In vitro Cell Wall Stress Assay for Fusarium oxysporum

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Molecular Plant Pathology
Aug 2015



In this protocol we describe a cell wall stress assay for the fungal pathogen F. oxysporum, based on exposure to the two anionic dyes Calcofluor White (CFW) and Congo Red (CR). Both compounds have been used to exert stress upon the fungal cell wall in vitro (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Leach et al., 2012; Heilmann et al., 2013; Garcia et al., 2015). CFW perturbs chitin assembly, whereas CR interferes with β-glucan synthesis, resulting in cell wall-weakening and activation of the cell wall stress response (Ram and Klis, 2006; Kopecka and Gabriel, 1992; Roncero and Duran, 1985). Presumably, the signaling pathways and cell wall changes associated with this response reflect cell wall homeostasis during normal growth as well as cell wall remodeling events in response to stresses encountered during the fungus-host interactions. The conditions for preparation of CFW and CR culture medium specified in this protocol are based on the paper by Ram and Klis entitled “Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red”, published in Nature protocols (Ram and Klis, 2006). This paper established the optimum conditions for preparation of CFW and CR stock solutions and suggested maintaining the culture medium at a constant pH to avoid acidification, protonation and precipitation of these dyes. This cell wall stress assay has been widely used in our group for the characterization of F. oxysporum mutants in mitogen activated protein kinase (MAPK) signaling pathway genes involved in cell wall integrity (Perez-Nadales and Di Pietro, 2015; Perez-Nadales and Di Pietro, 2011; Turra et al., 2014).

Materials and Reagents

  1. Round (90 mm diameter) or square (120 x 120 mm) Petri dishes
  2. Autoclavable, filtration material [e.g., Miracloth (Merck Millipore, catalog number: 475855 )]
  3. Calcofluor white fluorescent brightener (Sigma-Aldrich, catalog number: F-3543 )
  4. Congo red (Sigma-Aldrich, catalog number: 860956 )
  5. Distilled water
  6. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
  7. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  8. Potatoes
  9. Cloth strainer
  10. Potato dextrose agar (PDA) (Scharlab, catalog number: 01-483-500 )
  11. Yeast extract (Merck Millipore, catalog number: 1119261000 )
  12. Peptone (Merck Millipore, catalog number: 1119311000 )
  13. Glucose (VWR, Normapur®, catalog number: 50-99-7 )
  14. Agar (BD, BactoTM, catalog number: 214010 )
  15. MgSO4·7H2O (Merck Millipore, catalog number: 1058865000 )
  16. KH2PO4 (Merck Millipore, catalog number: 1048731000 )
  17. KCl (Merck Millipore, catalog number: 1049330500 )
  18. NaNO3 (Merck Millipore, catalog number: 1065371000 )
  19. Sucrose (Merck Millipore, catalog number: 1076875000 )
  20. Agar (Thermo Fisher Scientific, OxoidTM, catalog number: LP0011 )
  21. 2-(N-morpholino) ethanesulfonic acid (MES), monohydrate (Sigma-Aldrich, catalog number: 69892 )
  22. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
  23. Potato dextrose broth (PDB) (see Recipes)
  24. CFW and CR stock solutions (see Recipes)
    1. CFW stock solutions (20 mg/ml)
    2. CR stock solutions (50 mg/ml)
  25. Fungal growth agar medium (see Recipes)
    1. YPD agar medium
    2. PDA agar medium
    3. Puhalla’s minimal agar medium (MM)

Note: CFW and CR are hazardous and potentially carcinogenic so caution must be taken to avoid skin contact or inhalation of these compounds.


  1. Protection gloves and coat
  2. Flasks and magnetic stirrer for preparation of solutions
  3. Neubauer chamber for cell counting
  4. Microwave
  5. Autoclave
  6. Fungal growth incubator
  7. Water bath
  8. Laminar flow hood
  9. Heat plate and cooking pot to boil potatoes
  10. pH meter

Note: No other special equipment is required.


  1. SPSS 15.0 for Windows® (LEAD Technologies Inc.)


A major requirement for this protocol is to experimentally establish the optimum concentration of CFW or CR to be used. This may be influenced by several parameters, including size of the inoculum, genetic background of the strains and composition of the growth medium. The wild type strain used in our laboratory was Fusarium oxysporum f.sp. lycopersici (strain 4287/FGSC 9935). Appropriate CFW and CR concentration should be sublethal for the wild-type or reference strain used in the experiment. We typically define this parameter by inoculating serial dilutions of conidia in the form of spots on plates containing CFW and CR, with concentrations in the range of 10-200 μg/ml of CFW and 5-600 μg/ml of CR.

  1. Production of fresh microconidia (3-4 d)
    Inoculate 50 µl of a -80 °C stock microconidial suspension (in 30% glycerol) into 50 ml of liquid potato dextrose broth (PDB) (see Recipe 1). Grow cultures for 3-4 d at 28  °C with shaking at 170 rpm (Di Pietro and Roncero, 1998). Filter microconidia through one layer of Miracloth, centrifuge and wash with sterile water. Count conidia using a neubauer chamber and adjust to a final concentration of 1 x 107 conidia/ml.
  2. Preparation of stock solutions of CFW (20 mg/ml) and CR (50 mg/ml) (1 h) (see Recipe 2)
  3. Preparation of growth medium (1-2 h)
    Prepare growth agar medium and autoclave (20 min at 120 °C). Cool the medium down to 50-60 °C in a water bath before adding CR and CFW to avoid any potentially harming fumes. Then, add CFW or CR to reach the desired concentrations. This can be done in a laminar flow hood. For F. oxysporum, we suggest to start by testing 4-5 different dye concentrations, in the following range: 10 to 200 μg/ml for CFW and 10 to 600 µg/ml for CR. Additionally, prepare a control plate without the dyes. The plates can be stored overnight, at room temperature and in the dark, to be used the next day.
  4. Preparation of a serial dilution of microconidia (1-2 h)
    Prepare a 10-fold serial dilution of freshly-obtained microconidia starting with the 1 x 107 conidia/ml stock solution generated in step 1 (1 x 107, 1 x 106, 1 x 105 and 1 x 104 conidial/ml). Inoculate 2 µl drops of this series on plates containing CFW or CR and a control plate (final concentrations: 2 x 104, 2 x 103, 2 x 102 and 2 x 10 conidia/ml). We typically use square (120 x 120 mm) Petri dishes, to allow sufficient space for growth of the fungal colonies.
  5. Incubation of plates (2-3 d)
    Incubate plates at 28 °C in the dark for 2-3 d.
  6. Interpreting and monitoring results
    1. Sensitivity to CFW and CR should be determined by comparing the extent of colony formation of parental and mutant strains in control and CFW or CR plates. Colony diameter may be measured for quantification of differences in colony growth of the strains tested. For this, inoculate each single strain (a 5 µl drop of a 1 x 107 microconia/ml suspension) on the control and cell wall stress plates, with 5 replicates each. We use round (90 mm diameter) Petri dishes. Incubate the plates at 28 °C for 5 d before scanning and measuring colony diameter.
    2. Data from three independent experiments, each with five replicates, can then be analyzed with a statistic software such as SPSS 15.0 for Windows®. Kruskal-Wallis analysis of variance (ANOVA) and Mann-Whitney test are used to assess statistically relevant differences among strains at P ≤ 0.05.

Representative data

Figure 1 shows the sensitivity of several F. oxysporum MAPK pathway mutants to CFW and CR (Perez-Nadales and Di Pietro, 2011). In F. oxysporum, the Fmk1 MAPK pathway is essential for plant infection (Di Pietro et al., 2001). This experiment revealed that Msb2, a signaling mucin receptor functioning upstream of Fmk1 and also the Fmk1 MAPK are required for cell wall integrity in F. oxysporum under these conditions.

Figure 1. Cell wall stress assay for F. oxysporum. A. Plates containing YPD medium or YPD medium supplemented with CR or CFW were spot-inoculated with the indicated strains and amounts of microconidia, incubated for three days at 28 °C and photographed. B. Colony diameter of the indicated media was measured at day 3 post-inoculation and plotted relative to the wild-type strain (100%). Error bars represent standard deviations calculated from five independent plates. Values with the same letter are not significantly different according to Mann–Whitney test (P ≤ 0.05).


  1. During handling, CFW and CR solutions should always be protected from light by wrapping the containers in aluminum foil. As a precaution to prevent possible inactivation of CFW and CR by light, the plates should be incubated in the dark.


  1. Potato dextrose broth (PDB)
    1. Boil 200 g of pealed potatoes in 1 L of distilled water for 60 min 
    2. Filtrate through a cloth strainer
    3. Add 20 g of glucose and add up to 1 L distilled water
    4. Sterilize by autoclaving 20 min at 120 °C
  2. CFW and CR stock solutions
    1. CFW stock solutions (20 mg/ml)
      We use the following diluting solution (Ram and Klis, 2006):
      20 mg/ml CFW
      0.5% (w/v) KOH
      83% (v/v) glycerol
      Note: We recommend preparing 20-30 ml of this solution prior to the experiment and store it at room temperature. On the day of the experiment, CFW can be freshly added to this solution reach a final stock concentration of 20 mg/ml.
    2. CR stock solutions (50 mg/ml)
      Note: CR is in the di-sodium form and is readily dissolved in water.
      Prepare CR in sterile double-distilled water at a stock concentration of 50 mg/ml
    Note: It is important to ensure that CFW and CR are completely dissolved by checking that no dye powder is visible in the stock solution after gentle mixing (i.e., in a rotatory shaker).
  3. Fungal growth agar medium
    The composition of the growth medium for this cell wall stress test may vary depending on the purpose of the experiment. We have used in our assays complete media such as YPD or potato dextrose agar (PDA) and Puhalla’s minimal medium (Puhalla, 1985). All media are prepared in distilled water.
    1. YPD agar medium
      3 g/L yeast extract
      10 g/L peptone
      20 g/L glucose
      15 g/L Bacto agar
    2. PDA agar medium
      3.9% PDA, w/v
    3. Puhalla’s minimal agar medium (MM)
      0.5 g/L MgSO4·7H2O
      1 g/L KH2PO4
      0.5 g/L KCl
      2 g/L NaNO3
      30 g/L Sucrose
      Add 15 g/L of oxoid agar
    Note: For this assay, the growth medium is buffered to pH 5.5-7.0 before autoclaving (20 min at 120 °C) (Ram and Klis, 2006). For this, we add 50 mM MES to the growth medium solution and adjust to pH 6.5 with NaOH (a 10 N NaOH solution can be prepared in advance and be added slowly to the MES-buffered medium, until the desired pH is reached). After this, we add the agar (except for PDA), top up the solution to the desired volume with distilled water and autoclave it.


This research was supported by the SIGNALPATH Marie Curie Research Training Network (MRTN-CT-2005-019277) and by grants BIO2008-04479-E, EUI2009-03942, and BIO2010-15505 from the Spanish Ministerio de Ciencia e Innovación.


  1. Di Pietro, A. and Roncero, M. I. (1998). Cloning, expression, and role in pathogenicity of pg1 encoding the major extracellular endopolygalacturonase of the vascular wilt pathogen Fusarium oxysporum. Mol Plant Microbe Interact 11(2): 91-98.
  2. Di Pietro, A., Garcia-MacEira, F. I., Meglecz, E. and Roncero, M. I. (2001). A MAP kinase of the vascular wilt fungus Fusarium oxysporum is essential for root penetration and pathogenesis. Mol Microbiol 39(5): 1140-1152.
  3. Garcia, R., Botet, J., Rodriguez-Pena, J. M., Bermejo, C., Ribas, J. C., Revuelta, J. L., Nombela, C. and Arroyo, J. (2015). Genomic profiling of fungal cell wall-interfering compounds: identification of a common gene signature. BMC Genomics 16: 683.
  4. Heilmann, C. J., Sorgo, A. G., Mohammadi, S., Sosinska, G. J., de Koster, C. G., Brul, S., de Koning, L. J. and Klis, F. M. (2013). Surface stress induces a conserved cell wall stress response in the pathogenic fungus Candida albicans. Eukaryot Cell 12(2): 254-264.
  5. Kopecka, M. and Gabriel, M. (1992). The influence of Congo red on the cell wall and (1----3)-beta-D-glucan microfibril biogenesis in Saccharomyces cerevisiae. Arch Microbiol 158(2): 115-126.
  6. Leach, M. D., Budge, S., Walker, L., Munro, C., Cowen, L. E. and Brown, A. J. (2012). Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast. PLoS Pathog 8(12): e1003069.
  7. Perez-Nadales, E. and Di Pietro, A. (2015). The transmembrane protein Sho1 cooperates with the mucin Msb2 to regulate invasive growth and plant infection in Fusarium oxysporum. Mol Plant Pathol 16(6): 593-603.
  8. Perez-Nadales, E. and Di Pietro, A. (2011). The membrane mucin Msb2 regulates invasive growth and plant infection in Fusarium oxysporum. Plant Cell 23(3): 1171-1185.
  9. Puhalla, J. E. (1985). Classification of strains of Fusarium oxysporum on the basis of vegetative compatibility. Can J Bot 63(2):179-183.
  10. Ram, A. F. and Klis, F. M. (2006). Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat Protoc 1(5): 2253-2256.
  11. Roncero, C. and Duran, A. (1985). Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization. J Bacteriol 163(3): 1180-1185.
  12. Turra, D., Segorbe, D. and Di Pietro, A. (2014). Protein kinases in plant-pathogenic fungi: conserved regulators of infection. Annu Rev Phytopathol 52: 267-288.


在该协议中,我们描述了真菌病原体F的细胞壁应激测定。 ,基于暴露于两种阴离子染料Calcofluor White(CFW)和刚果红(CR)。两种化合物都已经用于在体外对真菌细胞壁施加应激(Perez-Nadales和Di Pietro,2015; Perez-Nadales和Di Pietro,2011; Leach等人, 2012; Heilmann等人,2013; Garcia等人,2015)。 CFW干扰几丁质装配,而CR干扰β-葡聚糖合成,导致细胞壁弱化和细胞壁应激反应的激活(Ram和Klis,2006; Kopecka和Gabriel,1992; Roncero和Duran,1985)。据推测,与该反应相关的信号通路和细胞壁变化反映了正常生长期间的细胞壁稳态以及响应于真菌 - 宿主相互作用期间遇到的应力的细胞壁重塑事件。本方案中规定的CFW和CR培养基的制备条件是基于Ram和Klis的论文"Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red",published in Nature protocols(Ram and Klis,2006)。本文建立了制备CFW和CR储备溶液的最佳条件,并建议将培养基保持在恒定的pH,以避免这些染料的酸化,质子化和沉淀。这种细胞壁应激测定已广泛用于我们的组中表征F。 (Perez-Nadales和Di Pietro,2015; Perez-Nadales和Di Pietro,2011; Turra等人,2004);在有丝分裂原活化蛋白激酶(MAPK)信号转导途径基因中, em>,2014)。


  1. 圆形(90mm直径)或方形(120×120mm)培养皿
  2. 可高压灭菌的过滤材料[例如,Miracloth(Merck Millipore,目录号:475855)]
  3. Calcofluor白色荧光增白剂(Sigma-Aldrich,目录号:F-3543)
  4. 刚果红(Sigma-Aldrich,目录号:860956)
  5. 蒸馏水
  6. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P5958)
  7. 甘油(Sigma-Aldrich,目录号:G5516)
  8. 土豆
  9. 布过滤器
  10. 马铃薯葡萄糖琼脂(PDA)(Scharlab,目录号:01-483-500)
  11. 酵母提取物(Merck Millipore,目录号:1119261000)
  12. 蛋白胨(Merck Millipore,目录号:1119311000)
  13. 葡萄糖(VWR,Normapur ®,目录号:50-99-7)
  14. 琼脂(BD,Bacto TM ,目录号:214010)
  15. MgSO 4·7H 2 O(Merck Millipore,目录号:1058865000)

  16. (Merck Millipore,目录号:1048731000)
  17. KCl(Merck Millipore,目录号:1049330500)
  18. NaNO 3(Merck Millipore,目录号:1065371000)
  19. 蔗糖(Merck Millipore,目录号:1076875000)
  20. 琼脂(Thermo Fisher Scientific,Oxoid TM ,目录号:LP0011)
  21. 2-(N-吗啉代)乙磺酸(MES)一水合物(Sigma-Aldrich,目录号:69892)
  22. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S8045)
  23. 马铃薯葡萄糖肉汤(PDB)(见配方)
  24. CFW和CR储备溶液(见配方)
    1. CFW储备溶液(20mg/ml)
    2. CR储备溶液(50mg/ml)
  25. 真菌生长琼脂培养基(见Recipes)
    1. YPD琼脂培养基
    2. PDA琼脂培养基
    3. Puhalla的最小琼脂培养基(MM)



  1. 保护手套和外套
  2. 用于制备溶液的烧瓶和磁力搅拌器
  3. 用于细胞计数的Neubauer室
  4. 微波
  5. 高压灭菌器
  6. 真菌生长孵化器
  7. 水浴
  8. 层流罩
  9. 加热板和蒸煮锅煮土豆
  10. pH计



  1. SPSS 15.0 for Windows ®(LEAD Technologies Inc.)


该方案的主要要求是实验确定要使用的CFW或CR的最佳浓度。这可能受几个参数影响,包括接种物的大小,菌株的遗传背景和生长培养基的组成。在我们的实验室中使用的野生型菌株是尖镰孢(Fusarium oxysporum)f.sp。 (菌株4287/FGSC 9935)。对于实验中使用的野生型或参照菌株,适当的CFW和CR浓度应是亚致死的。我们通常通过以板上斑点的形式接种连续稀释的分生孢子来定义该参数 含有CFW和CR,浓度范围为10-200μg/ml CFW和5-600μg/ml CR。

  1. 生产新鲜微小分生孢子(3-4天)
    接种50μl的-80°C股票微锥体悬浮液(在30%甘油)到50ml液体马铃薯葡萄糖肉汤(PDB)(见配方1)。在28℃下以170rpm振荡生长培养物3-4天(Di Pietro和Roncero,1998)。通过一层Miracloth过滤小分生孢子,离心并用无菌水洗涤。使用neubauer室计数分生孢子并调整至1×10 7分生孢子/ml的终浓度。
  2. 制备CFW(20mg/ml)和CR(50mg/ml)(1小时)的储备溶液(见配方2)
  3. 生长培养基的制备(1-2小时)
    准备生长琼脂培养基和高压釜(在120℃下20分钟)。在加入CR和CFW之前,将介质在水浴中冷却至50-60℃,以避免任何潜在的危害烟雾。然后,添加CFW或CR以达到所需的浓度。这可以在层流罩中进行。对于 F。我们建议从以下范围内测试4-5种不同的染料浓度开始:CFW为10至200μg/ml,CR为10至600μg/ml。另外,制备没有染料的对照板。板可以储存过夜,在室温和在黑暗中,第二天使用。
  4. 制备微小分生孢子的连续稀释液(1-2小时)
    制备新鲜获得的小分生孢子的10倍系列稀释液,从步骤1中产生的1×10 7个分生孢子/ml储液开始(1×10 7个,1× 10×10 6个,1×10 5个和1×10 4个分生孢子/ml)。将2μl该系列滴在含有CFW或CR的板上和对照板(终浓度:2×10 4,2×10 3,2×10 6, 2×2和2×10 5分生孢子/ml)。我们通常使用方形(120×120mm)培养皿,以便有足够的空间用于真菌菌落的生长。
  5. 平板培养(2-3天)
  6. 解释和监测结果
    1. 对CFW和CR的敏感性应通过比较对照和CFW或CR板中亲本和突变菌株的集落形成的程度来确定。可以测量菌落直径以定量测试的菌株的菌落生长的差异。为此,在对照和细胞壁应激平板上接种每个单个菌株(5μl的1×10 7个微孔/ml悬浮液滴),每个重复5次。我们使用圆形(90mm直径)培养皿。孵育板在28℃下5天,然后扫描和测量菌落直径
    2. 然后可以使用统计软件(例如用于Windows 的SPSS 15.0)分析来自三次独立实验的数据,每次实验重复五次。使用Kruskal-Wallis方差分析(ANOVA)和Mann-Whitney检验在P ≤0.05时评估菌株之间的统计学相关差异。


图1示出了几个F的灵敏度。尖孢镰孢MAPK通路突变体对CFW和CR的影响(Perez-Nadales和Di Pietro,2011)。在 F。尖孢镰孢,Fmk1 MAPK途径对于植物感染是必需的(Di Pietro等人,2001)。该实验揭示了在Fm中细胞壁完整性需要Msb2(一种在Fmk1上游起作用的信号转导粘蛋白受体以及Fmk1 MAPK)。 oxysporum 在这些条件下。

图1.F的细胞壁应力测定。 A.含有YPD培养基或补充有CR或CFW的YPD培养基的平板用指定的菌株和微量分生孢子点接种,在28℃温育3天并拍照。 B.在接种后第3天测量指定培养基的菌落直径,并相对于野生型菌株(100%)作图。误差棒表示从五个独立平板计算的标准偏差。根据Mann-Whitney检验,相同字母的值没有显着差异( P <0.05)。


  1. 在处理过程中,CFW和CR解决方案应始终通过用铝箔包装容器来防止光照。 作为防止可能的光灭活CFW和CR的预防措施,板应该在黑暗中孵育。


  1. 马铃薯葡萄糖肉汤(PDB)
    1. 将200g的剥皮马铃薯在1L蒸馏水中煮沸60分钟
    2. 通过滤布过滤器
    3. 加入20克葡萄糖,加入1升蒸馏水
    4. 在120℃下高压灭菌20分钟灭菌
  2. CFW和CR储备溶液
    1. CFW储备溶液(20mg/ml)
      20 mg/ml CFW
      83%(v/v)甘油 注意:我们建议在实验前准备20-30ml的这种溶液,并储存在室温下。 在实验当天,可以将CFW新鲜加入到该溶液中,达到20mg/ml的最终母液浓度。
    2. CR储备溶液(50mg/ml)
  3. 真菌生长琼脂培养基
    1. YPD琼脂培养基
      20g/L葡萄糖 15g/L Bacto琼脂
    2. PDA琼脂培养基
    3. Puhalla的最小琼脂培养基(MM)
      0.5g/L MgSO 4·7H 2 O·h/v 1g/L KH 2 PO 4 sub/
      0.5g/L KCl
      2g/L NaNO 3
      30 g/L蔗糖
    注意:对于该测定,在高压灭菌(120℃下20分钟)之前将生长培养基缓冲至pH 5.5-7.0(Ram和Klis,2006) 。为此,我们向生长培养基溶液中加入50 mM MES,并用NaOH调节至pH 6.5(可以预先制备10 N NaOH溶液,并缓慢加入MES缓冲的培养基中,直至达到所需的pH)。之后,我们加入琼脂(PDA除外),用蒸馏水将溶液加满至所需体积,并高压灭菌。


这项研究由SIGNALPATH Marie居里研究培训网络(MRTN-CT-2005-019277)和授权BIO2008-04479-E,EUI2009-03942和BIO2010-15505(来自西班牙部长会议)支持。


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引用:Pérez-Nadales, E. and Di Pietro, A. (2016). In vitro Cell Wall Stress Assay for Fusarium oxysporum. Bio-protocol 6(17): e1915. DOI: 10.21769/BioProtoc.1915.