Lcb 4.1 Verticillium dahliae – Freeze blotter 2017-05-23T17:23:15+00:00

National Seed Health System

Lcb 4.1   Verticillium dahliae – Freeze blotter

DATE: 12/2012
PATHOGEN: Verticillium dahliae
HOST: spinach (Spinacia oleracea)
COMMON NAME: Verticillium wilt
METHOD: Lcb 4.1 Freeze Blotter Method (modified from duToit et al,. 2005)
SAMPLE: 600 seeds


Complete a surface-sterilization step using four replicates of non-treated seeds of each seed lot (600 seeds/replicate to be surface-sterilized). Do not use the surface-sterilization step for fungicide-treated seeds! Handle all fungicide-treated seeds with gloves and other appropriate safety measures. Surface-sterilize 10-15 more seeds than you need for plating that week, and complete the surface-sterilization step 1 day prior to plating the seeds.

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. To sterilize each clear acrylic square box [4” x 4” (= 10 cm x 10 cm), Hoffman Manufacturing, Inc.], spray the box and lid with 70% ethyl alcohol or isopropyl alcohol in a laminar flow hood (do not autoclave the boxes and lids), and let each box and lid dry in the hood.

9. Place a sterilized (autoclaved) Steel blue germination blotter (Anchor Paper Co.) in each acrylic box using flame-sterilized forceps and sterile technique in a laminar flow hood or biological safety cabinet. The blotters are custom-cut to fit the boxes. Prior to setting up the assay, the blotters must be sterilized by autoclaving at 15 psi for 20 to 25 minutes, cooling the blotters for 24 hours, and then autoclaving a second time at 15 psi for 20 to 25 minutes.

10. Add 12 ml sterile, de-ionized (or distilled) water to the blotter in each box. Allow the blotter to absorb all of the water. Excess water can lead to problems with bacterial growth on spinach seeds. Inadequate water may result in the blotter drying before the seed assay is complete, halting development of fungi.

11. Place spinach seeds of the appropriate treatment in each box using sterilized forceps. Forceps should be flame-sterilized at regular intervals (at least between boxes), and cooled before picking up the next seed to avoid heat-killing fungi on/in the seed picked up. Use 3 boxes for each replicate of 100 seeds: 34 seeds in each of two boxes and 32 seeds in the third box. 34 + 34 + 32 = 100 seeds. Place 6 seeds in each of 5 rows, and 4 seeds in the top row to identify the top left corner of each box for examining seeds three times. In the third box, place 4 seeds in both the top and bottom rows, and 6 seeds in the middle 4 rows. In the drawing below (not to scale), locations of the ‘white seeds’ are not included in the third box. Count seeds left to right, and top to bottom row for recording results on the data sheets:

12. Label the seed lot, replication, treatment, date, and other pertinent information on the side of the box. Parafilm is not needed with the acrylic boxes as the lids fit tight enough to prevent blotters from drying over the duration of the assay.

13. Cover the boxes with a towel to incubate the seeds in the dark at lab temperature (approximately 22-24oC) for 25 hours to imbibe water from the blotters. Do not stack the boxes more than three high because this can affect uniformity of freezing of the seeds.

14. Place the seeds at -20oC for 25 hours to kill the embryos. A few seeds may still germinate after 25 hours of freezing.

15. Place the boxes of seeds in numeric order of the treatments for that replication, in an incubator set at 24oC with a 12 hour/12 hour day/night cycle with both near-UV (= black) light and cool white fluorescent light by day. The combination of cool white and near-UV light promotes formation of conidia by fungi such as Stemphylium botryosum. For each replicate of the treatments, place the NP-10 agar and freeze-blotter assay boxes for each seed lot and seed treatment combination in a randomized order on the shelves in the incubator. Do not stack the boxes in the incubator. Keep the lids on the boxes.

16. Using a dissecting microscope, examine the seeds in numeric order of the plots for development of fungi (8 to 100x magnification) 5, 9, and 14 days after plating; examine the fungicide-treated seeds again 21 days after plating because fungicides slow development of fungi on the seeds. The lid of each box must be removed to examine the seeds microscopically. Record results of each reading on the spreadsheet provided. By 14 days, some fungi may have spread between seeds so this reading is primarily for identification of slow- growing or immature fungal cultures.

17. When the assay is completed, autoclave the seeds and blotters for appropriate disposal of infected plant material. Acrylic boxes can be washed and re-sterilized to be used again.


du Toit, L. J. and Hernandez-Perez, P. 2005. Efficacy of hot water and chlorine for eradication of Cladosporium variabile, Stemphylium botryosum, and Verticillium dahlia from spinach seed. Plant Disease. 89:1305-1312.