Determination of Intracellular ATP Levels in Mycelium of Fusarium oxysporum

Materials and Reagents

1. Monodur nylon filters 15 µm diameter (Filtravibracion S.L., Spain, catalog number: Nylon-15 )
   
2. Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150 )
   
3. Eppendorf tube (2 ml)
   
4. 5 mm-diameter glass beads (Sigma-Aldrich, USA, catalog number: 18406-500G )
   
5. Microtiter plates (TermoFisher Scientific, catalog number: 2205 )
   
6. Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type and mutant strains.
   Note: Strains used in this study are wild type strain 4287 (race 2) and Δcon7-1 and cΔcon7-1 mutant strains.
   
7. Glycerol (Merck, catalog number: 104092 )
   
8. Potato dextrose broth (PDB) (Scharlau, Spain, catalog number: 01483 )
   
9. Sterile dH₂O
   
10. Trichloroacetic acid (Sigma-Aldrich, catalog number: T6399 )
    
11. EDTA (Amresco, catalog number: 0322 )
    
12. MgSO₄·7H₂O (Merck, catalog number: 1058865.5000 )
    
13. KH₂PO₄ (Merck, catalog number: 104873.1000 )
    
14. KCl (Merck, catalog number: 104933.0500 )
    
15. NH₄NO₃ (Merck, catalog number: 101187.1000 )
    
16. FeSO₄ (Merck, catalog number: 103965.0500 )
    
17. ZnSO₄·7H₂O (Merck, catalog number: 108883 )
    
18. MnSO₄ monohydrate (Merck, catalog number: 105941 )
    
19. Glucose (Coger SAS, catalog number: 24379.363 )
    
20. ATP determination kit (Thermo Fischer Scientific, Molecular Probes^®, catalog number: A22066 ).
    Kit contents:
1. D-Luciferin (Component A, MW 302, blue cap), 5 vials, each containing 3 mg of lyophilized powder
   
2. Luciferase, firefly recombinant (Component B, red cap) 40 μl of a 5 mg/ml solution in 25 mMTris-acetate, pH 7.8, 0.2 M ammonium sulfate, 15% (v/v) glycerol and 30% (v/v) ethylene glycol
   
3. Dithiothreitol (DTT) (Component C, MW 154, black cap) 25 mg
   
4. Adenosine 5’-triphosphate (ATP) (Component D, Green cap), 400 μl of a 5 mM solution in TE buffer
   
5. 20x Reaction Buffer (Component E) 10 ml of 500 mM
   
6. Tricine buffer, pH 7.8, 100 mM MgSO₄, 2 mM EDTA and 2 mM sodium azide
   
21. Extraction buffer (see Recipes)
    
22. Potato dextrose broth (PDB) medium (see Recipes)
    

Equipment

1. Sterile spatula (Fisher Scientific, catalog number: S50821 )
   
2. Mini-BeadBeater-16 homogenizer (BioSpec Products)
   
3. Hemocytometer Thoma (Marienfeld, Germany, catalog number: 06 407 10 )
   
4. Orbital incubator (Infors, Multitron Pro, Switzerland)
   
5. Microcentrifuge (Eppendorf, MiniSpin plus) (Fisher Scientific)
   
6. Fluorimeter (TECAN, SpectraFluorPlus, catalog number: InfiniteM200PRO )
   

Procedure