Mating and Progeny Isolation in the Corn Smut Fungus Ustilago maydis

Materials and Reagents

1. Plastic sterile syringes: 3 ml (BD, catalog number: 309657 ) and 10 ml (BD, catalog number: 309604 )
   
2. Needles: 22 gauge (BD, Eclipse^TM, catalog number: 305768 ) and 27 gauge (BD, Eclipse^TM, catalog number: 305758 )
   
3. Conical tubes: Falcon^® 50 ml (Corning, catalog number: 352070 )
   
4. Test tubes
   
5. 1.5 ml Microfuge tubes (Thermo Fisher Scientific, catalog number: 05-408-130 )
   
6. Haploid Ustilago maydis tester strains of known mating type
   
7. Seeds for maize seedling assay: Variety Golden Bantam (Rich Farm Garden Supply)
   
8. Seeds for maize ear inoculation: Variety Tom Thumb (Seed Savers Exchange)
   
9. Sterile distilled water (sdH₂O)
   
10. CuSO₄ solution (1%) (Sigma-Aldrich, catalog number: 451657 )
    
11. Casamino acids (Becton, Dickinson and Company, catalog number: 223050 )
    
12. Ammonium nitrate (Sigma-Aldrich, catalog number: A9642 )
    
13. Yeast extract (Sigma-Aldrich, catalog number: Y1625 )
    
14. Activated charcoal (Sigma-Aldrich, catalog number: C9157 )
    
15. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P5655 )
    
16. Sodium sulfate (Na₂SO₄) (Sigma-Aldrich, catalog number: 239313 )
    
17. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: 223506 )
    
18. Boric acid (H₃BO₃ ) (Sigma-Aldrich, catalog number: B6768 )
    
19. Manganese(II) chloride (MnCl₂) (Sigma-Aldrich, catalog number: 244589 )
    
20. Zinc chloride (ZnCl₂ ) (Sigma-Aldrich, catalog number: 746355 )
    
21. Sodium molybdate dehydrate (Na₂MoO₄·2H₂O) (Sigma-Aldrich, catalog number: 331058 )
    
22. Iron(III) chloride (FeCl₃) (Sigma-Aldrich, catalog number: 157740 )
    
23. Potato dextrose agar (PDA) 2% agar (Sigma-Aldrich) (see Recipes)
    
24. Potato dextrose broth (PDB) (Sigma-Aldrich) (see Recipes)
    
25. Charcoal mating plate medium (see Recipes)
    
26. U. maydis salt solution (Sigma-Aldrich) (see Recipes)
    
27. Trace element solution (Sigma-Aldrich) (see Recipes)
    

Equipment

1. Glass stirring rods
   
2. Pipettes
   
3. Ceramic mortars and pestles
   
4. Sterile cheesecloth
   
5. Hemocytometer or automated cell counter
   
6. Light microscope
   
7. Centrifuge holding 50 ml conical tubes (Beckman Coulter, model: J25I ) with a JS7.5 rotor (Beckman Coulter)
   
8. Growth chamber (Conviron, model: E15 )
   
9. Laminar flow hood
   
10. Rotational Incubator (VWR International, New Brunswick model: 12500KC )
    
11. Stationary incubator (Precision^TM, model: 815 )
    

Procedure