Title: ELISA ANALYSIS FOR FUSARIUM IN BARLEY: DEVELOPMENT OF METHODOLOGY AND FIELD ASSESSMENT Authors
|Hill, N - UNIV OF GA, ATHENS GA|
|Schwarz, P - N DAKOTA UNIV, FARGO ND|
|Neate, S - N DAKOTA UNIV, FARGO ND|
|Horsley, R - N DAKOTA UNIV, FARGO ND|
Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 30, 2006
Publication Date: November 21, 2006
Citation: Hill, N.S., Schwarz, P., Dahleen, L.S., Neate, S., Horsley, R., Glenn, A.E., O Donnell, K. 2006. Elisa analysis for Fusarium in barley: Development of methodology and field assessment. Crop Science. 46:2636-2642. Interpretive Summary: Fusarium head blight (FHB) of barley and other small grain cereals poses a serious constraint to their production due to reduction in yields and price discounts. Moreover, FHB-contaminated grains are often contaminated with toxins and estrogens. In this study, an antibody specific to Fusarium was developed so that we could more accurately quantify the amount of FHB in barley. Development of this novel test called ELISA was prompted by the fact that visual inspection of barley is often a poor indicator of toxin contamination. Mononclonal antibodies raised to the primary FHB pathogen, Fusarium graminearum, were tested for cross-reactivity with other Fusarium spp. and shown to be specific to members of this genus. Results of the ELISA test demonstrated that it is a more accurate predictor of toxin contamination than visual inspection of FHB of barley. These results will benefit barley producers and processors by providing a method that more accurately predicts toxin contamination than visual inspection for FHB.
Technical Abstract: Screening for Fusarium head blight (FHB) involves inoculating barley (Hordeum vulgare L.) seed heads with Fusarium graminearum [teleomorph Gibberella zeae] followed by visual observation of disease progression and analysis for the mycotoxin deoxynivalenol (DON). Disease symptoms and DON are not uniformly expressed and DON production varies with environment. DON and FHB are not highly correlated with one another. The objective of this study was to develop an enzyme linked immunosorbant assay (ELISA) for quantification of FHB in barley. Mononclonal antibodies raised to Fusarium graminearum were tested for cross-reaction with other Fusarium spp. Antibodies from cell line IF8 cross-reacted with all Fusarium spp. but not other ascomycota tested. A laboratory method was developed using seed lots with no, low, and high levels of DON. Varying quantities of seed, extraction buffer, and agitation were tested and Fusarium quantified with ELISA. The best protocol for ELISA used 5 g seed, a 1:6 (w/v) ratio of seed:extraction buffer for 1 h. Five genotypes each for high, medium, and low ELISA values were selected from a field experiment using a doubled-haploid mapping population in 2003. The lines were grown in 2004 and scored for FHB, DON and ELISA. ELISA had lower error than FHB or DON and lines low, medium, and high ELISA in 2003 had low, medium, and high DON, FHB, and ELISA in 2004. ELISA and DON were correlated in both controlled studies (r=0.51) and samples selected from grain elevators in 1993-2003 (r=0.71). The correlation among ELISA and DON in the grain elevator samples indicates naturally occurring Fusarium spp. outside the B clade had no effect on the performance of the ELISA analysis.