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Assays for Oxidative Responses of Fusarium graminearum Strains to Superoxide Radicals

YW Yan-Zhang Wang
YG Yan Guo
Wei-Hua Tang Wei-Hua Tang

Published: Sep 5, 2018 DOI: 10.21769/BioProtoc.2997 Views: 4677

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Abstract

The ascomycete fungus Fusarium graminearum is a major causal agent of Fusarium head blight (FHB), a devastating disease affecting small grains cereals worldwide. To better understand the pathogenesis of this fungus, we provide here an easy-to-use protocol to examine the sensitivity of the wild-type and mutant strains of F. graminearum to oxidative stress from superoxide anions (O2•-) generated by menadione. Similarly, this assay can also be used to detect other stress responses of different fungal strains to various stress agents. The change in stress response of a mutant can offer a clue for the biological function of mutated genes.

Keywords: Fusarium graminearum search Mutant search Mycelial disc search Sensitivity search Oxidative stress search

Background

The ascomycete fungus Fusarium graminearum (previously also called Gibberella zeae for its sexual state) is not only the major causal agent of Fusarium head blight and seedling blight on wheat and barley, but also one of the important causal agents of Gibberella stalk rot on maize (Dal Bello et al., 2002; Bai and Shaner, 2004; Kazan et al., 2012). Apart from causing a huge yield loss of cereals, this fungus also produces mycotoxins which affect human and animal health. Therefore, this fungus has received extensive attention, and ranked the fourth among all investigated plant pathogenic fungi (Dean et al., 2012).

F. graminearum overwinters on dead organic matter, particularly on infected crop residues of small grains and corn. To survive such a wide range of environment, F. graminearum has evolved the capacity to confront various stresses. Numerous genes in F. graminearum have been explored for their roles in counteracting stress treatment, and the resulting mutants exhibited diverse responses to the stresses, which indicated the association of the stress responses and pathogenicity (Son et al., 2011). Some of the frequently used stress agents that act on cell wall or cell membrane of fungi include oxidative stress agents (e.g., menadione and H2O2), cell wall-perturbing agents (e.g., Congo red), and membrane stress agents (e.g., SDS). Detection of the stress responses of a fungal mutant strain could provide a clue for further investigating pathogenetic function. Thus, we take oxidative stress treatment as an example to describe a reliable protocol to assess stress response of F. graminearum to a superoxide radical generating agent menadione (Kawamura et al., 2006). This protocol can be used to detect other stress responses of a fungal strain in an analogous procedure.

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog number: T-300-R-S )
  2. 9 cm Petri plates
  3. Sterile toothpick
  4. Parafilm (Bemis, catalog number: PM996 )
  5. Fungal strains: F. graminearum wild-type strain PH-1 (NRRL 31084), and its deletion mutant ∆sod1 (Yao et al., 2016)
  6. 75% alcohol (Sinopharm Chemical Reagent, catalog number: 80176961 )
  7. Menadione (also named Vitamin K3, Sangon Biotech, catalog number: A502486-100g )
  8. V8 vegetable juice (CAMPBELL, V8® ORIGINAL)
  9. Absolute ethanol (Sinopharm Chemical Reagent, catalog number: 10009259 )
  10. CaCO3 (Acros Organics, catalog number: 403811000 )
  11. Double distilled water
  12. Agar powder (Oxoid, catalog number: LP0011 )
  13. NaNO3 (Sinopharm Chemical Reagent, catalog number: 10019918 )
  14. KH2PO4 (Sinopharm Chemical Reagent, catalog number: 10017618 )
  15. MgSO4•7H2O (Sinopharm Chemical Reagent, catalog number: 10013018 )
  16. KCl (Sinopharm Chemical Reagent, catalog number: 10016318 )
  17. Sucrose (Sinopharm Chemical Reagent, catalog number: 10021418 )
  18. N-Z Amine (Casein acid hydrolysate) (Macklin, catalog number: C822594 )
  19. Yeast extract (Oxoid, catalog number: LP0021 )
  20. Inositol (Sinopharm Chemical Reagent, catalog number: 63007734 )
  21. Ca pantothenate (TCI Shanghai, catalog number: P0012 )
  22. Choline•Cl (Sinopharm Chemical Reagent, catalog number: 69008560 ）
  23. Thiamine (Shanghai Bo'ao Biological Technology, catalog number: H1230 )
  24. Pyridoxine (Shanghai Bo'ao Biological Technology, catalog number: H0970 )
  25. Nicotinamide (Shanghai Bo'ao Biological Technology, catalog number: H0850 ) 
  26. Ascorbic acid (Sinopharm Chemical Reagent, catalog number: 10004014 )
  27. Riboflavin (Sinopharm Chemical Reagent, catalog number: 67001734 )
  28. p-aminobenzoic acid (Sinopharm Chemical Reagent, catalog number: 31000116 )
  29. Folic acid (Shanghai Bo'ao Biological Technology, catalog number: H0550 ) 
  30. Biotin (Shanghai Bo'ao Biological Technology, catalog number: H50 ) 
  31. Citric acid (TCI Shanghai, catalog number: C1949 )
  32. ZnSO4•7H2O (Sinopharm Chemical Reagent, catalog number: 10024018 )
  33. CuSO4•5H2O (Sinopharm Chemical Reagent, catalog number: 10008218 )
  34. Fe(NH4)2(SO4)2•6H2O (Beijing Ouhe Technology, catalog number: 01000313 )
  35. MnSO4 (Sinopharm Chemical Reagent, catalog number: 10013418 )
  36. H3BO3 (Sinopharm Chemical Reagent, catalog number: 10004808 )
  37. Na2MoO4•2H2O (Sinopharm Chemical Reagent, catalog number: 10019818 )
  38. 30 mM menadione stock solution (see Recipes)
  39. V8 juice agar medium (Yao et al., 2016; see Recipes)
  40. CM medium (Yao et al., 2016; see Recipes)
  41. Vitamin stock solution (see Recipes)
  42. Trace element solution (see Recipes)

Equipment

  1. 500 ml flask
  2. Pipettes (Eppendorf)
  3. Incubator (Yiheng, model: MJ-150I )
  4. Biological safety cabinet (ESCO Micro, model: FHC1200A )
  5. Camera (Canon, model: EOS 7D )
  6. Autoclave (Zealway Instrument, model: GI54DWS )
  7. Ruler
  8. Microwave oven (Galanz, model: G70F23N1P-M8(SO) )

Software

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

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Microbe-host interactions > Fungus

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