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Quantification of Ralstonia solanacearum Colonization of Potato Germplasm Using Luminescence
发光法定量测定马铃薯种质中青枯菌的定植   

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Abstract

We have developed an unsophisticated, non-disruptive and accurate method for evaluation of pathogen colonization in planta. In this protocol we use a Ralstonia solanacearum (R. solanacearum) UY031 strain genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. This system allows bacterial quantification in a high-throughput manner, avoiding time-consuming and tedious bacterial dilution plating and colony counting. In addition, this system could be especially useful in plant breeding programs to detect bacterial latent growth in symptomless parental lines before their inclusion in long-term disease resistance breeding programs.

Materials and Reagents

  1. Ralstonia solanacearum UY031 strain transformed with the reporter plasmid pRCG-Ppslux, which bears the chloroplast promoter PpsbA and the entire LuxCDABE operon (Figures 1 and 2)
    Note: Plasmid and strain available under material transfer agreement (MTA) from Marc Valls’ laboratory.
  2. Four-week-old Solanum tuberosum (S. tuberosum) and Solanum commersonii (S. commersonii) plants
    Note: They were grown in a TREF universal potting soil mix, in a greenhouse, with 12 h light and temperatures maintained between 22 to 25 °C (50 to 60% relative humidity -RH) for three weeks and then transferred for an additional week into a growth chamber at 27 °C and 65% RH with a photoperiod of 12 h of light.
  3. Gentamicin (75 µg/ml in solid and 5 µg/ml in liquid cultures) (Sigma-Aldrich, catalog number: G1264 )
  4. Bacto-peptone (BactoTM, catalog number: 211677 )
  5. Yeast extract (BactoTM, catalog number: 210929 )
  6. Tryptone (BactoTM, catalog number: 211699 )
  7. Casamino acids (BactoTM, catalog number: 223050 )
  8. Dextrose Glucose (DifcoTM, catalog number: 215510 )
  9. Bacto-agar (BactoTM, catalog number: 214030 )
  10. Triphenyltetrazolium chloride (TTC) (DifcoTM, catalog number: 231121 )
  11. Sodium phosphate dibasic heptahydrated (Na2HPO4.7H2O) (Sigma-Aldrich, catalog number: S9390 )
  12. Potassium phosphate (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  13. Sodium chloride (NaCl) (VWR International, catalog number: 443824T )
  14. Ammonium chloride (NH4Cl) (VWR International, catalog number: 0621 )
  15. Magnesium sulfate (MgSO4) (VWR International, catalog number: 0338 )
  16. Calcium chloride (CaCl2) (VWR International, catalog number: 1.02391.1000 )
  17. L-glutamate (Sigma-Aldrich, catalog number: G1626-100G )
  18. Rich B medium (see Recipes)
  19. Boucher’s minimal medium (MM) (see Recipes)
  20. Luria and Bertani broth (LB) (see Recipes)
  21. 1 M magnesium sulfate (MgSO4) (see Recipes)
  22. 20% glucose (see Recipes)
  23. 1 M calcium chloride (see Recipes)
  24. 20 mM L-glutamate (see Recipes)
  25. To prepare 1 L of 1x MM (see Recipes)

Equipment

  1. LAS4000 Chemiluminescence and Fluorescence Imaging System (Fujifilm Life Science)
  2. Luminometer Berthold FB 12 (Titertek-Berthold, catalog number: 11010102 )
  3. Spectrophotometer (Shimadzu Kyoto, model: UV-1603 visible spectrophotometer)
  4. Incubator that can be set at 30 °C containing shaker for Erlenmeyer and tubes
  5. Tabletop centrifuge (Eppendorf, model: 5418R )
  6. Culture tubes (overflow volume 22 ml) (VWR International, catalog number: 47729-580 )
  7. Sterile 150 ml flasks
  8. 2 ml Eppendorf tubes
  9. Razor blade


    Figure 1. Vector map of pRCG-Pps-lux, designed to generate the luminescent strain for detection of bacterial colonization and growth in planta


    Figure 2. Visualization of Ralstonia solanacearum transformed with the pRCG-Pps-lux plasmid under white light A or chemiluminiscence B using the LAS4000 light imager

Procedure

  1. Measuring lux reporter expression in culture
    1. Plate bacterial strain from -80 °C frozen stock in a petri plate containing solid rich B medium, with TTC and selecting antibiotics (in this case gentamicin) overnight at 30 °C.
    2. Next day, get one colony from the plate, and grow bacterial strain overnight in a culture tube with 5 ml of rich B liquid medium (do not add TTC) in a shaker at 30 °C.
    3. The next day, make a ¼ dilution of the bacterial culture and measure its concentration using a UV visible spectrophotometer.
    4. Prepare minimal medium (MM) (20 ml per sample) into 150 ml flasks.
    5. Dilute overnight bacterial cultures in fresh MM adjusting bacterial concentration to a final OD600 of 0.1 in MM (approximately 108 cells/ml).
    6. Grow bacterial cultures at 30 °C with shaking at 200 rpm.
    7. Take 1 ml samples of the bacterial cultures every hour during 8 h starting at 0 h (control) for a total of 9 samples.
    8. For each sample, measure bacterial OD600 using the UV visible spectrophotometer and luminescence using a luminometer (express as Relative Luminescence Units or RLU). We have not used different luminometers; however, using different machines should not have an effect, because what matters are the relative measurements. Each machine should be calibrated with the control (not inoculated tissue) and all subsequent measurements are made with respect to the control for that machine.
    9. Establish background levels of luminescence.
    10. Create a correlation between bacterial concentration OD600 and RLU units (Figure 3).


      Figure 3. Correlation between cell number and luminescence in in vitro cultures of UY031 Pps-lux. Overnight cultures of the bacterial strain carrying the PpsbA::luxCDABE gene fusion were diluted in Minimal Medium and bacterial growth and reporter output were measured, respectively through OD600 and luminescence emission of 1 ml aliquots taken over an eight hour time-course. RLU= Relative luminescence units

  2. Plant inoculation and disease rating
    1. Plate bacterial strain carrying the pRCG-Pps-lux plasmid, from -80 °C frozen stock in a petri plate containing solid rich B medium, with TTC and selecting antibiotics (in this case gentamicin) overnight at 30 °C.
    2. Next day, get one colony from the plate and grow bacterial strain overnight in a 20 ml culture tube with 5 ml of rich B liquid medium (do not add TTC) in a shaker at 200 rpm at 30 °C.
    3. Pellet bacterial cells by centrifugation on a tabletop centrifuge at 5,914 x g for 2 min.
    4. Re-suspend the bacterial cells in 2 ml of water and spectrophotometrically adjust the concentration to 100 CFU/ml (corresponding to 0.01 OD600).
    5. Slightly damage the roots of four-week-old plants (S. commesonii and S. tuberosum in our case) by making three holes in the soil of each pot with a 1-ml pipette tip (2 cm deep) around the plant stem prior to inoculation. See Video 1 for more detail.

      Video 1. Drench inoculation of potato with Ralstonia solanacearum

    6. Drench-inoculate plants with 40 ml of a bacterial suspension at a concentration of 100 CFU/ml (corresponding to 0.01 OD600) per 12 cm 3 pots.
    7. After inoculation, keep plants in a growth chamber at 27 to 28 °C (65% relative humidity) with a 12 h photoperiod.
    8. Record disease development daily using an ordinal disease index scale ranging from 0 (no wilting symptoms) to 4 (all leaves wilted) (Winstead and Kelman, 1952) (Figure 4).


      Figure 4. Visual assessment of disease development by rating plant wilting phenotype: zero: No wilting; two: 50% of plant wilted; four: All leaves wilted (Zuluaga et al., 2014)

  3. Bacterial visualization in planta
    Bacterial visualization: Non-disruptive method using LAS4000 light imager system.
    1. Inoculate plants as described above.
    2. Visualize bacterial colonization of the whole plants using a Fujifilm LAS4000 light imager system with the following settings: Chemiluminescence method, incremental exposure time of 2 min, sensitivity/resolution set to high binning (Figure 5).
    3. Evaluate plants daily to assess for pathogen colonization.


      Figure 5. Bacterial colonization of whole plants using the non-disruptive method with the LAS4000 light imager system (Zuluaga et al., 2014). Days after inoculation= dai

    4. Example of the applicability of the protocol: Using bacterial visualization in planta as a tool to select for resistant potato genotypes against R. solanacearum.
      Visualization of pathogen colonization in planta using LAS4000: Correlation between disease symptoms and bacterial colonization of the plant tissue is not always direct, due to potential latent growth. The pictures below illustrate that some plants with symptoms ratings of zero (no disease) are colonized by bacteria (Figure 6). Using a bacterial strain transformed with the pRCG-Pps-lux plasmid could be especially useful in plant breeding to detect latency in symptomless parental lines before their inclusion in long-term disease resistance breeding programs.


      Figure 6. High-throughput non-disruptive evaluation of potato germplasm using R. solanacearum Lux::CDABE strain to assist selection of resistant genotypes

  4. Bacterial quantification: Disruptive method using a luminometer (Berthold FB 12).
    1. To quantify bacteria present in plants in a high-throughput manner, and avoid bacterial dilution plating and colony counting, collect one-cm sections of the root system or the stem from 1 cm above or below ground depending on the tissue to evaluate (1 cm below ground in this case, since we are evaluating root colonization).
    2. Introduce cut section (1 cm root tissue) in a 2 ml Eppendorf tube.
    3. Measure luminescence directly from the cut sections with a luminometer (Berthold FB 12).
    4. Weight each sample.
    5. Correct luminescence readings for the amount of tissue present in each sample to express the final values in RLU per milligram of tissue (Figure 7).
    6. Use the RLU/OD600 curve generated (Figure 3) to estimate bacterial concentration.


      Figure 7. Pathogen colonization is presented for four different potato genotypes with contrasting resistance to R. solanacearum. Tissue colonization is measured as Relative Luminescence Units (RLU) divided by the wet weight of the roots in milligrams (mg). Non-inoculated (N. I.) plant was used as a negative control.

Notes

  1. This procedure is highly reproducible; however, because bacterial penetration and colonization of plants is a stochastic event, at least 15 plants per treatment should be inoculated to get reliable results.
  2. Slight damage of the roots, by making three holes in the soil with a 1 ml pipette tip (two-cm deep) around the plant stem is highly recommended before inoculation, since bacteria need an entry point. See Video 1 for more details.

Recipes

  1. Rich B medium
    Bacto-peptone 10 g
    Yeast extract 1 g
    Casamino acids 1 g
    Glucose 5 g
    Solid medium contained Bacto-agar 15 g and TTC 50 mg
    Add distilled water to 1 L and autoclave
  2. Boucher’s minimal medium (MM) (Boucher et al., 1985; Monteiro et al., 2012)
    5x M9 saline solution stock
    Dissolve in 800 ml of distilled water
    Sodium phosphate heptahydrated (Na2HPO4.7H2O) 64 g
    Potassium phosphate (KH2PO4) 15 g
    Sodium chloride (NaCl) 2.5 g
    Ammonium chloride (NH4Cl) 5.0 g
    Stir until dissolved and then adjust volume to 1 L with distilled water and autoclave
  3. Luria and Bertani broth (LB) (Sambrook et al., 2000)
    Yeast extract 5 g
    Tryptone 10 g
    NaCl 10 g
    Add distilled water to 1 L and autoclave
  4. 1 M magnesium sulfate (MgSO4)
    Autoclaved
  5. 20% glucose
    Filter sterilized
  6. 1 M calcium chloride (CaCl2)
    Autoclaved
  7. To prepare 1 L of 1x MM (the final medium where R. solanacearum is grown)
    200 ml saline solution stock M9 (5x)
    800 ml distilled water
    Autoclave
    Once the medium is autoclaved, let it cool down to 60-65 °C and then add:
    2 ml of 1 M MgSO4
    0.1 ml of 1 M CaCl2
    20 mM L-glutamate as a carbon source (filter sterilized)

Acknowledgments

This work was supported by grants SGR0052 and CONES2010-0030 from Comissionat per Universitats i Recerca of the Catalan Government (Generalitat de Catalunya), AGL2010-21870 from the Ministerio de Economía of the Spanish Government, and FMV_2009_1_3045 from the National Research Council in Uruguay (ANII). We thank I. van DijK and F. Monteiro for constructing original plasmids (pG-Pps, pRCG-GWY and pRCGent-Peplux) used as sources of inserts for cloning, F. Vilaró and M. González for providing the S. commersonii genotypes used in this study and S. Balcells for lending us a luminometer.

References

  1. Boucher, C. A., Barberis, P. A. and Demery, D. A. (1985). Transposon mutagenesis of Pseudomonas solanacearum: isolation of Tn5-induced avirulent mutants. Journal of Gen Microbiol 131(9): 2449-2457.
  2. Monteiro, F., Genin, S., van Dijk, I. and Valls, M. (2012). A luminescent reporter evidences active expression of Ralstonia solanacearum type III secretion system genes throughout plant infection. Microbiology 158(Pt 8): 2107-2116.
  3. Sambrook, J., Fritsch, E. F. and Maniatis, T. (2000) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press.
  4. Winstead, N. N. and Kelman, A. (1952). Inoculation techniques for evaluating resistance to Pseudomonas solanacearum. Phytopathology 42: 628-634.
  5. Zuluaga, A. P., Ferreira, V., Pianzzola, M. J., Siri, M. I., Coll, N. S. and Valls, M. (2014). A novel, sensitive method to evaluate potato germplasm for bacterial wilt resistance using a luminescent Ralstonia solanacearum reporter strain. Mol Plant Microbe Interact 27(3): 277-285.

简介

我们已经开发了一种不成熟的,非破坏性和准确的方法用于评估植物中的病原体定居。 在该方案中,我们使用经基因修饰以从稳定插入的合成的luxCDABE 操纵子组成性地产生光的(R)solanacearum 其染色体。 该系统允许以高通量方式进行细菌定量,避免耗时和繁琐的细菌稀释电镀和菌落计数。 此外,该系统可以在植物育种程序中特别有用,以在其包括在长期疾病抗性育种程序中之前检测无症状亲本细胞中的细菌潜伏生长。

材料和试剂

  1. 用携带叶绿体启动子PpsbA和整个luxCDABE操纵子的报道质粒pRCG-Ppslux转化的UY031菌株(图1和图2) )
    注意:根据材料转让协议(MTA)从Marc Valls实验室获得的质粒和菌株。
  2. 四周龄的马铃薯( S. tuberosum br /> 注意:将它们在温室中的TREF通用盆栽土壤混合物中生长,持续12小时光照并且温度保持在22至25℃(50至60%相对湿度-RH),然后转移至在27℃和65%RH的生长室中再进行一周,光周期为12小时光照。
  3. 庆大霉素(固体75μg/ml,液体培养物5μg/ml)(Sigma-Aldrich,目录号:G1264)
  4. 细菌蛋白胨(Bacto TM ,目录号:211677)
  5. 酵母提取物(Bacto TM ,目录号:210929)
  6. 胰蛋白胨(Bacto TM ,目录号:211699)
  7. 酪氨酸(Bacto TM ,目录号:223050)
  8. 葡萄糖葡萄糖(Difco TM ,目录号:215510)
  9. 细菌琼脂(Bacto TM ,目录号:214030)
  10. 三苯基四唑氯化物(TTC)(Difco TM ,目录号:231121)
  11. 将磷酸氢二钠七水合物(Na 2 HPO 4)(Sigma-Aldrich,目录号: S9390)
  12. 磷酸钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  13. 氯化钠(NaCl)(VWR International,目录号:443824T)
  14. 氯化铵(NH 4 Cl)(VWR International,目录号:0621)
  15. 硫酸镁(MgSO 4)(VWR International,目录号:0338)
  16. 氯化钙(CaCl 2)(VWR International,目录号:1.02391.1000)
  17. L-谷氨酸(Sigma-Aldrich,目录号:G1626-100G)
  18. Rich B介质(参见配方)
  19. Boucher的最小培养基(MM)(参见食谱)
  20. Luria和Bertani肉汤(LB)(参见食谱)
  21. 1 M硫酸镁(MgSO 4)(参见配方)
  22. 20%葡萄糖(见配方)
  23. 1 M氯化钙(见配方)
  24. 20 mM L-谷氨酸(参见配方)
  25. 准备1升1x MM(参见配方)

设备

  1. LAS4000化学发光和荧光成像系统(Fujifilm Life Science)
  2. 发光计Berthold FB 12(Titertek-Berthold,目录号:11010102)
  3. 分光光度计(Shimadzu Kyoto,型号:UV-1603可见分光光度计)
  4. 孵育器可设置在30°C,包含锥形瓶和管子
  5. 台式离心机(Eppendorf,型号:5418R)
  6. 培养管(溢流容积22ml)(VWR International,目录号:47729-580)
  7. 无菌150 ml烧瓶
  8. 2 ml Eppendorf管
  9. 剃刀刀片


    图1. pRCG-Pps-lux的载体图谱,旨在产生用于检测细菌在植物中的细菌定殖和生长的发光应变。


    图2.在白光下用pRCG-Pps-lux质粒转化的Ralstonia solanacearum的可视化A或使用LAS4000光成像仪的化学发光B

程序

  1. 测量培养物中的lux记者表达
    1. 将细菌菌株从培养皿中的-80℃冷冻储存液中平板培养 含有富含B的固体培养基,用TTC和选择抗生素 这种情况下庆大霉素)在30℃下过夜
    2. 第二天,得到一个 菌落,并在培养物中培养菌株过夜 管与5毫升富B液体培养基(不要加TTC)在摇床上 30℃。
    3. 第二天,对细菌培养物进行1/4稀释,并使用紫外可见分光光度计测量其浓度
    4. 制备基本培养基(MM)(每个样品20ml)到150ml烧瓶中。
    5. 在新鲜MM调节细菌中稀释过夜细菌培养物 浓度至MM(约10 8个细胞/ml)中的0.1的最终OD 600。
    6. 在30℃下以200rpm振荡培养细菌培养物
    7. 从0小时(对照)开始,在8小时内每小时取1ml细菌培养物样品,共9个样品。
    8. 对于每个样品,使用UV可见光测量细菌OD 600 分光光度计和发光计使用发光计(express as 相对发光单位或RLU)。 我们没有使用不同的 光度计; 但是,使用不同的机器不应该有 效果,因为重要的是相对测量。 每台机器   应该用对照(不是接种的组织)和所有 相对于其的控制进行后续测量 机。
    9. 建立发光的背景水平。
    10. 创建细菌浓度OD 600和RLU单位之间的相关性(图3)

      图3. UY031Pps-lux的体外培养物中细胞数量和发光之间的相关性。细菌菌株的过夜培养物 携带ppsbA :: luxCDABE基因融合体在最小培养基中稀释 并分别测量细菌生长和报告输出 通过OD 600和1ml等分试样的发光发射 八小时的时程。 RLU =相对发光单位

  2. 植物接种和疾病等级
    1. 平板细菌菌株携带pRCG-Pps-lux质粒,从-80℃ 在含有固体富含B培养基的培养皿中的冷冻原液,用TTC 并在30℃下选择抗生素(在这种情况下是庆大霉素)过夜
    2. 第二天,从板中得到一个菌落并生长细菌菌株 在20ml培养管中用5ml富B液体培养基过夜 不加TTC)在振荡器中在200rpm,30℃下进行
    3. 通过在台式离心机上以5,914×g离心2分钟沉淀细菌细胞。
    4. 重悬细菌细胞在2毫升的水和 分光光度法将浓度调节至100CFU/ml (对应于0.01OD 600)。
    5. 轻微损害根 四周龄植物(在我们的情况下为 S. commesonii 和 S. tuberosum ) 在每个锅的土壤中用1-ml移液管尖端(2cm)制造三个孔   深)在植物茎附近。 有关详情,请参阅视频1 细节。

      视频1.用洋葱Ralstonia solanacearum 灌注马铃薯
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    6. 在40℃下用40ml细菌悬浮液对植物进行离体接种 浓度为100CFU/ml(相当于0.01OD 600)/12cm 3 壶。
    7. 接种后,将植物保持在27-28℃(65%相对湿度),12小时光周期的生长室中。
    8. 使用顺序疾病指数记录疾病发展 规模从0(无枯萎症状)到4(所有叶枯萎) (Winstead和Kelman,1952)(图4)

      图4.视觉评估 的疾病发展通过评级植物萎ting表型:零:不 枯萎 两个:50%的植物枯萎; 四:所有叶枯萎(Zuluaga et al al。,2014)

  3. 在植物中的细菌可视化
    细菌可视化:使用LAS4000光成像仪系统的无中断方法
    1. 接种如上所述的植物
    2. 可视化细菌 使用Fujifilm LAS4000光成像仪对整个植物进行定殖 系统具有以下设置:化学发光法, 增量曝光时间为2分钟,灵敏度/分辨率设置为高 (图5)。
    3. 每天评估植物以评估病原体的建群

      图5.整个植物的细菌定居 使用LAS4000光成像仪系统的非破坏性方法(Zuluaga等人, al。,2014)。接种后天= dai

    4. 示例 方案的适用性:使用细菌在植物中的可视化作为用于选择针对抗性R的抗性马铃薯基因型的工具。 solanacearum 。
      病原体定植的可视化在植物中使用  LAS4000:疾病症状与细菌的相关性 植物组织的定居并不总是直接的,由于电位 潜伏生长。下面的图片说明了一些植物 症状评级为零(无疾病)被细菌定殖(图 6)。使用用pRCG-Pps-lux质粒转化的细菌菌株 可以在植物育种中特别有用以检测延迟 无症状的父母系,然后才纳入长期疾病 抗性育种计划。


      图6.高吞吐量 使用R的土豆种质的非破坏性评价。 solanacearum Lux :: CDABE菌株以帮助选择抗性基因型

  4. 细菌定量:使用光度计(Berthold FB 12)的破坏性方法
    1. 以高通量方式定量存在于植物中的细菌,和 避免细菌稀释电镀和菌落计数,收集一厘米 根系或茎的截面高于或低于1cm 地面取决于组织评价(在地下1厘米在这 case,因为我们正在评估根殖民化)。
    2. 在2ml Eppendorf管中引入切割切片(1cm根组织)
    3. 使用光度计(Berthold FB 12)直接测量切割部分的发光。
    4. 重量每个样品。
    5. 针对存在的组织的量的正确的发光读数 每个样品表示以每毫克组织的RLU为单位的最终值 (图7)。
    6. 使用产生的RLU/OD <600>曲线(图3)估计细菌浓度

      图7.针对四种不同的马铃薯呈现病原体定殖 具有对抗R的基因型。 solanacearum 。组织 定殖作为相对发光单位(RLU)除以   根的湿重以毫克(mg)表示。 未接种(N.I.) 植物用作阴性对照。

笔记

  1. 这个程序是高度可重复的; 然而,因为植物的细菌渗透和定居是随机事件,每次处理至少应接种15株植物以获得可靠的结果。
  2. 由于细菌需要一个入口点,在接种前强烈建议在植物茎附近用1ml移液管尖(两厘米深)在土壤中形成三个孔,以轻微损坏根。 有关详情,请参阅视频1

食谱

  1. Rich B媒介
    细菌用蛋白胨10g 酵母提取物1 g
    酪蛋白氨基酸1 g
    葡萄糖5克
    固体培养基含有15g的细菌琼脂和45mg的TTC 加入蒸馏水至1升,高压釜
  2. Boucher的基本培养基(MM)(Boucher等人,1985; Monteiro等人,2012)
    5x M9盐水溶液仓
    溶于800ml蒸馏水中
    磷酸钠七水合物(Na 2 HPO 4)0.75g/g H 2 O)64g
    磷酸钾(KH 2 PO 4)15g
    氯化钠(NaCl)2.5g
    氯化铵(NH 4 Cl)5.0g
    搅拌直至溶解,然后用蒸馏水和高压釜将体积调节至1L
  3. Luria和Bertani肉汤(LB)(Sambrook et al。,2000)
    酵母提取物5 g
    胰蛋白胨10g
    NaCl 10g
    加入蒸馏水至1升,高压釜
  4. 1M硫酸镁(MgSO 4)
    高压灭菌
  5. 20%葡萄糖 过滤灭菌
  6. 1M氯化钙(CaCl 2)
    高压灭菌
  7. 准备1L的1×MM(生长茄科植物的最终培养基)
    200ml盐水溶液储液M9(5x)
    800毫升蒸馏水
    高压灭菌器
    一旦培养基高压灭菌,让它冷却到60-65°C,然后添加:
    加入2ml 1M MgSO 4 0.1ml的1M CaCl 2·6H 2 O 20mM L-谷氨酸作为碳源(过滤灭菌)

致谢

这项工作得到了来自加泰罗尼亚政府(Generalitat de Catalunya)的大学奖学金的SGR0052和CONES2010-0030,西班牙政府的经济部长的AGL2010-21870和乌拉圭国家研究委员会的FMV_2009_1_3045 ANII)。我们感谢I.van DijK和F.Orangeiro构建用作克隆插入片段的原始质粒(pG-Pps,pRCG-GWY和pRCGent-Peplux),F.Vilaró和M.González用于提供。本研究中使用的基因型和用于给我们发光度计的S. Balcells的基因型。

参考文献

  1. Boucher,C.A.,Barberis,P.A.and Demery,D.A。(1985)。 假单胞菌solanacearum的转座子诱变:分离Tn5诱导的无毒力突变体。 Journal of Gen Microbiol 131(9):2449-2457。
  2. Monteiro,F.,Genin,S.,van Dijk,I.and Valls,M。(2012)。 发光的记者证实了Ralstonia solanacearum III型分泌系统基因的活性表达整个植物感染。 微生物学 158(第8页):2107-2116
  3. Sambrook,J.,Fritsch,E.F.and Maniatis,T。(2000)Molecular cloning:A laboratory manual。 Cold Spring Harbor Laboratory Press 。
  4. Winstead,N.N。和Kelman,A。(1952)。 用于评估对假单胞菌的抗性的接种技术。 < em> Phytopathology 42:628-634。
  5. Zuluaga,A.P.,Ferreira,V.,Pianzzola,M.J.,Siri,M.I.,Coll,N.S.and Valls,M。(2014)。 一种评估马铃薯种质的新颖灵敏方法 使用发光的Ralstonia solanacearum 报道菌株进行细菌性青枯病抗性。 Mol Plant Microbe Interact 27(3):277-285。
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引用:Zuluaga, A. P., Coll, N. S. and Valls, M. (2015). Quantification of Ralstonia solanacearum Colonization of Potato Germplasm Using Luminescence . Bio-protocol 5(9): e1461. DOI: 10.21769/BioProtoc.1461.
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