Screening for Disease Resistance in Faba Beans
General Laboratory Techniques
- Isolation of Fungi from Infected Plant Tissue

Infected plant parts are brought to the laboratory in plastic bags.

Method:

a. Wash specimens in tap water.
b. Cut small portions (0.5 - 1.0 cro) of tissue from the advancing margin of a lesion with a sterile knife.
c. Surface sterilize these portions with any of the following disinfectants for 2-3 minutes:
      0.1 % mercuric chloride
      0.1 % silver nitrate
      0.5% sodium hypochlorite 70% alcohol
      5% formalin
      3 % hydrogen peroxide
      2% potassium permanganate
d. Dip in sterile water, then plate on the appropriate medium. Rinsing in water is not necessary if sodium hypochlorite solution is used.
e. Woody parts may be dipped in 70% alcohol and flamed. The surface is then cut away with a flamed knife and the tissue beneath is plated.
f. Incubate plates at 22°C and examine after 5-7 days.
- Purification of Fungal Cultures
Several saprophytes may also be present in infected plant tissues and they may grow into the medium with the principal pathogen. To obtain a pure culture of the pathogen, take a small sample of the growing edge of a colony with a flamed loop and streak over the surface of a prepoured plate. As the streak progresses over the agar, fungal spores are separated until single spores are obtained from which separate colonies will grow. Repeat this procedure until pure cultures are obtained (Fig. 2).
Fig. 2. Pure culture of Botrytis fabae.
- Single Spore Isolation
Single spore isolations are made for various studies, particularly to investigate pathogenic variabilities. Place an inoculum of spores in a tube containing 10 mls of sterile water. Streak this spore suspension along a marked line on the surface of a thin tap water agar medium, and incubate at 22°C. After 24 hrs incubation, select germinated spores using a stereoscopic microscope and transfer one spore at a time to another agar plate.
 
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