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Published online 21 November 2006
Published in Crop Sci 46:2636-2642 (2006)
© 2006 Crop Science Society of America
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SEED PHYSIOLOGY, PRODUCTION & TECHNOLOGY

ELISA Analysis for Fusarium in Barley

Development of Methodology and Field Assessment

N. S. Hilla,*, P. Schwarzb, L. S. Dahleenc, S. M. Neated, R. Horsleyb, A. E. Glenne and K. O'Donnellf

a Dep. Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602
b Plant Sciences Dep., North Dakota State Univ., Fargo, ND 58105
c USDA-ARS, Cereal Crops Research Unit, Fargo, ND 58105
d Dep. of Plant Pathology, North Dakota State Univ., Fargo, ND 58105
e USDA-ARS, Toxicology & Mycotoxin Research Unit, Russell Research Center, Athens, GA 30604
f USDA-ARS, Microbial Genomics Research Unit, National Center for Agricultural Utilization Research, Peoria, IL, 61604

* Corresponding author (nhill{at}uga.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Evaluating Fusarium head blight (FHB) involves inoculating barley (Hordeum vulgare L.) with Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schwein.) Petch] followed by visual observation of disease and analysis for deoxynivalenol (DON). Disease symptoms and DON are not always correlated because both are affected by environmental variables. The objective of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for quantification of FHB in barley. Antibodies to F. graminearum were tested for reaction with other Fusarium spp. Antibodies from cell line IF8 reacted with Fusarium spp. tested, but not other Ascomycota. The ELISA method was developed using seed lots with no, low, and high levels of DON. Quantity of seed, volume of extraction buffer, and agitation time were tested and Fusarium quantified with ELISA. 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. Lines selected for low, medium, and high ELISA in 2003 had low, medium, and high ELISA values in 2004. ELISA and DON were correlated in both field experiments (r = 0.51). ELISA and DON were correlated (r = 0.71) in samples selected from grain elevators in 1993 through 2003 indicating naturally occurring Fusarium spp. outside the B clade had no effect on the performance of the ELISA analysis.

Abbreviations: DON, deoxynivalenol • ELISA, enzyme-linked immunosorbent assay • FHB, Fusarium head blight


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
INFECTION of barley by F. graminearum causes a destructive disease known as Fusarium head blight (FHB) (Rudd et al., 2001). Numerous variables—cultural practices, spike morphology, canopy density, plant height, rainfall, relative humidity, temperature, and plant genetics—affect fungal colonization of the spike. Screening for FHB involves inoculation of the developing spike followed by visual screening of disease development. Because disease development is dependent on environmental conditions, sequential observations are necessary to assess the disease (Rudd et al., 2001). Chemical analysis for DON is also performed since disease development and toxin production can be under independent control (De la Pena et al., 1999). In uniform evaluation nurseries, correlations of head blight scores across locations among elite barley germplasms and cultivars ranged between –0.27 and 0.69 in 2002 www.scabusa.org/pdfs_dbupload/02_NABSEN_Rep.pdf), and –0.45 to 0.73 in 2004 (Neate and Gross, 2004). Correlations across locations for DON ranged from 0.00 and 0.83 in the 2002 nurseries and from 0.09 to 0.73 in the 2004 nurseries. Experimental error for both of these Fusarium indices are typically high (CVs ranging from 40 to 70%) suggesting that lack of correlation among lines from location to location is a combination of experimental error and genotype x environment interaction. Thus, a standardized quantitative screening technique is needed to assess Fusarium head blight in barley. The objectives of this study were to (i) test existing antibodies for cross reaction with phylogenetically distinct Fusarium spp. and other Ascomycota, (ii) assess extraction conditions necessary for ELISA quantification of Fusarium spp. in barley expressing symptoms of head blight, and (iii) evaluate the performance of the ELISA method in both inoculated and naturally infested grain via comparison with FHB and DON analyses.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Experiment 1: Cross Reaction of Monoclonal Antibodies from Cell Line IF8 with Fungal Proteins from Fusarium and Other Ascomycota Species
Antibodies from UGA cell line IF8 were tested for cross reaction with six Fusarium spp. within the B clade (O'Donnell et al., 2004), six Fusarium spp. which are phylogenetically distinct from the B clade, and 10 stock cultures of Ascomycoteous fungi other than Fusarium spp. Isolates from each were inoculated in Shenk–Hildebrandt medium (Sigma-Aldrich, St. Louis, MO) and agitated on a rotary shaker set at 150 rpm. After 5 d of continuous agitation, mycelium was separated from the media using commercial coffee filters and the mycelium washed with three equivalent volumes of distilled water. Ten milligrams wet mycelium was placed into a 1.5-mL microcentrifuge tube, 0.5 mL of 0.15 M NaCl solution was added, and the mixture was incubated overnight at 45°C. The following day it was vortexed for 3 min and centrifuged at 10000 g for 4 min and 1 µL of the supernate was dotted onto two nitrocellulose membranes. The membranes were air dried for 1 h and placed into a 10-cm plastic petri dish. With the exception of monoclonal antibodies from cell line IF8, all reagents used to develop the immunoblot were kindly provided by Agrinostics Ltd. Co. (Watkinsville, GA) The petri dishes were placed onto a rotary shaker at 50 rpm and, following addition of 10 mL of blocking solution, incubated for 30 min at ambient temperature. Five hundred microliters of hybridoma media containing antibodies from cell line IF8 was added to 10 mL of the blocking solution and poured into one of the two petri dishes containing the nitrocellulose membranes. Ten milliliters of blocking solution was added to the second immunblot to serve as a control. The petri dishes were returned to the shaker for 60 min, after which the solutions were decanted from the Petri dishes, and washed for 6 min twice using the blocking solution. Ten milliliters of blocking solution containing rabbit anti-mouse antibody with an alkaline phosphatase conjugate was added to each membrane and the petri dishes were returned to the shaker for 60 min and washed. After the final wash, 8 mL of liquid phosphatase substrate was added and membranes were incubated for 15 min. Each membrane was washed with distilled water and the spots containing fungal proteins scored for affinity to the antibodies from IF8. Affinity of the antibodies to the fungal proteins was considered positive if a dark blue-black spot developed where the protein had been placed on the membrane and negative if there was no color development.

Experiment 2: Assessing Extraction Conditions Necessary for ELISA Quantification of Fusarium spp. in Barley Expressing Symptoms of Head Blight
Three seed lots of barley were selected for this study. The seed lots had previously been tested and had DON contents of 0, 1.3, and 24.7 µg g–1 and respectively had 0, 3, and 24% damaged kernels typical of Fusarium infection. Samples of 1, 3, 5, and 10 g from each seed lot were weighed and extraction buffer was added in ratios of 1:3, 1:6, and 1:12 (w/v) (Agrinostics Ltd. Co., Watkinsville, GA). Samples were mixed horizontally on a rotary shaker (Eberbach Labtools, Ann Arbor, MI) at 150 rpm and a 100-µL subsample removed with a micropipette at 0, 0.33, 0.67, 1, 2, 5, and 10 h after shaking. Four hundred microliters of cold acetone was added to each extraction subsample and incubated at –20°C overnight to precipitate proteins. The subsamples were centrifuged the next day, the proteins resuspended in 0.5 mL of plate coating buffer, and Fusarium quantified by ELISA (see method below). The experiment was arranged in a split plot assignment with a factorial of seed weight and volume of extraction buffer as whole plots and the time of incubation as subplots. Treatments were replicated four times.

Data were analyzed using the PROC MIXED subroutine of the SAS version 8.2 (SAS, 2002). Mean ELISA values were separated using a Fisher's protected LSD (P ≤ 0.05). The ideal set of Fusarium extraction conditions for ELISA analysis was estimated by selecting the treatment with the lowest coefficient of variation.

Experiment 3: Field Evaluation of ELISA for Fusarium Quantification in Inoculated and Naturally Infected Grain for Comparisons with Head Blight Scores and DON
Evaluation of the ELISA method in a field-inoculated trial used a set of 71 doubled-haploid lines from a Zhedar2/ND9712/Foster mapping population. The doubled-haploid lines were planted in hill plots spaced 0.304 m apart with 15 seeds per plot at Osnabrock, ND, in May 2003. The lines, parents, and the resistant cultivar Chevron were grown in a randomized complete block design with two replications. Field inoculation with F. graminearum was conducted by infesting autoclaved barley and maize (Zea mays L.) grain with a mixture of five F. graminearum isolates (isolates 172, 173, 176, 582, and 672) and spreading infested grain 50 g m–2 in the experimental plots approximately 1 wk before heading and a second time when 50% of the heads had emerged from the flag leaf. Overhead misting for 30 s every half hour from 0400 to 0800 h and again from 1800 to 2000 h was used to enhance infection in the nursery. The rate of misting was approximately 2300 L ha–1 min–1. Grain was harvested at physiological maturity and threshed, and 5 g of seed from each plot was placed into a 50-mL disposable polypropylene centrifuge tube. Thirty milliliters of extraction buffer was added to each and the tubes were placed horizontally on a rotary shaker set at 150 rpm for 60 min. One hundred microliters of buffer was pipetted into a microcentrifuge tube and 400 µL of cold acetone was added. The tubes were incubated at –20°C overnight to precipitate proteins and centrifuged at 10000 rpm in a Marathon Micro A benchtop centrifuge (Fisher Scientific, Norcross, GA). The liquid was decanted from the microcentrifuge tubes, 0.5 mL of ELISA plate coating solution was added, the tubes were vortexed to dissolve the proteins, and the Fusarium spp. antigen was quantified by ELISA.

ELISA data were used to select five of the doubled-haploid lines, each of which tested high, medium, or low for Fusarium. The high, medium, and low lines were planted at Osnabrock, ND, in May 2004 using a randomized complete block design with eight replications. Growing conditions and field inoculation with F. graminearum were as described above. Heading date was recorded as days after planting when 50% of the spikes had completely emerged from the flag leaf sheath, and plant height was measured at maturity. Ten spikes from each plot were randomly selected at the soft-dough stage and scored for severity of F. graminearum infection by determining the percentage of the seed expressing damage typical of Fusarium infestation. The grain was harvested at maturity, threshed, analyzed for DON by GC–MS (Tacke and Casper, 1996), and Fusarium extracted for subsequent ELISA quantification as previously described.

Data were analyzed as a randomized complete block design using the PROC ANOVA subroutine of the SAS version 8.2 (SAS, 2002). Data from randomly selected replications were analyzed as 2-, 3-, 4-, 5-, 6-, 7-, and 8-replicate data sets and the experimental coefficients of determination plotted for each data set. Within the 8-replicate data set, means were calculated for the haploid lines and separated using a Fisher's protected LSD (P ≤ 0.05). The number of replications necessary to show specific differences between two genotypes was calculated for each method of FHB assessment using the following formula modified from Mendenhall and Schaeffer (1973):

Formula 1[1]
where:

t{alpha}/2is the tabular t value with a probability value of 0.05
tßis the t value associated with accepting a false null hypothesis (ß = 0.25)
{sigma}2the error variance
d2the difference between genotypic means for DON, FHB score, or Fusarium antigen concentration (ELISA values) expressed as a percentage of the mean.

Heading date, plant height, FHB, DON, and ELISA means of the haploid lines were assigned as random replications within their original high, medium, or low ELISA class, and analysis of variance was conducted to determine differences among the classes using a completely random design.

Eighty-nine barley grain samples were gathered from county grain elevators to use in a second study to determine whether ELISA and other disease parameters were associated under conditions of natural infection. Barley samples were collected at harvest throughout all barley growing regions of North Dakota and Minnesota as part of regional crop surveys from 1993 to 2003 (Barr et al., 2000). The number of samples collected per county was based on production, with a greater number of samples being collected in counties with higher projected barley production. For the years 2001 through 2003, every sample collected was tested for DON, for an average testing of one sample for each 159 Mg of production. In years previous to 2001, every second to third sample collected was tested for DON, for an average testing frequency of one sample for each 358 Mg of production. The DON data were used to estimate the proportion of the crop within each crop district that fell within the following DON ranges: ≤ 0.5, 0.5 to 0.9, 1.0 to 2.9, and >3.0 µg g–1. FHB incidence was visually assessed on 100 or 200 randomly selected kernels from each harvested sample. Kernels with greater than 25% of their surface covered with lesions were considered blighted. FHB incidence was calculated using Eq. [2]

Formula 2[2]
The PROC CORR subroutine of the SAS version 8.2 (SAS, 2002) was used to determine the statistical association of ELISA, DON, and %FHB data.

ELISA Quantification of Fusarium spp.
Fusarium spp. were quantified on harvested seed using genus-specific monoclonal antibodies in an ELISA format. All extraction and dilution buffers and chromogenic reagents for immunoquantification of F. graminearum were provided by Agrinostics Ltd. Co. (Watkinsville, GA). Fusarium proteins were extracted by weighing and placing 5 g of whole grain into 50 mL disposable centrifuge tubes. Thirty milliliters of fungal extraction buffer was added using a Filamatic vial filler (National Instrument Co., Baltimore, MD). The tubes were placed into styrofoam racks, positioned horizontally on a rotary shaker (Eberbach Labtools, Ann Arbor, MI) at 150 rpm for 1 h. The extraction buffer was decanted from the seed, a 1-mL subsample centrifuged at 10000 g to remove particulate matter, and the particle-free subsample diluted 1:6 in a microtiter plate coating buffer. Fifty microliters of the diluted samples was placed into wells of a Dynex Immulon IV (Thermo Labsystems, Franklin, MA) microtiter plate and incubated for 2 h at room temperature. Wells of the microtiter plates were washed and blocked with 100 µL of ELISA blocking buffer for 30 min at room temperature. The plates were washed and media from hybridoma cell line IF8 was diluted 1:20 in antibody dilution buffer and 50 µL was added to each well. The plates were incubated for 2 h at room temperature and washed. Rabbit anti-mouse antibody with an alkaline phosphatase conjugate was diluted 1:500 in antibody dilution buffer and 50 µL was added to each well. The plates were incubated for 2 h at room temperature and washed with ELISA wash buffer. Five milligrams of dry chromogen was dissolved in 6 mL chromgenic bufer, 50 µL of the solution was added to each well, the plates were incubated for 10 min at room temperature, and 50 µL chromogenic stop solution was added to halt color development. Diluted purified F. graminearum standard antigens were placed into wells of each microtiter plate and used to calculate concentrations of antigen present in the samples. All samples were analyzed in duplicate.

Deoxynivalenol Determination
Deoxynivalenol analysis of all samples was conducted immediately after harvest each crop year. Deoxynivalenol concentration was determined by column cleanup and gas chromatography with electron-capture detection according to Tacke and Casper (27). The limit of quantitation was 0.1 µg DON g–1 barley.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Experiment 1: Cross Reaction of Monoclonal Antibodies from Cell Line IF8 with Fungal Proteins from Fusarium spp. and Other Ascomycota spp.
Monoclonal antibodies from hybridoma cell line IF8 had affinity to all Fusarium spp. tested (Table 1). The causal agent of FHB was initially identified as F. graminearum, but recent evidence indicates FHB is caused by a complex of species in the B trichothecene toxin-producing clade in addition to F. graminearum (O'Donnell et al., 2000, 2004). Six of the Fusarium isolates cross reacting with monoclonal antibodies from cell line IF8 were within the B clade (Table 1) but the antibodies cross reacted with the six phylogenetically diverse Fusarium spp. outside the B clade as well. There were no reactions with any Fusarium protein extracts on the membrane that did not receive antibody IF8 (data not shown) nor did antibodies from hybridoma cell line cross react with other Ascomycota tested (Table 1). Thus, the antibodies were specific to the Fusarium genus, but not specific to the B clade. This suggests that an antibody-based test for head blight using IF8 could potentially cross react with Fusarium spp. not associated with the head blight. Nonetheless, a goal of developing immunochemically based methods for quantification of Fusarium within the head blight complex will necessitate antibody cross reaction with a number of Fusarium species to accurately assess fungal presence. The cross reactivity of antibodies from cell line IF8 with Fusarium spp. outside the B clade certainly necessitates testing under field conditions to determine the validity of such a method where potential exists for spurious contamination with other Fusarium spp. Most research trials investigating FHB utilize laboratory-produced inoculum as a source of contamination, thus testing the ELISA method of Fusarium spp. quantification under field conditions with both artificial and natural inoculum is necessary.


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  		Table 1. Affinity of Fusarium-specific monoclonal antibodies (mAb) from UGA cell line IF8 to Fusarium spp. and common ascomycota.

 
Experiment 2: Assessing Extraction Conditions Necessary for ELISA Quantification of Fusarium in Barley Expressing Symptoms of Fusarium Head Blight
Analysis of variance indicated main effects of seed lot, extraction time, and proportion of extraction buffer were significantly different but there were no interactions among the treatments. There was no effect of sample size on the amount of Fusarium extracted, but the coefficient of variation decreased as sample size increased (Table 2). This implies that variation was not uniform among treatments, a violation of the assumptions of analysis of variance. Based on the coefficients of variation, we concluded that a 5-g sample of seed was sufficient to minimize sampling error. Thus, the data for the 1-, 3-, and 10-g sample sizes were eliminated from the data set and the data from the 5-g samples were re-analyzed for differences among treatments. There was a seed lot x time interaction for the amount of Fusarium antigen extracted for ELISA analysis (Fig. 1 ). No antigen was extracted from the seed lot which had no symptoms of Fusarium infestation and no DON, but the other seed lots had increasing amounts of antigen extracted between 0 and 1 h, and a slight decrease in antigen in the extract thereafter. Although not tested, the reduction in antigen in the extract after 1 h likely resulted from protease activity in the sample. In subsequent studies, we found protein extracts from pure cultures of F. graminearum to be unstable over time and have added a fungal protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO; cat. no. P 8215) to the extracts which eliminated the effect.


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  		Table 2. Coefficients of variation for enzyme-linked immunosorbent assay (ELISA) quantification of Fusarium graminearum when varying amounts of seed were used in the sample.

 

Figure 1
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  		Fig. 1. Effect of extraction time on Fusarium antigen recovered from different barley seed lots measured in the enzyme-linked imunosorbent assay (ELISA) reaction. Bars represent standard deviations for each data point. DON, deoxynivalenol.

 
There was an interaction among seed lots when varying amounts of buffer solution were added to extract Fusarium antigens (Table 3). There was no detectable antigen in treatments with the zero DON/FHB seed lot, but antigen extraction was greater when the seed/buffer ratio of 1:6 or 1:12 was used in the two seed lots with Fusarium infestation. There were no differences when the seed/buffer ratio was 1:6 or 1:12 regardless of seed source. Thus, we determined that the optimal laboratory conditions for extraction of Fusarium antigen from whole seed for ELISA quantification was to add 5 g of whole seed to 30 mL extraction buffer, and to agitate the mixture for 60 min at 150 rpm.


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  		Table 3. Effect of varying amount of buffer on antigen extraction for enzyme-linked immunosorbent assay (ELISA) quantification of Fusarium infection of barley seed.

 
Experiment 3: Field Evaluation of ELISA for Fusarium Quantification and Comparison with Head Blight Scores and Deoxynivalenol
The 2003 growing season was drier than normal and symptoms developed after the final visual evaluation of FHB was made at the soft dough stage and when the seed was harvested. Thus, FHB scores were low. Nevertheless, FHB scores and ELISA values were different among the doubled-haploid lines, but variability of DON data combined with the limited number of replications resulted in no differences among the haploid lines (Table 4). Samples selected for the respective ELISA classes and grown in 2004 tended to rank similarly in both years for FHB, ELISA, and DON. The analysis of variance indicated there were also significant effects for heading date, plant height, and lines which were selected for high, medium, and low ELISA (Table 5). It is important to note, however, that differences occurred for heading date, plant height, FHB score, and DON within the lines that had been selected for high, medium, and low ELISA (Table 6), suggesting that pleiotropic effects of plant architecture (Zhu et al., 1999) may not necessarily be associated with low Fusarium infection. It is also important to note that low FHB scores did not necessarily portend low DON or low ELISA, nor did low DON necessarily portend low ELISA values. Therefore, an obvious concern is which more accurately predicted disease. Environmental conditions can affect DON production independent of fungal growth and, therefore DON is not always indicative of fungal mass (Champeil et al., 2004) or kernel characteristics (Liu et al., 1997). It also appears that visual symptoms of FHB are not always indicative of Fusarium infection and that FHB can be asymptomatic even though DON is present. For example, line F103–123 had a relatively low FHB score but had high DON and ELISA values. Given that the antibody used in the ELISA system is specific to Fusarium spp., ELISA may be a more accurate assessment of Fusarium infection than FHB because visual evidence of the disease can be asymptomatic.


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  		Table 4. Analysis of variance for Fusarium assessment methods among 81 doubled hapoid lines of barley grown in Osnobrock, ND, in 2003.{dagger}

 

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  		Table 5. Analysis of variance for agronomic traits and Fusarium assessment methods among 15 barley lines selected for high, medium, and low Fusarium graminearum ELISA from a doubled-haploid mapping population.{dagger}

 

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  		Table 6. Mean values for agronomic traits and Fusarium assessment methods among 15 barley doubled-haploid lines selected for high, medium, and low susceptibility to Fusarium graminearum by enzyme-linked immunosorbent assay (ELISA).

 
Certainly, one of the main objectives of researchers is to find ways to more efficiently screen plant materials in the field. Therefore, numbers of replications required to detect a specific difference between two treatment means were calculated for FHB, DON, and ELISA methods for Fusarium assessment using the error variances in Table 3. A sample calculation to determine the number of replications (Eq. [1]) for FHB is given below when {alpha} = 0.05, ß = 0.25, and d = 0.10 of the mean:

Formula 3[3]

Formula 4[4]
Coefficients of variation were lower and less variable among the ELISA data than FHB or DON, regardless of the number of replications used in the analysis of variance (Fig. 2 ). The lower coefficient of variation for ELISA resulted in a reduction of the number of replications required to detect a specific difference between two treatment means (Table 7). Attempting to detect differences of 10% of the mean appears to be unrealistic for any of the methods used to assess Fusarium infestation since the number replications necessary to do so is 91 or more. Moreover, it appears the number of replications necessary to detect differences of 50% precludes the utility of FHB or DON in assessing Fusarium infestation since the number of replications necessary to do so is 12 or more. These results indicate, however, that four replications are necessary to detect a difference of 50% among two treatment means using ELISA. Relative to the means, LSD values for ELISA data were consistently lower than those for FHB or DON (Fig. 3 ). Thus, ELISA assessment of the doubled-haploid lines would be expected to more effectively select for low infection than selecting for low FHB or DON. ELISA and DON data were correlated (r = 0.57) and a scattergram of the data indicates that samples with the lowest ELISA values also had low DON (Fig. 4 ). Conversely, samples with low DON did not necessarily have low ELISA values. Correlations between DON and FHB in grain are typically low in studies using inoculum from mixed cultures of F. graminearum strains (Bai et al., 2001; Liu et al., 1997; Mesterházy, 2002; Salas et al., 1999) because different strains of F. graminearum produce varying amounts of DON and differ in their capacity to elicit FHB symptomology (Salas et al., 1999). Environmental conditions may also affect DON production regardless of disease progression (Champeil et al., 2004; Liu et al., 1997). Five strains of F. graminearum were used to inoculate the field plots in this study. We have tested abundance of antigen (for which IF8 is specific) in mycelium of 10 Fusarium species. There was no difference for antigen abundance among those species tested (data not presented). Therefore, lower coefficients of variation would be expected for ELISA compared to DON because of a more uniform measurement of fungal presence. Data from barley samples gathered from grain elevators gave similar results as that from research plots (Fig. 5 ). It was expected that incidental Fusarium spp. contamination from outside the B clade would have inaccurately predicted FHB and a correlation of ELISA and DON would be significantly lower than in the controlled study. However, the correlation between ELISA and DON in the uncontrolled population of samples collected from grain elevators was higher than grain samples from the inoculated controlled plots. Therefore, incidental Fusarium spp. infection from outside the B clade was, in this case, of no consequence.


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  		Table 7. Number of replications required to show specific treatment differences for Fusarium head blight (FHB), deoxynivalenol (DON), or enzyme-linked immunosorbent assay (ELISA) between two plants whose genotypes were 10, 25, and 50% different from the mean performance.

 

Figure 2
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  		Fig. 2. Effect of number of replications in an experiment on coefficients of variation for three methods of Fusarium graminearum assessment in barley. FHB, Fusarium head blight; DON, deoxynivalenol; ELISA, enzyme-linked immunosorbent assay.

 

Figure 3
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  		Fig. 3. Least significant difference values expressed as a percentage of the mean for Fusarium head blight (FHB) scores, deoxynivalenol (DON), and enzyme-linked immunosorbent assay (ELISA) assessments of Fusarium infection in barley samples.

 

Figure 4
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  		Fig. 4. Scatter plot of enzyme-linked immunosorbent assay (ELISA) vs. deoxynivalenol (DON) accumulation in barley grain from a replicated field experiment using 71 doubled-haploid lines from a Zhedar2/ND9712/Foster mapping population.

 

Figure 5
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  		Fig. 5. Scatter plot of enzyme-linked immunosorbent assay (ELISA) vs. deoxynivalenol (DON) accumulation in 89 barley samples gathered from county grain elevators between 1993 and 2003.

 
Analysis of variance among the lines nested within their high, medium, and low groups shows that selection for low ELISA resulted in low DON and FHB scores (Table 8). Plants in the high class were shortest, but those in the low class were shorter than those in the medium class, suggesting plant height may not be as significant in disease development as previously thought (Zhu et al., 1999). Also, heading date was later as the ELISA classes progressed from high to low.


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  		Table 8. Means from the 2004 growing season for agronomic traits and Fusarium assessment methods among high, medium, and low classes of doubled-haploid lines selected for resistance to Fusarium graminearum using enzyme-linked immunosorbent assay (ELISA) in 2003.

 
In summary, a 1-h extraction using 5 g whole seed and 30 mL extraction buffer followed by ELISA provided repeatable quantification of Fusarium contamination in barley. Results from ELISA had lower CVs than FHB severity scores and DON, requiring less replication to detect genotypic differences among lines. Consistent performance of lines selected based on ELISA in 1 yr and performance of those lines in the subsequent year show the promise of ELISA as an analytical tool for researchers. The data presented herein suggest that even modest changes in amount of Fusarium quantified by ELISA can have a significant impact on head blight symptoms and DON.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
This material is based on work supported by the U.S. Dep. of Agriculture, under Agreement No. 59-0790-4-134. This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Dep. of Agriculture.

Received for publication March 28, 2006.


   REFERENCES
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 ABSTRACT
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N. S. Hill, S. M. Neate, B. Cooper, R. Horsley, P. Schwarz, L. S. Dahleen, K. P. Smith, K. O'Donnell, and J. Reeves
Comparison of ELISA for Fusarium, Visual Screening, and Deoxynivalenol Analysis of Fusarium Head Blight for Barley Field Nurseries
Crop Sci., July 1, 2008; 48(4): 1389 - 1398.
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Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome