National Seed Health System
Lcb 5.1 Cucumber Mosaic Virus
|PATHOGEN: Cucumber Mosaic Virus (CMV)|
|HOST: spinach (Spinacia oleracea)|
|METHOD: Lcb 5.1 Seedling growout and ELISA (Yang et al., 1997)|
|METHOD CLASS: STANDARD (A)|
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.
PBST 137 mM NaCl, 1.5 mM K2HPO4, 8 mM Na2HPO4, 2.7 mM KCl, 0.05% [vol/vol] Tween 20, pH 7.4
Yang, Y., Kim, K. S. and Anderson, E. 1997. Seed transmission of cucumber mosaic virus in spinach. Phytopathology. 87(9):924-931.