Screening for Disease Resistance in Faba Beans

Pathogenic fungi are normally grown as pure cultures in bottles, test tubes, or petri dishes containing a water-agar gel with added nutrients. Although such media may contain all known essential nutrients (carbohydrates, proteins, fats, amino acids, vitamins, minerals, and water), none is exactly the same as the natural substrate of the pathogen. However, plant pathologists must depend on such media until more is known about the nutrition and artificial cultivation of pathogenic fungi.

     Before preparing culture media it is always important to remember the following points:

a. Wipe the floor and laboratory benches with 0.5% sodium hypochlorite solution (10% Clorhox), to avoid microbial contamination of plates. This is particularly important in tropical regions.
b. Tap water contains useful trace elements, therefore it is better than distilled water for the growth of most  pathogens.
c. When preparing agar, dissolve it in half the quantity of water on a iow heat, the nutrients in the other half, then mix and autoclave.
d. Pathogenic fungi normally grow well at pH 6-6.5. Isolation media can therefore be acidified with 25% lactic acid (added at rate of 2-4 drops per petri dish after it is cooled) to inhibit bacterial contaminants which prefer pH7.
e. Carbohydrate - poor media help sporulation of several pathogens.
f. Bolrylis fabae does not sporulate well on potato-dextrose-agar.
g. Sterilization of media is normally done at 15 p.s.i. (121°C) for 20 minutes. The time required for sterilization however, varies with the volume of liquid in the container as shown below:

Commonly Used Media

ABA BEAN - DEXTROSE AGAR (FDA)

This medium is used for propagation of B. fabae, A. fabae, and A. tenuis.

Method:

a. Weigh out 200 g of faba bean seeds or 400 g of faba bean leaves in a 1.5 1 flask. Add 11 of water, and autoclave at 15 p.s.i. for 30 minutes.
b. Pass the autoclaved beans through a sieve, add 18 g of agar, heat, and stir till dissolved.
c. Add 20 g of dextrose,stir till dissolved, and make up the volume to 11 with tap water. d. Autoclave at 15 p.s.i. for 20 minutes, cool to about 40°C, and pour into petri dishes (normally 40 petri dishes/1).

POTATO - DEXTROSE AGAR (PDA)

This is useful for the isolation of several pathogens (2).

Method:

a. Scrub potatoes clean and cut into small cubes without peeling.
b. Weigh out 200 g, rinse rapidly in tap water, and place in a 1.51 flask. Add 11 of tap water and boil for 1 hour.
c. Fitter the boiled potatoes through a cheesecloth and add 17 g of agar to the filtrate. Heat and stir till dissolved.
d. Add 20 g of dextrose, stir till dissolved, and make up the volume to 11 with tap water, e. Autoclave at 15 p.s.i. for 20 minutes, cool, and pour into petri dishes.

TAP WATER AGAR (TWA)

This medium is used to induce sporulation of many pathogens (4).

Method:

a. Dissolve 18 g of agar in 11 of tap water in a 1.51 container.
b. Autoclave at 15 p.s.i. for 20 minutes.
c. Cool and pour into petri dishes.

SELECTIVE MEDIUM FOR Rhizoctonia solani

This medium is used for isolating R. solani from soil (2).

Method:

a. Weigh out the following ingredients:

 * Rose Bengal is an inhibitory chemical used to control fast growing fungi and inhibit many bacteria.

b. Place the agar in half the quantity of water (0.51) and dissolve over a low heat.
c. Place the other ingredients in the remaining water, dissolve, and add to the agar.
d. Autoclave at 15 p.s.i. for 20 minutes, cool, and pour into petri dishes.

SELECTIVE MEDIUM FOR Fusarium oxysporum OR F. solani:

Method:

a. Weigh out the following ingredients:

* Oxgall is an inhibitory chemical used to restrict fungal colony size to aid counting.
** PCNB is a fungicide which inhibits many fungi except Fusariurn s p. and Pythium sp. and Phytophthora. sp.

d. Pour the medium into petri dishes.