NC State and USDA Cucumber Disease Handbook

Belly Rot (Rhizoctonia solani Kuhn)

Inoculum Preparation:

1. PDA plates:

Make PDA following directions on can (Difco). Pour plates with 20ml of agar per plate. Allow to solidify before attempting to grow Rhizoc on the plates. Do not keep the fungus growing on PDA plates for any longer than 2 weeks. The longer it is kept on a plate the greater the probability of mutations.

2. Small amounts of oat grains:

Small amounts can be made by mixing equal amount of oats and water in the desired size flask. Autoclave for 1 1/2 hours for two consecutive days and allow to cool before inoculating with PDA disks or other oat grains. When working with small amounts I suggest drying the oats when the are fully colonized. This can be done under the hood in petri dishes. Once dry, the oats are fairly resistant to contamination and can be stored covered on the shelf. This is the recommended method or making oats for the damping-off tests.

3. Large amounts of oat grains:

a. Jenkins procedure:
crimped oats 325.0 ml
deionized water 200.0 ml
Put the oats and water in autoclavable bag or flask then plug with cotton
Autoclave for 1 hour on each of 2 consecutive days
Place mycelial plug into each bag of oats
Incubate at 20-24C for about 2 weeks
Squeeze bags every other day to distribute fungus over oats

b. Wehner/Abad procedure:
crimped oats 5 kg.
deionized water 4 liters
Put the oats and water in autoclavable trays (Nalgene Large Capacity - Fisher Cat. 13-359-20B)
Soak for 24 hours and mix
Autoclave for 1 hour on each of 3 consecutive days (or 1 1/2 hours for 2 consecutive days)
Place several PDA disks evenly spaced in the tray (may wish to tray using colonized oat grains)
Cover the tray immediately with sterility maintenance covers if it is possible (American Scientific A 9355-17)
Then incubate for about 4 weeks and store the inoculum at cool chamber (4-10°C)
The flats may be stacked at room temperature in a place relatively free from air movements.

c. Modified Wehner/Abad procedure:(most recent modifications-1992)
crimped oats 5 kg
deionized water 3 liters
Put the oats and water in autoclavable trays (Nalgene Large Capacity - Fisher Cat. 13-359-20B)
Soak for 24 hours and mix (optional)
Autoclave 1 1/2 hours for 2 consecutive days
When trays have cooled and 15-20 agar disks to the top of the tray, under sterile conditions
Cover the tray immediately with sterility maintenance covers if it is possible (American Scientific A 9355-17)
Then incubate for about 4-6 weeks and store the inoculum at cool chamber (4-10°C.)
The flats may be stacked at room temperature in a place relatively free from air movements
Oats may be dried in a seed drier
This makes them easy and nice to apply, but the drying process is very slow
You can only put two trays in the drier, spread between racks and for two days, or you risk major contamination

Inoculation procedure:

40 oat grains infested with R. solani weighs 2.8 g

For 1 flat of soil (10 L soil); need 28 g oats (60 ml oats)

These measurement should be checked each year as the weight may vary considerably

Oat grains:

kg/field

= 38 rows/field x 18 plots/row x 20 ft2/plot x 144 in2/ft2 x 4 grains/in2 x .000033 kg/grain

= 260 kg (572 lb)

These calculations should be checked yearly.

Test Methods:

Detached-fruit test:

1. Line bottom of trays with newspaper to prevent soil from falling through

2. Fill flats with two shovels of sterile field soil

3. Wet soil in flats with 1.5 liters water (The soil can also be wetted lightly before filling flats to get the right soil moisture)

4. Inoculate soil with 4 oat grains / in2 or 125 ml/flat

5. Dip fruits in bleach solution (10% Clorox) for 30 sec.

6. Do not rinse in water

7. Place fruits on inoculated soil in flats

8. Place flats in disease chamber (2 days 100 % RH)

9. Rate for percent damage (30% is entire belly) per fruit after 7-10 days

Field Inoculation with Rhizoctonia solani:

1. Grow large quantities of oats. (some fields may require as much as 500 lbs. dry weight of oats)

2. When plants reach tip-over, lightly rake two feet on each side of the row to help the oats stay in place.

3. Inoculate with 4 grains/square inch

4. Gently sweep the oats off the leave with a broom

Seedling Inoculation with Rhizoctonia solani:

5 methods have been tried in seedling tests.

1. PDA disks placed in the center of a leaf will cause symptoms if humidity is kept high. Disks should be between 3 and 7 days old, and can be secured to the leaf by smashing the disk with your thumb. Symptoms are necrotic lesions originating from the agar disk.

2. PDA disks placed on the hypocotyl at the soil line. Disks are the same as those used for the leaf method. Symptoms are cracking of the stem and brown lesions on the stem.

3. Leaf dip. Inoculum is made by combining on plate of Rhizoc growing on PDA with 100ml of water for 30 seconds in the blender. This is then poured in a beaker with one drop of spreader sticker. Leaves ma the be dipped in the solution. It will only work easily if plants are potted individually. Symptoms include many small lesions which may grow together with time.

4. A syringe may be used to inject a suspension into the stem. Inoculum is made by combining on plate of Rhizoc growing on PDA with 100ml of water for 30 seconds in the blender. Inoculum should be injected until it begins to ooze out of the wound. Symptoms may include necrotic lesions and cracking of the stem.

5. A soil drench may be applied by placing 1 ml of inoculum at the base of the stem. Inoculum is made by combining one 7-day-old plate of Rhizoc (Rs-143-N) growing on PDA with 100ml of water for 30 seconds in the blender. Symptoms are necrotic lesions and cracking of the stem. This method is similar to placing an agar disk on the hypocotyl, but it is probably a lot easier. Plant in 4" pots, peatlite with fertilizer (mixed into soil); inoculate at 2nd true leaf; read in 5 to 10 days.

Damping-off Test with Rhizoctonia solani:

1. Flats should be filled with a mix of 1 sand: 2 soil: 1 metro mix.

(The soil can also be wetted lightly before filling flats to get the right soil moisture)

2. Each flat should be divide into two halves with a piece of wood.

3. In the center of each half flat place 25 oat grains and cover with soil.

4. A template is used to punch twenty spaced holes in the soil mix. (see Echandi)

5. Plant one seed in each hole.

6. As plants emerge, mark them with toothpicks

7. Rate after 14 days (remember there used to be a plant at each toothpick).