Lcb 6.1 Stemphylium botryosum fsp spinacia2018-04-24T09:12:37-05:00

National Seed Health System

Lcb 6.1  Stemphylium botryosum fsp spinacia

DATE: 12/2012
PATHOGEN: Stemphylium botryosum f. sp. spinacia (sexual: Pleospora herbarum)
HOST: spinach (Spinacia oleracea)
COMMON NAME: leaf spot
METHOD: Lcb 6.1 Freeze blotter method (Hernandez-Perez and du Toit, 2006)
SAMPLE: : 400 seeds


1. Place seed into a mesh tea strainer (Model 101, Venalicia Tea, Neuss, Germany).

2. Put the tea strainer in 150 ml of 1.2% NaOCl and shake manually for 60 seconds.

3. Triple-rinse seed in sterile deionized water and dry on a sterile paper towel in a laminar flow hood.

4. Flame-sterilize forceps, then plate 20 seed onto a sterile steel blue germination blotter (8.25 cm diameter; Anchor Paper Co., St. Paul, MN) that has been moistened with 4 ml of sterile deionized water in a 10-cm-diameter petri dish.

5. Seal the petri dishes with parafilm (Pechiney Plastic Packaging, Menasha, WI).

6. Incubate in the dark at 24°C for 25 h to imbibe moisture.

7. Freeze at –20°C for 22 to 25 h to prevent further germination.

8. Place in an incubator (Model I30BLL, Percival Scientific, Perry, IA) set at 24°C for 12 to 14 days with a 12h/12h day/night cycle, with cool-white fluorescent light and near-ultraviolet light by day.

9. Examine seeds approximately 4, 7, and 14 days after plating, at 8X to 100X magnification using a dissecting microscope for S. botryosum.


Hernandez-Perez, P. and du Toit, L. J. 2006. Seedborne Cladosporium variabile and Stemphylium botryosum in spinach. Plant Dis. 90:137-145.