Lcb 4.2 Verticillium dahliae – NP-10 agar 2017-05-23T17:47:26+00:00

National Seed Health System

Lcb 4.2   Verticillium dahliae – NP-10 agar

VERSION: 1.0
DATE: 12/2012
PATHOGEN: Verticillium dahliae
HOST: spinach (Spinacia oleracea)
COMMON NAME: Verticillium wilt
METHOD: Lcb 4.2 NP-10 Agar Method (Kabir et al., 2004)
METHOD CLASS: STANDARD (A)
SAMPLE: 100 seeds

PROCEDURE:

1. For each treatment combination except the fungicide-treated seeds, surface-sterilize one replicate of ~110 seeds (a little more seeds than the 100 to be plated in case a few seeds are dropped during the setup).

2. Sterilize absorbent paper towels in a laminar flow hood or biological safety cabinet by spraying the paper towel thoroughly with 70% ethyl alcohol or isopropanol and letting the paper towel dry in hood, or by autoclaving the paper towels.

3. Place 110 seeds for each treatment combination in one of the tea-strainers provided. Prepare a 1.2% NaOCl solution and dispense enough of the solution into a small glass beaker to fully cover the tea-strainer when it is immersed in the solution. Place the tea-strainer with the 110 seeds in the NaOCl solution for 60 seconds with constant manual agitation of the strainer to keep the seeds swirling throughout the 60 seconds. NOTE: Use a new bottle of NaOCl for the ring test, and store the bottle in a refrigerator. Prepare the 1.2% NaOCl solution just prior to (within an hour of) treating the seed samples.

4. Immediately remove the tea-strainer from the NaOCl solution, shake off the solution, and immerse the tea-strainer and seeds in sterile de-ionized (or distilled) water in a small glass beaker for 30 seconds with constant agitation. Do not use sterile tap water. Use enough water to fully immerse the tea-strainer and seeds.

5. Repeat the rinse step two more times, using a new batch of sterile deionized or sterile distilled water for each rinse.

6. Spread the seeds onto dry, sterilized paper towel in a laminar flow hood or biological safety cabinet to dry thoroughly for 60 minutes.

7. Place the surface-sterilized, dried seeds in a sterile, disposable petri dish labeled clearly with the replication and treatment information. Store the seeds in the dark to be plated the next day.

8. Sterilize the acrylic boxes and lids prior to adding the agar medium, by spraying each box and lid with 70% isopropyl alcohol in a biological safety cabinet or laminar flow hood. Then air-dry the boxes and lids. If the BSC or laminar flow hood has UV light, expose the boxes and lids to UV light for 10 to 15 minutes for additional sterilization.

9. Dispense 35 ml molten NP-10 agar medium into the appropriate number of sterilized boxes (3 boxes x 2 seed treatments x 3 seed lots = 18 boxes per replicate). Once the medium has solidified, store the boxes at room temperature in the dark for up to 1 week.

10. Use flame-sterilized forceps and sterile technique in a laminar flow hood to plate spinach seeds onto NP-10 agar medium in each box (32 to 34 seeds in each box as shown above for the freeze-blotter assay). Press each seed into the agar medium slightly to prevent seeds from rolling around when the boxes are moved.

11. Place each box in an incubator set at 24oC under a 12 hour/12 hour day/night cycle with both near-UV (= black) light and cool white fluorescent light by day.

12. Examine the seeds microscopically 5, 9, and 14 days after plating as described above; in addition, examine the fungicide-treated seeds again 21 days after plating. 13. After completing the assay, autoclave the NP-10 agar medium and seeds to destroy the antibiotics in the medium for appropriate disposal. Use a spatula to remove the agar medium and seeds from the boxes. Do not autoclave the boxes. The boxes can be washed, re-sterilized, and re-used.

BUFFERS USED:

Modified Sorensen’s NP-10 agar medium
 
Ingredients: Amount:
Bottle A
Polygalacturonic acid, Na salt from orange, SIGMA Grade (P-3889) 5.0 g
NaOH pellets (0.025N) 1.2 g
Distilled or de-ionized water 500 ml

Bottle B
Agar (Difco Bacto) 15.0 g
KNO3 1.0 g
 KH2PO4 1.0 g
 KCI 0.5 g
 MgSO4.H2O 0.5 g
Tergitol NP-10 0.5 ml
Distilled or de-ionized water 500 ml

Antibiotics
Chloramphenicol  0.5 g
Streptomcyin sulfate  0.5 g
Chlortetracycline hydrochloride  0.5 g

• Prepare and autoclave Bottle A and Bottle B separately.

• Allow both bottles to cool to 50oC slowly using a hot water bath.

• Chloramphenicol and chlortetracycline dissolve more readily in methanol than ethanol.

• Streptomycin sulfate dissolves in water. Therefore, prepare a stock solution of chlortetracycline (15 mg/1 ml methanol), chloramphenicol (100 mg/1 ml methanol), and streptomycin sulfate (25 mg/1 ml deionized, sterilized water) and filter sterilize each. Store the stock solutions in a fridge. After adding the antibiotics to Bottle B, add the contents of Bottle A to Bottle B. Mix thoroughly using a magnetic stirrer and stir bar. Dispense the medium into the sterilized acrylic boxes.

REFERENCES:

Kabir, Z., Bhat, R. G. and Subbarao, K. V. 2004. Comparison of media for recovery of Verticillium dahliae from soil. Plant Disease. 1:49-55.