Screening for Disease Resistance in Faba Beans

2.3. Artificial Production of Epiphytotics in Faba Bean Disease Screening Nurseries

Procedure No.2 for preparation of the inoculum

This is a simpler procedure, suggested only for small-scale artificial inoculations in areas where laboratory facilities are not available.

a. Collect chocolate spot-infected leaves, flower parts, and stems having the aggressive stage of the disease (defoliated infected leaves from the field form a good source of inoculum).
b. Rinse infected plant parts in water, place in pans, then cover with plastic sheets to maintain high humidity.
c. Keep at room temperature for 3-5 days, until spores have formed.
d. Place sporulating plant parts on top of a sieve and wash vigorously with tap water, while collecting the leachates containing spores in a container.
e. Count spores in the leachates, if possible, and use for direct artificial inoculation in the field.

2.3.1.3. Artificial Inoculation in the Field

a. Use the spore suspension containing 400,000500,000 spores/ml for inoculation. This spore density has been standardized to kill the Syrian local cultivar BPL 1814 under Lattakia conditions.
b. Inoculate 10-12 week old plants in the field after sunset, with 10-15 ml of suspension per plant and using a knapsack sprayer.
c. Cover inoculated nurseries immediately with polythene sheets, supported by metal frames (2 X 6 X 1.5 m) to provide adequate humidity and a water film on the leaf surfaces. Next morning, uncover plants, spray with a fine mist of water, once every 3 hrs, then cover again in the evening to maintain high humidity.
d. Uncover, spray with water, and then cover again until the aggressive stage of chocolate spot is evident on local check lines (normally 1-2 weeks under Lattakia conditions).
e. Take disease readings 2-3 weeks after inoculation. Polythene sheets should be removed on sunny days and replaced after sunset. However, on continuous rainy days, as long as the leaf surface remains wet there is no need to cover the nursery with polythene sheets. The above procedure is also good for artificial inoculation in special moist chambers (Fig. 9) designed specifically for race studies.

Fig. 9. Special moist chambers designed for race studies in different foliar pathogens.

2.3.1.4 Detached Leaf Test in the Laboratory

This is a simple and rapid laboratory test (Fig. 8) designed to double check results from chocolate spot screening nurseries in the field.

a. Harvest fully-expanded leaflets of similar physiological age from the eighth node position.
b. Lay leaflets flat on a 2 cm thick moist sponge lining the bottom of 90 X 40 X 6 cm galvanized metal or plastic pans.
c. Inoculate the upper laminal surface of the leaves with 0.1 ml of a spore suspension containing about 500,000 spores of B. fabae/ml. One droplet on each half of each leaflet is enough.
d. Cover the pans immediately with polythene sheets and leave at room temperature (20 + 2°C).
e. Score disease development after 5 or 6 days.

2.3.2. Ascochyta Blight ( A.Fabae )

Procedure No.1 for inoculum preparation

This procedure should be used where pathologists and laboratory facilities are available.

2.3.2.1 Isolation of A.fabae

a. Surface sterilize infected faba bean seeds in a 0.5% sodium hypochlorite solution (10% Clorox) for 2-4 min, plate on FDA medium, and incubate at 221C.Do not sterilize for more than 1-2 min when infected leaf tissue is used for isolation.
b. After 5-8 days' incubation, take samples from the growing edge of A. fabae colonies and plate on FDA (Fig. 10).

c. Repeat step b until pure colonies are obtained.
 d. Incubate a pure culture at room temperature for 7-10 days until spores have formed, exposing plates to 12 hrs light from a 40W fluorescent tube and 12 hrs darkness to encourage sporulation.

Fig. 10. Growth of Ascocbyta fabae from infected faba bean seed in culture.

2.3.2.2 Propagation and Harvest of A.fabae Spores

a. Place 5-10 mls of sterilized tap water on the surface of pure colonies in petri dishes.
b. Leave at room temperature for 24 hrs, until spores have been released from the small black pycnidia which can be seen on the plate. To help spore release from pycnidia, the entire culture may be transferred to a flask and suspended for 3-6 hrs.
c. Inoculate fresh plates by transferring spore suspension from one plate to another, incubate, and expose to light and darkness as described for B. fabae.
d. Use cultures for field inoculations when they are 14-18 days old.
e. Transfer the contents of 100 isolation plates to a 101 plastic container.
f. Add 51 of tap water and keep at room temperature for 12 hrs.
g. Shake vigorously for 30 min and filter the contents of the container through a cheesecloth.
h. Dilute the filtrate with tap water until a spore suspension containing about 300,000 spores per ml is obtained.

Procedure No.2 for inoculum preparation

This is a simpler procedure, suggested only for small-scale artificial inoculations in areas where laboratory facilities are not available.

a. Collect ascochyta blight - infected leaves, stems, and pods (Fig. 11).
b. Fill 3/4 of a 201 plastic container with infected tissue and add tap water until all infected material is covered.
c. Leave at room temperature for 6 hrs, shake vigorously for 30 minutes, then decant into another container by filtering the contents through cheesecloth.
d. Count the spores in the filtrates if possible, and use for direct inoculation in the field.

Fig. 11. Ascochyta blight-infected leaves, stems and pods.

2.3.2.3. Artificial Inoculation in the Field

a. Use the spore suspension containing about 300,000 spores per ml for inoculation. This spore density has been standarized to kill the Syrian local cultivar BPL 1814 under Lattakia conditions.

b. Inoculate 10-12 week-old plants in the field, with 10-15 ml of suspension per plant and using a knapsack sprayer.
c. After 12 hrs, spray plants during the day, once every 3-4 hrs, with a fine mist of water.
d. Record disease ratings 2-3 weeks after inoculation. In the case of A. fabae, there is no need to cover plants with polythene sheets, unless weather conditions are not favorable for disease development.
 
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