禾谷镰孢的分子策略。 植物细胞 24(12):5159-5176。
  11. Zhang,Y.,He,J.,Jia,LJ,Yuan,TL,Zhang,D.,Guo,Y.,Wang,Y.and Tang,WH(2016)。  玉米秆病病发展过程中细胞跟踪和禾谷镰刀菌的基因谱分析其在宿主apoplast中面对磷限制的策略。

    12(3):e1005485。

  12. 正在加载...文档类型:doc纯文本预览:(跳过预览,直接下载格式良好doc版) "http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049495"target ="_ blank"> FgHOG1 途径调节菌丝生长,胁迫反应和植物 7(11):e49495。
  13. Zhou,X.,Heyer,C.,Choi,YE,Mehrabi,R.and Xu,JR(2010)。  CID1 细胞周期蛋白C样基因对于禾谷镰孢菌中的植物感染是重要的。 > Fungal Genet Biol 47(2):143-151。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. He, J., Yuan, T. and Tang, W. (2016). Fusarium graminearum Maize Stalk Infection Assay and Associated Microscopic Observation Protocol. Bio-protocol 6(23): e2034. DOI: 10.21769/BioProtoc.2034.
  2. Zhang, Y., He, J., Jia, L. J., Yuan, T. L., Zhang, D., Guo, Y., Wang, Y. and Tang, W. H. (2016). Cellular tracking and gene profiling of Fusarium graminearum during maize stalk rot disease development elucidates its strategies in confronting phosphorus limitation in the host apoplast. PLoS Pathog 12(3): e1005485.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。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或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。