Verticillium dahliae microsclerotia production
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Purpose
Ingredients
Minimal agar media
| Ingredient | 500 ml | 1 L |
|---|---|---|
| KH2PO4 | 0.7 g | 1.4 g |
| K2PO4 | 0.85 g | 1.7 g |
| Na2SO4 | 1.6 g | 3.2 g |
| Ca(NO3)2.4H2O | 0.2 g | 0.4 g |
| KNO3 | 2.3 g | 4.6 g |
| MgSO47H2O | 0.35 g | 0.7 g |
| NaCl | 0.25 g | 0.5 g |
| Minor element sol. | 0.5 ml | 1 ml |
| Sucrose | 10 g | 20 g |
| Agar | 10 g | 20 g |
| H2O | 500 ml | 1 L |
Minimal element solution
| Ingredient | 100 ml | 1 L |
|---|---|---|
| ZnSO4.7H2O | 0.1 g | 1 g |
| MnCl2.4H2O | 0.1 g | 1 g |
| H3BO3 | 1.6 g | 3.2 g |
| FeCl3.6H2O | 0.05 g | 0.5 g |
| CuSO4.5H2O | 0.01 g | 0.1 g |
| Kl | 0.01 g | 0.1 g |
| Na2MoO4.2H2O | 0.01 g | 0.1 g |
| H2O | 100 ml | 1 L |
Supplements
(Not used for microsclerotia production)
- Amino acids 100mg/L
- Nitrogenous bases 10mg/L
- Vitamins 10mg/L
Instructions
- Autoclave cellophane membranse and place on minimal agar media.
- Inoculate plates with conidial suspensions (harvested from on-week-old cultures grown on PDA)
- Seal plates in plastic bags and incubate in the dark at 24°C for 18 days.
- At harvest, cover cellophane membranse with microsclerotia.
- Blend each suspension in a Waring blender for 1 minute at high speed and determine the densities of microsclerotia with the aid of a hemocytometer.
Notes
- Cellophane membranes were scraped off using a scalpel and suspended in sterile water.
References
- Microsclerotia production From Puhalla, J. E., and Mayfield, J. E. 1974. The mechanism of heterokaryotic growth in Verticillium dahliae. Genetics 76:411-422.
Contributed by
Contributed by Sherrie Smith; the University of Arkansas
