Isolation and Culture Media

Isolation

• MATERIALS FOR ISOLATION
  
• ISOLATION FROM ROOTS
  
• ISOLATION FROM GRASS ROOT AND CROWN ROT
  
• ISOLATION FROM CROWN, AND VASCULAR TISSUE, STEM, AND STORAGE ORGANS
  
• ISOLATION FROM LEAVES AND AERIAL PARTS
  
• NOTES
  

Culture Media

• P10VP/H for Phytophthora
  
• PARPH for Phytophthora and Pythium (without hymexasol)
  
• PYTHIUM SELECTIVE
  
• NASH & SNYDER'S FUSARIUM SELECTIVE MEDIUM
  
• ALKALINE-WATER AGAR (AWA) for Colletotrichum, Cylindrocarpon, Fusarium, Phoma, Rhizoctonia, and other related fungi
  
• THIELAVIOPSIS BASICOLA SELECTIVE MEDIA
  
• ACIDIFIED POTATO DEXTROSE AGAR
  
• GENERAL CULTURE MEDIA
  
• TECHNIQUES FOR SPORULATION
  
• OBTAINING PURE CULTURES
  
• MICROSCOPIC EXAMINATION OF FUNGI
  
• ADDITIONAL REFERENCES
  

Materials for Isolation

• 2 Petri dishes with selective culture media/sample
  Phytophthora: Corn meal agar (CMA) + P10ARPH, CMA + P10VPH
  Pythium: CMA Pythium A+B, CMA + P10ARP
  Thielaviopsis: Thielaviopsis selective media
  Ascomycetes, Hypomycetes and others: Alkaline water agar (AWA), acidified Potato Dextrose Agar (acPDA)
  
• Scalpel, knife, razor, tweezers
  
• Scissors
  
• Paper towel
  
• Marker
  
• Alcohol 70%
  
• Distilled deionized sterile water
  
• Stereoscope
  
• Compound microscope
  

Isolation from Roots
Injuries in the root system can be expressed in the aerial part of plants as yellowing, stunting, or wilting. Observe disease symptoms (also signs) including the small-discolored areas, dead areas, root tail, and general death of the root system. Depending on the symptoms take: individual symptomatic roots (with part of the lesion and part of healthy tissue), or a group of affected roots.

1. Wash the tissue under sink water to release soil, debris and external     contamination.
2. Dry the tissue in paper towel.
3. Slice a group of roots into multiple pieces.
4. Leave tissue drying in the paper towel.
5. Using tweezers place samples in 5 points of the plate in at least
    two Petri dishes containing selective media (i.e. Phytophthora P10ARPH).     Some samples of root rot require transfer into two or more different
    selective media (Phytophthora selective, Pythium selective, AWA).
6. Incubate plates under dark at room temperature (25 C) for 24-48 hours.
7. Observe under compound microscope inverting plates.
8. Observe the area close to the sections for mycelial growth (i.e. coralloid     coenocytic mycelia for Phytophthora species) or fruiting bodies (i.e.
    sporangia, oospores, etc).
9. In many instances it is possible to make the identification of the
    organism associated to the disease at this stage. Other cases require
    of future transferences and special procedures for identification. For
    those cases mark areas of interest in original plates.
10. Transfer marked areas to new selective media (i.e. P10ARPH) and
    after 2-3 days to general media [i.e. Corn Meal Agar (CMA) for
    Phytophthora and Pythium].
11. Send samples to the PPIL for identification to species levels.

NOTE: To prevent contamination, use a clean tweezers, scalpels or razors. You can sterilize tweezers and scalpels by submerging in alcohol 70%, or by flaming. Remember to sterilize material every time you work with different samples.