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Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae
大丽轮枝菌的渗透测定、真菌恢复和致病性测定   

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Abstract

Verticillium dahliae is a soil-borne phytopathogenic fungus that infects host roots and proliferates in vascular tissues. The great loss of economically important crop caused by V. dahliae has raised worldwide concern, however, little is known about the mechanism of its pathogenicity (Klosterman et al., 2011; Yadeta and Thomma, 2013). Our recent work has shown that V. dahliae develops hyphopodium as an infection structure to breach plant root cell wall (Zhao et al., 2016). Here, we provide a detailed protocol to analyze the penetration ability and the pathogenicity of V. dahliae as well as recover fungal hyphae from infected cotton stems developed from our previous studies (Zhang et al., 2016a and 2016b; Zhao et al., 2016). Cellophane membrane has been used in inducing appressorium development of foliar pathogens but not root pathogens (Bourett and Howard, 1990). We adopted the method of using the cellophane membrane to induce and assess the development of hyphopodium. Hopefully, it will greatly promote the research of molecular events involved in recognition of the host that regulate infectious development. This protocol is also helpful to identify the key component controlling the pathogenicity of V. dahliae and widen our understanding of the mechanism of plant-microbe interaction.

Keywords: Verticillium dahliae(大丽轮枝菌), Penetration assays(渗透测定), Fungal recovery(真菌恢复), Pathogenicity assays(致病性测定)

Background

The cellophane membrane has been widely used to study the development of infection structure in foliar pathogens (Bourett and Howard, 1990; Kleemann et al., 2012; Gu et al., 2014), we firstly adopt this method to induce infection structure in root pathogen of V. dahliae, which is a simple and efficient method to study the hyphopodium development. Also, we previously developed a novel unimpaired root dip-inoculation method to assess the pathogenicity of V. dahliae in cotton (Gao et al., 2010). The regular procedure for infection of plants with the soil-borne pathogen is to uproot soil-grown plants, incubate the roots in a conidial suspension, and then replant the plants in fresh soil. Our inoculation method avoids damaging the roots and is convenient for operation, which combined the protocol of fungal recovery from stem facilitates the pathogenicity study of soil-borne pathogens that colonize the vascular tissues.

Materials and Reagents

  1. Protection gloves (Medicom, catalog number: 1174D )
  2. Protection coat (Medicom, catalog number: 8018 )
  3. Cellophane membrane (DINGGUO CHANGSHENG, catalog number: XH444-1 )
  4. Round (90 mm diameter) Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 101VIRR )
  5. Sterilized gauze (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22-415-469 )
  6. Pot for cotton growth (Shantou Xing Lv Yuan, dimensions: 300 x 200 x 100)
  7. Sterilized pipette tips (Corning, Axygen®, catalog number: TF-1005-WB-L-R-S )
  8. 50 ml conical tubes (Corning, catalog number: 430828 )
  9. Cotton seeds (Nongqishi Agricultural Institute, cv. Xinluzao NO.16)
  10. Potato tubers
  11. Glycerol
  12. Distilled water (Milli-Q) (EMD Millipore, catalog number: QTUM00ICP )
  13. 70% (v/v) ethanol (EtOH in ddH2O)
  14. 30% H2O2 (ALADDIN, catalog number: H112519 )
  15. Glucose (Sigma-Aldrich, catalog number: V900392 )
  16. Agar (BD, BactoTM, catalog number: 214010 )
  17. Sodium nitrate, NaNO3 (EMD Millipore, catalog number: 106537 )
  18. Potassium dihydrogen phosphate, KH2PO4 (EMD Millipore, catalog number: 104873 )
  19. Potassium phosphate dibasic, K2HPO4 (Sigma-Aldrich, catalog number: V900050 )
  20. Magnesium sulfate heptahydrate, MgSO4·7H2O (EMD Millipore, catalog number: 105886 )
  21. Potassium chloride, KCl (EMD Millipore, catalog number: 104933 )
  22. Sodium citrate (Sigma-Aldrich, catalog number: V900095 )
  23. Zinc sulfate heptahydrate, ZnSO4·7H2O (Sigma-Aldrich, catalog number: Z4750 )
  24. Iron(II) sulfate heptahydrate, FeSO4·7H2O (Sigma-Aldrich, catalog number: V900038 )
  25. Ammonium iron(III) sulfate dodecahydrate, NH4Fe(SO4)2·12H2O (Sigma-Aldrich, catalog number: V900032 )
  26. Copper(II) sulfate pentahydrate, CuSO4·5H2O (Sigma-Aldrich, catalog number: C7631 )
  27. Manganese(II) sulfate monohydrate, MnSO4·H2O (Sigma-Aldrich, catalog number: V900271 )
  28. Boric acid, H3BO3 (Sigma-Aldrich, catalog number: V900267 )
  29. Sodium molybdate dehydrate, Na2MoO4·2H2O (EMD Millipore, catalog number: 106521 )
  30. Sucrose (Merck Millipore, catalog number: 107687 )
  31. Murashige and Skoog (MS) medium including vitamins (Duchefa Biochemie, catalog number: M0222 )
  32. Potato dextrose agar (PDA) (see Recipes)
  33. TES (see Recipes)
  34. Minimal medium (MM) (see Recipes)
  35. MS liquid medium (see Recipes)
  36. Czapek-Dox medium (see Recipes)

Equipment

  1. Heat plate and cooking pot to boil potatoes (Supor, catalog number: SDHC8E15 )
  2. Flasks and magnetic stirrer for preparation of solutions (Thermo Fisher Scientific, Fisher Scientific, catalog number: S88850206 )
  3. Fungal growth incubator (SAIFU, catalog number: MJX-450S )
  4. 26 °C incubator shaker (Zhicheng, catalog number: ZHWY-2102C )
  5. Autoclave (Hirayama, catalog number: HVE-50 )
  6. Clean bench (Zhicheng, catalog number: ZHJH-C1209B )
  7. Hemocytometer for conidia counting (Qiujing, catalog number: QJ1102 )
  8. Rotator (Qilinbeier, catalog number: QB-208 )

Procedure

  1. Penetration assays
    1. Pipette 20 μl conidial suspension (3 x 107 conidia per ml) of V. dahliae stored at -80 °C in 20% glycerol and plate the suspension culture onto the PDA plate and incubate for 5 days at 26 °C in the dark.
    2. Prepare Minimal medium (MM medium) and autoclave 20 min at 113 °C. Pour plates after cooling the medium down to 50-60 °C and dry the plates in a clean bench.
    3. Cut the flat sheet membranes of cellophane into circles of 90 mm diameter and autoclave 20 min at 113 °C. Lay one circle on each MM plate prepared in step A2.
    4. Use a sterilized pipette tip to inoculate the fungi from step A1 onto the cellophane membrane overlaid on the MM medium. Grow cultures for 3 days at 26 °C in the dark. Then remove the membrane and incubate the medium for 7 days to observe hyphae in the underlying medium for determination of breach of cellophane (Figure 1).


      Figure 1. Penetration assay on the cellophane membrane. A. Uncovering the membrane from the MM plates with a colony of V. dahliae at 3 days post inoculation (dpi). B. The colony of V. dahliae on the underlying medium indicating the penetration of cellophane membrane.

  2. Pathogenicity assays
    Susceptible upland cotton (cv. Xinluzao NO. 16) and strain V592 of V. dahliae were used in our lab for pathogenicity assays.
    1. Sterilize cotton seeds for 15 min and rinse three times with distilled water. Soak the seeds overnight at room temperature. Germinate seeds in pots filled with wet soil and cover with plastic dome for one week to grow seedlings until two cotyledons are fully expanded.
    2. Transplant 12 seedlings per pot with MS liquid medium and grow at 26 °C with a 16 h light (8,000 lux)/8 h dark cycle for about 2 weeks before inoculation of V592.
    3. Streak V592 onto fresh PDA plates (from older PDA plates or glycerol stocks) and incubate for 1 week. Transfer one plate (90 mm) of mycelia by a sterilized blade into 200 ml Czapek-Dox medium with shaking at 200 rpm for 3-4 days at 26 °C in the dark to obtain conidia.
    4. Prepare the conidial suspension through filtrating the fungal cultures with 4 layers of sterilized gauze to remove mycelia. Measure the concentration of conidia in the suspension using a hemocytometer and adjust to approximately 1 x 107 conidia per ml with Czapek-Dox medium.
    5. Immerse the roots of seedlings with two true leaves to the conidial suspension from step B4 for 1 h (Figure 2A). Put back the inoculated seedlings to MS liquid medium and supply water once a week.
    6. Count disease incidence by the percentage of cottons that showed wilting symptom at 30 dpi (Figure 2B). The infection assay was repeated at least three times with 3 pots of cotton each trial.


      Figure 2. Pathogenicity assay of V. dahliae. A. Root-dip inoculation of cotton seedling with conidia suspension. B. Representative disease symptom in cotton at 30 dpi of V. dahliae.

  3. Fungal recovery
    To identify the colonization of V. dahliae in vascular tissues, we isolate the inoculated cotton stems to recover the fungi.
    1. Cut the stem above cotyledons of cotton at 30 days post inoculation (Figure 3).
    2. Sterilize the stem sections for 1 min in 70% ethanol within a 50 ml conical tube followed by 60 min in 40 ml of 10% (v/v) H2O2 (dilute the 30% [v/v] H2O2 with distilled water). Revolve the tube on the rotator at 20 rpm during this process.
    3. Rinse the stems three times with sterilized water for 3 min each time and culture at 26 °C on PDA medium in the dark. The colonies of V. dahliae can grow on the stems within 1 week (Figure 3).


      Figure 3. Recovery of V. dahliae from the infected stem of cotton

Data analysis

Data from at least three biological repeats were obtained and one-way ANOVA was used to analyze the disease incidence with Microsoft Excel (Table 1).

Table 1. Analysis of disease incidence. Disease incidence was counted by the percentage of diseased cottons in 3 pots of infected cottons (3 x 12, total 36 cottons). Assay was repeated 3 times.

Notes

  1. Each of cellophane membranes should be separated by a filter paper to prevent adhesion during autoclaving.
  2. To prevent shrinkage of the cellophane membrane, it is soaked into sterilized water firstly before putting on MM plate.

Recipes

  1. Potato dextrose agar (PDA)
    Boil 200 g of peeled potatoes in 1 L of distilled water for 20 min
    Filtrate through 4 layers of gauze
    Add 20 g of glucose and 20 g of agar
    Distilled water to 1 L
    Sterilize by autoclaving 20 min at 113 °C
  2. TES
    50 g/L citrate
    50 g/L ZnSO4·7H2O
    50 g/L FeSO4·7H2O
    13 g/L NH4Fe(SO4)2·12H2O
    2.5 g/L CuSO4·5H2O
    0.5 g/L MnSO4·H2O
    0.5 g/L H3BO3
    0.5 g/L Na2MoO4·2H2O
    Stored at 4 °C
  3. Minimal medium
    2 g/L glucose
    2 g/L NaNO3
    1 g/L KH2PO4
    0.5 g/L MgSO4·7H2O
    0.5 g/L KCl
    0.02% (v/v) TES
    20 g/L agar
    Sterilize by autoclaving 20 min at 113 °C
  4. MS liquid medium
    0.1% (w/v) MS
  5. Czapek-Dox medium
    30 g/L sucrose
    3 g/L NaNO3
    1 g/L K2HPO4
    0.5 g/L MgSO4·7H2O
    0.5 g/L KCl
    100 mg/L FeSO4·7H2O
    Sterilize by autoclaving 20 min at 113 °C

Acknowledgments

This protocol was developed from the following published paper: Bourett and Howard, 1990. This work was supported by grant from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB11040500) and the China Transgenic Research and Commercialization Key Special Project (2014ZX0800908B).

References

  1. Bourett, T. M. and Howard, R. J. (1990). In vitro development of penetration structures in the rice blast fungus Magnaporthe grisea. Can J Bot 68(2): 329-342.
  2. Gao, F., Zhou, B. J., Li, G. Y., Jia, P. S., Li, H., Zhao, Y. L., Zhao, P., Xia, G. X. and Guo, H. S. (2010). A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity. PLoS One 5(12): e15319.
  3. Gu, S. Q., Li, P., Wu, M., Hao, Z. M., Gong, X. D., Zhang, X. Y., Tian, L., Zhang, P., Wang, Y., Cao, Z. Y., Fan, Y. S., Han, J. M. and Dong, J. G. (2014). StSTE12 is required for the pathogenicity of Setosphaeria turcica by regulating appressorium development and penetration. Microbiol Res 169(11): 817-823.
  4. Kleemann, J., Rincon-Rivera, L. J., Takahara, H., Neumann, U., Ver Loren van Themaat, E., van der Does, H. C., Hacquard, S., Stuber, K., Will, I., Schmalenbach, W., Schmelzer, E. and O'Connell, R. J. (2012). Sequential delivery of host-induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen Colletotrichum higginsianum. PLoS Pathog 8(4): e1002643.
  5. Klosterman, S. J., Subbarao, K. V., Kang, S., Veronese, P., Gold, S. E., Thomma, B. P., Chen, Z., Henrissat, B., Lee, Y. H., Park, J., Garcia-Pedrajas, M. D., Barbara, D. J., Anchieta, A., de Jonge, R., Santhanam, P., Maruthachalam, K., Atallah, Z., Amyotte, S. G., Paz, Z., Inderbitzin, P., Hayes, R. J., Heiman, D. I., Young, S., Zeng, Q., Engels, R., Galagan, J., Cuomo, C. A., Dobinson, K. F. and Ma, L. J. (2011). Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog 7(7): e1002137.
  6. Yadeta, K. A. and Thomma, B. P. (2013). The xylem as battleground for plant hosts and vascular wilt pathogens. Front Plant Sci 4(97): 1-12.
  7. Zhang, T., Jin, Y., Zhao, J. H., Gao, F., Zhou, B. J., Fang, Y. Y. and Guo, H. S. (2016a). Host-induced gene silencing of the target gene in fungal cells confers effective resistance to the cotton wilt disease pathogen Verticillium dahliae. Mol Plant 9(6): 939-942.
  8. Zhang, T., Zhao, Y. L., Zhao, J. H., Wang, S., Jin, Y., Chen, Z. Q., Fang, Y. Y., Hua, C. L., Ding, S. W. and Guo, H. S. (2016b). Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nat Plants 2(10): 16153.
  9. Zhao, Y. L., Zhou, T. T. and Guo, H. S. (2016). Hyphopodium-specific VdNoxB/VdPls1-dependent ROS-Ca2+ signaling is required for plant infection by Verticillium dahliae. PLoS Pathog 12(7): e1005793.

简介

大丽轮枝菌属是土壤传播的植物病原真菌,其感染宿主根并在血管组织中增殖。经济上重要的作物造成的巨大损失。大丽花已经引起了全世界的关注,然而,其致病性机制知之甚少(Klosterman等人,2011; Yadeta和Thomma,2013)。我们最近的工作表明,大丽花开发hyphopodium作为感染结构以破坏植物根细胞壁(Zhao等人,2016)。在这里,我们提供了一个详细的方案来分析渗透能力和致病性。大丽花,以及从以前的研究(Zhang等人,2016a和2016b; Zhao等人,2016年)开发的感染棉花茎中恢复真菌菌丝)。玻璃纸膜已被用于诱导叶面病原体的发育,而不是根系病原体(Bourett和Howard,1990)。我们采用玻璃纸膜的方法诱导和评估hyphopodium的发展。希望能大力促进调控传染性发育的宿主的分子生物学研究。该协议还有助于识别控制V的致病性的关键成分。大丽花,并扩大了我们对植物与微生物相互作用机理的理解。

背景 玻璃纸膜被广泛用于研究叶病原体感染结构的发展(Bourett和Howard,1990; Kleemann等人,2012; Gu等人, 2014),我们首先采用这种方法诱导V型根病原体的感染结构。大丽花,这是一种简单有效的研究hyphopodium开发的方法。此外,我们以前开发了一种新颖的未受损的根浸种接种方法来评估V的致病性。棉花中的大丽花(Gao等人,2010)。用土壤传播的病原体感染植物的常规程序是根除土壤种植的植物,将根部分散在分生孢子悬浮液中,然后在新鲜土壤中补充植物。我们的接种方法避免了根部的破坏,操作方便,结合茎的真菌回收方案,有利于定植于血管组织的土传病原体的致病性研究。

关键字:大丽轮枝菌, 渗透测定, 真菌恢复, 致病性测定

材料和试剂

  1. 防护手套(Medicom,目录号:1174D)
  2. 保护外套(Medicom,目录号:8018)
  3. 玻璃纸(DINGGUO CHANGSHENG,目录号:XH444-1)
  4. 圆形(90mm直径)培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:101VIRR)
  5. 灭菌纱布(Thermo Fisher Scientific,Fisher Scientific,目录号:22-415-469)
  6. 棉花种植罐(汕头兴乐园,尺寸:300 x 200 x 100)
  7. 灭菌移液器吸头(Corning,Axygen ®,目录号:TF-1005-WB-L-R-S)
  8. 50ml锥形管(Corning,目录号:430828)
  9. 棉花种子(农业农业研究所,新路寨NO.16)
  10. 土豆块茎
  11. 甘油
  12. 蒸馏水(Milli-Q)(EMD Millipore,目录号:QTUM00ICP)
  13. 70%(v/v)乙醇(ddH 2 O中的EtOH)
  14. 30%H 2 O 2(ALADDIN,目录号:H112519)
  15. 葡萄糖(Sigma-Aldrich,目录号:V900392)
  16. 琼脂(BD,Bacto TM ,目录号:214010)
  17. 硝酸钠,NaNO 3(EMD Millipore,目录号:106537)
  18. 磷酸二氢钾,KH 2 PO 4(EMD Millipore,目录号:104873)
  19. 磷酸氢二钾,K 2 HPO 4(Sigma-Aldrich,目录号:V900050)
  20. 硫酸镁七水合物,MgSO 4·7H 2 O(EMD Millipore,目录号:105886)
  21. 氯化钾,KCl(EMD Millipore,目录号:104933)
  22. 柠檬酸钠(Sigma-Aldrich,目录号:V900095)
  23. 硫酸锌七水合物,ZnSO 4·7H 2 O(Sigma-Aldrich,目录号:Z4750)
  24. 硫酸铁(II)七水合物,FeSO 4·7H 2 O(Sigma-Aldrich,目录号:V900038)
  25. 硫酸铁铵(III)十二水合物,NH 4 Fe(SO 4 H 2)2·12H 2 O(Sigma -Aldrich,目录号:V900032)
  26. 硫酸铜(II)五水合物,CuSO 4·5H 2 O(Sigma-Aldrich,目录号:C7631)
  27. 硫酸锰(II)一水合物,MnSO 4 H 2 O(Sigma-Aldrich,目录号:V900271)
  28. 硼酸,H 3 BO 3(Sigma-Aldrich,目录号:V900267)
  29. 钼酸钠脱水,Na 2 MoO 4·2H 2 O(EMD Millipore,目录号:106521)
  30. 蔗糖(Merck Millipore,目录号:107687)
  31. Murashige和Skoog(MS)培养基,包括维生素(Duchefa Biochemie,目录号:M0222)
  32. 马铃薯葡萄糖琼脂(PDA)(见食谱)
  33. TES(见食谱)
  34. 最小介质(MM)(见配方)
  35. MS液体介质(见配方)
  36. Czapek-Dox培养基(参见食谱)

设备

  1. 加热板和煮锅煮土豆(苏泊尔,目录号:SDHC8E15)
  2. 用于制备溶液的烧瓶和磁力搅拌器(Thermo Fisher Scientific,Fisher Scientific,目录号:S88850206)
  3. 真菌生长培养箱(SAIFU,目录号:MJX-450S)
  4. 26℃孵化器(志诚,目录号:ZHWY-2102C)
  5. 高压釜(平山市,目录号:HVE-50)
  6. 洁台(智城,目录号:ZHJH-C1209B)
  7. 血细胞计数器用于分生孢子计数(邱静,目录号:QJ1102)
  8. 旋转器(Qilinbeier,目录号:QB-208)

程序

  1. 穿透试验
    1. 移取V型20μl分生孢子悬浮液(3×10 7 /分钟/ml)。大丽花储存在-80℃的20%甘油中,并将悬浮培养物平板化到PDA板上,并在26℃下在黑暗中孵育5天。
    2. 制备最小培养基(MM培养基),并在113℃下高压灭菌20分钟。将介质冷却至50-60°C后倒入板中,并在干净的工作台上干燥板。
    3. 将玻璃纸的平片膜切成90mm直径的圆圈,并在113℃下高压灭菌20分钟。在步骤A2中制备的每个MM板上放一圈。
    4. 使用灭菌的移液管接头将来自步骤A1的真菌接种到覆盖在MM培养基上的玻璃纸上。在黑暗中在26°C培养3天。然后去除膜并孵育培养基7天,以在底层培养基中观察菌丝以测定玻璃纸的破裂(图1)。


      图1.在玻璃纸膜上的渗透测定。 A.用具有V氏菌落的MM板展开膜。 dahliae 在接种后3天(dpi)。 B.殖民地。大丽花在底层介质上,表示玻璃纸的渗透
  2. 致病性分析
    敏感高地棉(cv。新鲁仓第16号)和菌株V V592。大丽花在我们的实验室中用于致病性测定。
    1. 灭菌棉籽15分钟,用蒸馏水冲洗3次。在室温下将种子浸泡过夜。在充满湿土的盆中发芽种子,用塑料圆顶覆盖一周,种植幼苗直到两个子叶完全展开。
    2. 在每个盆中用MS液体培养基移植12个幼苗,并在接种V592之前用26小时光(8,000lux)/8小时黑暗循环生长大约2周。
    3. 将条纹V592连接到新鲜的PDA板上(来自较旧的PDA板或甘油储备),并孵育1周。通过灭菌的叶片将一个板(90mm)的菌丝体转移到200ml Czapek-Dox培养基中,在26℃下在黑暗中以200rpm摇动3-4天以获得分生孢子。
    4. 通过用4层灭菌纱布过滤真菌培养物来准备分生孢子悬浮液以去除菌丝体。使用血细胞计数器测量悬浮液中的分生孢子的浓度,并用Czapek-Dox培养基调节至约1×10 7 /分钟/ml的分生孢子。
    5. 将具有两个真叶的幼苗的根部浸入步骤B4的分生孢子悬浮液中1小时(图2A)。将接种的幼苗放回MS液体培养基,每周供水一次
    6. 以30dpi显示萎缩症状的棉花百分比计算疾病发病率(图2B)。每次试验,用3盆棉花重复感染测定至少三次。


      图2. V的致病性测定。大丽花。 A.用分生孢子悬浮液对棉苗进行根浸接种。 B.在30dpi的V代表棉花中的代表性疾病症状。大丽花。

  3. 真菌恢复
    识别V的殖民化。大丽花在血管组织中,我们分离接种的棉花茎以回收真菌。
    1. 在接种后30天切割棉花子叶上方的茎(图3)
    2. 在50ml锥形管中,在70%乙醇中将茎切片灭菌1分钟,然后在40ml 10%(v/v)H 2 O 2 O 2/(用蒸馏水稀释30%[v/v] H 2 O 2 O 2)。在这个过程中,旋转器上的旋转器以20 rpm的速度旋转
    3. 用无菌水冲洗茎三次,每次3分钟,并在26摄氏度的PDA培养基上在黑暗中培养。殖民地。大丽花可以在1周内在茎上生长(图3)

      图3.恢复V。来自受感染的棉花茎的大丽花

数据分析

获得至少三个生物重复数据,并使用单因素方差分析法用Microsoft Excel分析疾病发生率(表1)。

表1.疾病发病率分析疾病发病率以3盆感染棉花(3×12,共36只棉花)中的病棉百分比计算。测定重复3次

笔记

  1. 每个玻璃纸应通过滤纸分离,以防止在高压灭菌过程中粘附
  2. 为了防止玻璃纸膜的收缩,首先将其浸入灭菌水中,然后再放入MM板上。

食谱

  1. 马铃薯葡萄糖琼脂(PDA)
    将200克去皮土豆在1升蒸馏水中煮沸20分钟
    通过4层纱布渗透
    加入20 g葡萄糖和20 g琼脂 蒸馏水至1升
    用113℃高压灭菌20分钟灭菌
  2. TES
    50克/升柠檬酸盐
    50g/L ZnSO 4·7H 2 O
    50g/L FeSO 4·7H 2 O
    13g/L NH4Fe(SO4)2·12H2 O
    2.5g/L CuSO 4·5H 2 O
    0.5g/L MnSO 4·H 2 O
    0.5g/L H 3/3> 3
    0.5g/L Na 2 MoO 4·2H 2 O
    储存于4°C
  3. 最小媒体
    2 g/L葡萄糖
    2g/L NaNO 3
    1g/L KH 2 PO 4
    0.5g/L MgSO 4·7H 2 O
    0.5克/升KCl
    0.02%(v/v)TES
    20 g/L琼脂
    用113℃高压灭菌20分钟灭菌
  4. MS液体介质
    0.1%(w/v)MS
  5. Czapek-Dox媒体
    30克/升蔗糖
    3g/L NaNO 3
    1 g/L K 2 HPO 4
    0.5g/L MgSO 4·7H 2 O
    0.5克/升KCl
    100mg/L FeSO 4·7H 2 O
    用113℃高压灭菌20分钟灭菌

致谢

该协议是从以下发表的论文:Bourett和Howard,1990年开发的。这项工作得到了中国科学院战略重点研究计划(XDB11040500)和中国转基因研究与商业化重点专项项目(2014ZX0800908B)的资助, 。

参考文献

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhao, Y., Zhang, T. and Guo, H. (2017). Penetration Assays, Fungal Recovery and Pathogenicity Assays for Verticillium dahliae. Bio-protocol 7(4): e2133. DOI: 10.21769/BioProtoc.2133.
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