Lcb 5.1 Cucumber Mosaic Virus 2017-05-20T17:32:18+00:00

National Seed Health System

Lcb 5.1   Cucumber Mosaic Virus

VERSION: 1.0
DATE: 12/2012
PATHOGEN: Cucumber Mosaic Virus (CMV)
HOST: spinach (Spinacia oleracea)
COMMON NAME: 
METHOD: Lcb 5.1 Seedling growout and ELISA (Yang et al., 1997)
METHOD CLASS: STANDARD (A)
SAMPLE: 

PROCEDURE:

1. Germinate seeds from suspected infected plants in large flats containing Redi-Earth 3CF potting mixture (Grace Sierra, Milpitas, CA).

2. Grow plants in a growth chamber with a 10-h day at 21°C or in a greenhouse with a 9-11h day at temperatures ranging from 20 to 30°C. The plants need to be carefully monitored to ensure they remain free of aphids.

3. When seedlings reach the 5-leaf stage harvest ~1.5 g of mature leaf tissue.

4. Extract plant sap and dilute 1:10 with phosphate buffered saline-Tween buffer.

5. Use protein A sandwich indirect ELISA (PAS-ELISA) to detect CMV. (CMV polyclonal antiserum (1:10,000 in PBST)).

6. Add alkaline phosphatase substrate (p-nitrophenyl phosphate, di-sodium; Sigma Chemical Co., St. Louis).

7. To quantify the reaction, determine absorbance at 405 nm with a microplate reader (Cambridge Technology Inc., Watertown, MA).

8. A sample is considered positive for CMV infection when the mean absorbance of two replicates is three times greater than the absorbance of the uninfected plant controls.

RECIPE:

PBST 137 mM NaCl, 1.5 mM K2HPO4, 8 mM Na2HPO4, 2.7 mM KCl, 0.05% [vol/vol] Tween 20, pH 7.4

REFERENCES:

Yang, Y., Kim, K. S. and Anderson, E. 1997. Seed transmission of cucumber mosaic virus in spinach. Phytopathology. 87(9):924-931.