Assays for Oxidative Responses of Fusarium graminearum Strains to Superoxide Radicals   

Abstract

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

Note: This procedure should be carried out following aseptic techniques, working in a biological safety cabinet.

1. Preparation of F. graminearum mycelial discs
   Pick up a bit mycelia of the wild-type strain PH-1 and the mutant ∆sod1 and deposit into the middle of a V8 juice agar plate, respectively. Seal the plates with Parafilm and incubate in a 25 °C incubator for 3 days.
   Note: Operations in Step 1 should be carried out in a biological safety cabinet that has been sterilized under UV-light for at least 20 min.
2. Preparation of 30 mM menadione stock solution (see Recipe 1).
3. Prepare the complete medium (CM) beforehand as the recipe provided below, and store at room temperature for further use. Thaw CM agar medium in flask completely in microwave oven, and cool to 50 °C. Then add the menadione to a final concentration of 30 μM into the flask and shake to make menadione well-distributed in the medium. Subsequently pour plates. At the same time, pour the control CM plate without adding menadione as the control.
   Note: To keep the activity of menadione, make sure that CM agar medium cool to 50 °C before adding it. Alternatively, consider to examine oxidative stress responses at different concentrations of menadione.
4. Use a yellow 200-μl pipette tip as a cork borer, then hold the tip of a sterile 200-μl pipette tip with fingers to cut mycelial discs (0.5 cm diameter) from the edge of an actively growing colony of PH-1 or ∆sod1 from V8 agar plates (Figures 1A-1C). Place one mycelial disc in the center of a CM agar plate amended with 30 μM menadione as the experimental treatment (Figure 2A), and without the addition of menadione as the control. And make sure that the mycelial side of the disc should be in contact with the surface of the medium (Figure 2B). Lastly, incubate in a 25 °C incubator for 4 days. At least three CM agar plates in the experiment treatment or the control are performed for each strain.
   
   
   Figure 1. Preparation of F. graminearum mycelial discs. Cut mycelial discs (0.5 cm) aseptically using a sterile 200-μl pipette tip as a cork borer from the periphery of a 3-day-old colony grown in V8 agar plates (A, B and C).
   
   
   Figure 2. Transfer a mycelial disc into the center of a CM agar plate. A. Pick up a mycelial disc with a pipette tip. B. Place a mycelial disc in the center of the plate and make the mycelial side of the disc attached to the surface of the medium.
   
   
5. The cultures should be checked daily. When the growth of the colony on the control approaches the edge of the plate, photograph these plates with a ruler as a reference, as shown in Figure 3.
   
   
   Figure 3. Images of the colony of wild type (WT) grown for 4 days on the control and menadione-treated plates. When the growth of the colony on the control nears the edge of the plate, photograph plates with a ruler as a reference.

Data analysis

1. The colony diameter in each plate was measured using ImageJ software (http://rsbweb.nih.gov/ij/index.html). If a colony is not perfectly round, measure its diameter at several points and use the mean in subsequent calculation. Open the photo in ImageJ (Figure 4A) and choose straight line option to measure 1 cm of the ruler in the photo as scale, then set the length as 1 cm (Analyze → Set Scale → known distance and unit) (Figure 4B). Lastly, measure colony diameter of wild type and mutant successively (Figure 4C) and export the data to Excel sheet (Figure 5).
   
   
   Figure 4. Measurement of colony diameter using ImageJ software. A. ImageJ software window shows the image of the colony of wild type (WT) grown for 4 days on the control and menadione-treated plates. B. Set scale using the ruler in the image. C. Measurement of the colony diameter.
   
   
   Figure 5. Data of colony diameter of wild type (PH-1) and mutant (∆sod1) grown on the control and menadione-treated plates
   
   
2. Assess the sensitivity of the wild type and mutant to 30 μM menadione by analyzing the percentage of mycelial radial relative growth inhibition (RGI) of treated colony, as of control. For the relative growth inhibition is calculated using the following equation:
   
   RGI = [(Dc - Dt)/(Dc - 0.5)] x 100
   
   where Dc is the mean colony diameter for the control and Dt is the mean colony diameter for the experimental treatment. 0.5 cm should be subtracted from the diameter of each colony because this was the original mycelial disc diameter at the beginning of the procedure. Student's t-test was used to analyze the difference in relative growth inhibition of wild type and mutant with Microsoft Excel (Figure 6). Statistical analysis showed that there is significant difference in RGI of PH-1 and ∆sod1 at P < 0.05 level, suggesting that the mutant is more sensitive than wild type to superoxide anions (Figure 7).
   
   
   Figure 6. Calculation of relative growth inhibition (RGI) of wild type (PH-1) and mutant (∆sod1) by 30 μM menadione
   
   
   Figure 7. Relative growth inhibition of wild type (PH-1) and mutant (∆sod1) was shown by Excel bar graph

Notes

To obtain enough discs of the same size, prepare at least two V8 agar plates for culturing each strain (Step 1).

Recipes

1. 30 mM menadione stock solution
   Dissolve 516.54 mg of menadione in a total volume of 100 ml absolute ethanol, and store this stock at 4 °C away from light
2. V8 juice agar medium
   168 ml V8 vegetable juice
   1 g CaCO₃
   Add up to 1 L with double distilled water
   Divide it into small aliquots, and add 15 g agar powder
   Autoclave at 121 °C for 20 min
3. Complete medium (CM)
   2 g NaNO₃
   1 g KH₂PO₄
   0.5 g MgSO₄•7H₂O
   0.5 g KCl
   30 g Sucrose
   2.5 g N-Z Amine (Casein acid hydrolysate)
   1 g Yeast extract
   10 ml Vitamin stock solution
   0.2 ml Trace element solution
   Add up to 1 L with double distilled water
   Divide it into small aliquots, and add 20 g agar powder
   Autoclave at 121 °C for 20 min
4. Vitamin stock solution
   4 g Inositol
   200 mg Ca pantothenate
   200 mg Choline•Cl
   100 mg Thiamine
   75 mg Pyridoxine
   75 mg Nicotinamide
   50 mg Ascorbic acid
   30 mg Riboflavin
   5 mg p-aminobenzoic acid
   5 mg Folic acid
   5 mg Biotin
   Add up to 1 L with 50:50 ethanol:H₂O, and store at 4 °C in the dark
5. Trace element solution
   5 g Citric acid (dissolve into water first)
   5 g ZnSO₄•7H₂O
   1 g Fe(NH₄)₂(SO₄)₂•6H₂O
   250 mg CuSO₄•5H₂O
   50 mg MnSO₄
   50 mg H₃BO₃
   50 mg Na₂MoO₄•2H₂O
   Add up to 1 L with double distilled water, and store at 4 °C in the dark
   

Acknowledgments

We thank Mr. Gregory Brumberg for English editing. This protocol was modified from the previous method by Yao et al. (2016). This work was supported by the Ministry of Science and Technology of China (Grant 2016YFD0100600), the Natural Science Foundation of China (Grant 31730077) and the Ministry of Agriculture of China (Grant 2016ZX08009-003). The authors declare that there is no conflict of interests.

References

1. Bai, G. and Shaner, G. (2004). Management and resistance in wheat and barley to Fusarium head blight. Annu Rev Phytopathol 42: 135-161.
2. Dal Bello, G. M., Mónaco, C. I., Simón, M. R. (2002). Biological control of seedling blight of wheat caused by Fusarium graminearum with beneficial rhizosphere microorganisms. World J Microb Biot 18: 627-636.
3. Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J. and Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13(4): 414-430.
4. Kawamura, F., Hirashima, N., Furuno, T. and Nakanishi, M. (2006). Effects of 2-methyl-1,4-naphtoquinone (menadione) on cellular signaling in RBL-2H3 cells. Biol Pharm Bull 29(4): 605-607.
5. Kazan, K., Gardiner, D. M. and Manners, J. M. (2012). On the trail of a cereal killer: recent advances in Fusarium graminearum pathogenomics and host resistance. Mol Plant Pathol 13(4): 399-413.
6. Son, H., Seo, Y. S., Min, K., Park, A. R., Lee, J., Jin, J. M., Lin, Y., Cao, P., Hong, S. Y., Kim, E. K., Lee, S. H., Cho, A., Lee, S., Kim, M. G., Kim, Y., Kim, J. E., Kim, J. C., Choi, G. J., Yun, S. H., Lim, J. Y., Kim, M., Lee, Y. H., Choi, Y. D. and Lee, Y. W. (2011). A phenome-based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum. PLoS Pathog 7(10): e1002310.
7. Yao, S. H., Guo, Y., Wang, Y. Z., Zhang, D., Xu, L. and Tang, W. H. (2016). A cytoplasmic Cu-Zn superoxide dismutase SOD1 contributes to hyphal growth and virulence of Fusarium graminearum. Fungal Genet Biol 91: 32-42.