Submitted to: Crop Science
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/15/2006
Publication Date: 11/21/2006
Citation: Hill, N.S., Dahleen, L.S., Schwartz, P., Neate, S., Horsley, R. 2006. Elisa analysis for fusarium in barley: development of methodology and field assessment. Crop Science. 46:2636-2642. Interpretive Summary: Fusarium head blight (FHB) and the deoxynivalenol (DON) toxin it produces have been a continuing problem for barley since the early 1990s. Rating FHB in the field usually involves counting the number of infected kernels per spike from multiple samples per plot. Quantifying DON contamination involves grinding grain samples, extracting toxins and identifying them using GC-MS. Both these methods are labor and time intensive, and often lack repeatability across environments. We developed an enzyme linked immunosorbant assay (ELISA) protocol to quantify Fusarium infection in barley. Compared to field ratings and DON quantification, the ELISA protocol was more accurate, with lower coefficients of variation, and required less replications to detect differences between genotypes. This suggests that ELISA is a better system for quantifying Fusarium infection in barley than field FHB ratings and DON tests.
Technical Abstract: Screening for Fusarium head blight (FHB) involves inoculating developing barley (Hordeum vulgare L.) spikes with Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.)] followed by visual observation of disease progression, and analysis for the mycotoxin deoxynivalenol. Disease symptoms and toxins are not always expressed and mycotoxin production is under environmental influences. Hence, these two methods of Fusarium assessment are not highly correlated with one another, or from one environment to the next. The objective of this study was to develop an enzyme linked immunosorbant assay (ELISA) protocol for quantification of Fusarium in barley. Laboratory method development was conducted using three seed lots of barley testing either 0.0, 1.3, or 24.7 ng g-1 deoxynivalenol, and 0, 3, and 24% kernel damage indicative of FHB, respectively. An ELISA protocol was used to quantify the extracted proteins. The best extraction protocol for ELISA analysis used 5 g seed, extracted with a 1:6 (w/v) ratio of seed:buffer solution for 1 h. The method was used to select five genotypes each for high, medium, and low ELISA values within a doubled-haploid mapping population inoculated with F. graminearum and grown at Osnabrock and Langdon, ND in 2003. The lines were scored for FHB and harvested grain analyzed for DON and F. graminearum with ELISA. Coefficients of variation and numbers of replications necessary to detect specific differences in FHB, DON, and ELISA were calculated. FHB and DON had higher CV’s than ELISA and required more replications (15, 12, and 4 respectively) to detect differences equal to 50% of the means. Lines selected as low, medium, and high using ELISA in 2003 had low, medium, and high DON, FHB, and ELISA in 2004. These data suggest ELISA is a better system for F. graminearum quantification than DON or FHB.