Euphytica 139: 123–132, 2004. C 2004 Kluwer Academic Publishers. Printed in the Netherlands.
123
Estimation of deoxynivalenol (DON) content by symptom rating and exoantigen content for resistance selection in wheat and triticale T. Miedaner1,∗ , N. Heinrich1 , B. Schneider1 , G. Oettler1 , S. Rohde2 & F. Rabenstein2 1
State Plant Breeding Institute, University of Hohenheim, D-70593 Stuttgart, Germany; 2 Federal Center for Breeding Research on Cultivated Plants, Institute of Resistance Research and Pathogen Diagnostics, P.O. Box 1505, D-06435 Aschersleben, Germany; (∗ author for correspondence: e-mail:
[email protected])
Received 4 March 2004; accepted 24 August 2004
Key words: deoxynivalenol, exoantigens, Fusarium culmorum, immunoassay, mycotoxin, resistance breeding, triticale, wheat
Summary Fusarium head blight (FHB) in wheat and triticale leads to contamination of the grain with the mycotoxin deoxynivalenol (DON) that is harmful to animal and man. A fast, low-cost, and reliable method for quantification of the DON content in the grain is essential for selection. We analysed 113 wheat and 55 triticale genotypes for their symptom development on spikes, Fusarium exoantigen (ExAg) and DON content in the grain after artificial inoculation with a highly aggressive isolate of F. culmorum in three (wheat) and six (triticale) location-by-year combinations. Additionally, in triticale the amount of Fusarium damaged kernels (FDK) was assessed. ExAg content was analysed by a newly developed Fusarium-specific plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) and DON content by an immunoassay. A moderate disease severity resulted in an ExAg content of 0.87 optical density (OD) units in wheat and 1.02 OD in triticale. DON content ranged from 12.0 to 105.2 mg kg−1 in wheat and from 24.2 to 74.0 mg kg−1 in triticale. Genotypic and genotype-by-environment interaction variances were significant (P < 0.01). Coefficient of phenotypic correlation between DON content analysed by the immunoassay and ExAg content was r = 0.86 for wheat and r = 0.60 for triticale. The highest correlation between DON content and symptom rating was found by FHB rating in wheat (r = 0.77) and by FDK rating in triticale (r = 0.71). In conclusion, selection for reduced FHB symptoms should lead to a correlated selection response in low fungal biomass and low DON content in the grain. Abbreviations: DON, deoxynivalenol; ExAg, Exoantigens; FDK, Fusarium damaged kernels; FHB, Fusarium head blight
Introduction Fusarium head blight (FHB) caused by Fusarium graminearum or Fusarium culmorum (W.G. Sm.) Sacc. is a devastating disease of wheat (Triticum aestivum L.), triticale (×Triticosecale Wittmack), and other cereals. It causes not only yield and quality losses but also contamination of the grain with mycotoxins (Leonard & Bushnell, 2003). The trichothecene deoxynivalenol (DON) was found in 69–96% and its derivative 3-acetyl deoxynivalenol (3-ADON) in 17–62% of all wheat
samples from commercial farms in southwest Germany during a six-year study (M¨uller et al., 1997). These toxins are the most prevalent in Germany (Schollenberger et al., 2002) and worldwide (Placinta et al., 1999). Storage of cereals under warm and humid conditions may further increase mycotoxin content even when field infections were only slight to moderate (Homdork et al., 2000). In Germany, about 60% of the wheat and the total harvest of triticale is used for feeding. Grain contaminated by mycotoxins is harmful to humans and animals. DON may cause emesis, depressed feed intake,
124 and feed refusal in pigs (D’Mello et al., 1999). It is therefore of utmost importance to reduce DON contamination already in the field by applying appropriate agronomical measures and growing more resistant varieties. FHB resistance is quantitatively inherited in wheat and triticale and no source of complete resistance is known yet (Snijders, 1990; Mesterh´azy, 1995; Miedaner, 1997; Oettler & Wahle, 2001). Genotypes differ in the level of disease severity; environmental conditions greatly affect resistance differentiation (Bai et al., 2000; Miedaner et al., 2001a; Hollins et al., 2003). For both, resistance selection and phytopathological studies, a quantification of disease severity is, therefore, indispensable. Selection for FHB resistance in wheat can already be applied in early segregating generations on plot basis (Miedaner et al., 2003). This is advantageous for the breeder, because a high genetic variation can be used at this early stage of variety development, and the size of breeding populations may be reduced effectively. Such a procedure, however, requires a rapid, cheap, and reliable screening method. Artificial spray inoculation with an aggressive, DON-producing isolate at flowering followed by several ratings of disease symptoms is a powerful method that allows screening for more resistant germplasm (Miedaner, 1997; Rudd et al., 2001). DON determination may not be feasible for routine application in breeding owing to its high costs and technical requirements (Bai et al., 2001). The available chromatographic methods are highly sensitive and can detect up to eight trichothecenes (Schollenberger et al., 1998), but they are expensive, time-consuming, and require a highly equipped chemical laboratory. The detection of DON by commercially available immunoassays allows a rapid screening with low investment costs for the breeder, and the results are highly correlated to DON analysis by gas chromatography (GC) in wheat (Sinha & Savard, 1996; Hart et al., 1998) and rye (Miedaner et al., 2003). The costs are, however, still high when large populations have to be screened in breeding programmes each year. An alternative might be the detemination of Fusarium exoantigens (ExAgs) by a newly developed ELISA (Rabenstein, 2002). ExAgs are a mixture of water soluble extracellular fungal products. Kaufman & Standard (1987) were the first to use ExAg analysis and demonstrated its usefulness. Abramson et al. (1998) found linear correlations between ExAg levels detected by an indirect enzyme immunoassay using F. sporotrichioides antisera and DON
content in hard and soft wheats (r = 0.76 and 0.80, respectively). Generally, the more resistant entries have less symptoms and a lower DON content at harvest in wheat, especially when highly resistant non-adapted materials are included in the test (Mesterh´azy et al., 1999; Mesterh´azy, 2002). In these studies, FDK rating showed a high covariation with the DON content. It is, however, not clear if this also applies to the current central European wheat breeding materials and to triticale, a crop that has not yet been improved for FHB resistance systematically. In winter rye, the correlation between FHB rating and DON content was only moderate and varied with the environment (Miedaner et al., 2003). The aim of this study was to analyze phenotypic and genotypic correlations among symptom ratings, ExAg content, and DON content, and to elucidate the possibility of indirectly select for low DON content.
Materials and methods Plant materials Our study comprised 113 winter wheat and 55 winter triticale genotypes. In wheat, 107 F4 lines from a breeding programme were tested together with their six parents, i.e. varieties released in Germany (Ambras, Kontrast, Pegassos, Piko, Ronos) and Switzerland (Arina; Miedaner et al., 2001a). In triticale, 45 F1 crosses and their 10 parents were used. Seven of the parents are varieties released in Germany (Alamo, Binova, Lasko, Modus, Trimaran) and Poland (Malno, Moreno), the remainder are breeding strains (Oettler & Wahle, 2001). Besides the parents, no other genotypes were previously tested for their FHB resistance or DON content. Experimental design Wheat genotypes were grown in 2001 at two locations in southwest Germany: Hohenheim (HOH) near Stuttgart (400 m above sea level, 8.8 ◦ C mean annual temperature, 697 mm mean annual precipitation) and Eckartsweier (EWE) near Kehl/Rhein (141 m above sea level, 9.9 ◦ C mean annual temperature, 726 mm mean annual precipitation) and in 2003 at Hohenheim only. Triticale genotypes were grown in 1999 and 2000 at the same locations and additionally at Oberer Lindenhof (OLI) near Reutlingen (700 m above sea level, 6.6 ◦ C mean annual temperature, 952 mm mean annual precipitation). Thus, wheat was tested in three
125 environments (location-by-year combinations) and triticale in six environments. Genotypes of wheat were planted in two-row plots with 0.21 m between rows and 1.2 m length at a seed density of approximately 350 kernels m−2 . In triticale, the plots comprised 10 plants in two rows of 1.2 m length with 0.25 m between rows. To avoid infection by other pathogens, all plots were sprayed once against Blumeria graminis and Puccinia recondita (Opus TopTM ; epoxiconazol 126 g L−1 ha−1 + fenpropimorph 375 g L−1 ha−1 , BASF, Ludwigshafen, Germany) shortly before heading of the respective crop.
of visually discoloured and degenerated kernels (Chelkowski, 1989) in a sample of 200 g using a similar scale as for FHB rating.
Inoculation
Exoantigen (ExAg) analysis
A single-spore isolate of F. culmorum (FC 46) was used for inoculation in all experiments. FC 46 was originally isolated from winter wheat in the Netherlands in 1966 and was first described as IPO 39-01 by Snijders & Perkowski (1990). Preparation of inoculum and inoculation procedure were as previously reported in detail (Miedaner & Perkowski, 1996). In brief, conidia were produced on wheat-grain medium. A suspension of 5 × 105 spores mL−1 was applied in the evening at a rate of 100 mL m−2 at flowering with a portable sprayer. The spraying device covered the whole plot and was equipped with a portable compressor to give a standardized pressure of 3 bar. All genotypes were inoculated simultaneously at three (wheat) or two (triticale) dates to allow for variation in flowering date.
A serological method developed to quantify fungal biomass in grain samples was used (Rabenstein, 2002). For sample preparation, 0.1 g grain flour was ground in 2 mL extraction buffer (phosphate buffered saline (PBS)) without Tween, containing 0.01 M ethylenediaminetetraacetic acid disodium salt (EDTA, Sigma code: E 5513) in a mortar and pestle and incubated overnight at 4 ◦ C in a refrigerator. Each sample was measured at least twice in each of two replications in an indirect ELISA format already described for the detection of Rhynchosporium antigens in barley leaves (Foroughi-Wehr et al., 1995). The methods for antiserum production in rabbits and purification of immunoglobulin (IgG) are essentially as described by Foroughi-Wehr et al. (1995). Altogether, eight polyclonal antisera (PAS) against surface washings and/or mycelium homogenates from cultures of Fusarium culmorum (Fc) and F. graminearum (Fg) were raised in rabbits and the antisera were characterized using different ELISA variants (Banks & Cox, 1992; Danks et al., 2001) and Western blot analysis (Gan et al., 1997). Antiserum PAS Fc 7/2 to soluble ExAg fractions of F. culmorum showed in ELISA a strong reaction with mycelia extracts of all tested cereal-infecting Fusarium species and was, therefore, chosen for the development of the test. This antiserum had no cross-reactions with mycelia extracts of non-Fusarium species. In Western blotting experiments, specific glycoprotein bands in artificially Fusarium-infested wheat grains were detected (Rabenstein, 2002). The plate-trapped antigen (PTA)-ELISA was performed in NUNC PolySorb ELISA plates (code: 475094) using polyclonal IgG of antiserum PAS Fc 7/2 as follows: A 100 µL antigen extract per well was incubated for 2 h at 37 ◦ C (plates were emptied without washing). A 200 µL blocking solution per well was added (1% nonfat dry milk
Resistance traits Recording of FHB ratings commenced at the onset of symptom differentiation. To determine disease progress, all entries were rated on a whole-plot basis at three (wheat) and four (triticale) successive dates in terms of infected spikelets per plot using the following rating classes: 1, no symptoms visible; 2, 95% of all spikelets per plot diseased. Ratings were recorded in intervals of three to five days according to disease progress. Correlations between ratings were very high (r > 0.9, P < 0.01). Arithmetic means of all individual ratings with significant (P < 0.01) genotypic differentiation were used to further calculate values across replicates and environments. These means shall, therefore, not include the variation in flowering date on an individual plot. In triticale, additionally the amount of Fusarium damaged kernels (FDK) was estimated as the proportion
Sample preparation for immunotests Plots were harvested by a combine (Walter and Wintersteiger, Austria) with a minimum of forced air. Grain samples were dried and carefully winnowed. A grain sample (≈100–200 g) of each plot was ground to a particle size of about 1 mm with a laboratory mill and stored at −20 ◦ C until required for analyses.
126 powder (TM) in PBS), further incubated for 1 h at 37 ◦ C, and subsequently washed three times with PBS-Tween 20. A 100 µL per well IgG of PAS Fc 2/7 (concentration 1 µg mL−1 ) was added in blocking solution and incubated for 1 h at 37 ◦ C and washed four times with PBS-Tween. A 100 µL per well alkaline phosphatase-conjugated goat anti-rabbit IgG (H + L) (DIANOVA, Hamburg, Germany, code: 111-055-003) diluted 1:2000 in 0.05 M Tris–HCl-buffer (pH 8.0) containing 1% TM was added and incubated for 1 h at 37 ◦ C, washed four times with PBS-Tween. Finally, 200 µL substrate per well was incubated with pnitrophenyl phosphate (1 mg mL−1 in substrate buffer pH 9.6) for 1 h at room temperature. Absorbance was measured at 405 nm with TECAN “Rainbow” ELISA reader (TECAN SLT Lab Instruments, Crailsheim, Germany) and is given as optical density (OD). DON immunoassay DON content of all samples was analysed by a commercially available enzyme immunoassay (RIDASCREENTM FAST DON, R-Biopharm GmbH, Darmstadt, Germany). This competitive immunoassay detects DON and 3-ADON (cross reactivity: 213%) and has no cross reactivity to other trichothecenes such as 15-acetyl DON, triacetyl DON, nivalenol, triacetyl nivalenol, and fusarenon-X. The detection limit is 0.222 mg kg−1 . The measurement was made with a microtiter-plate spectrophotometer (Spectra Basic, TECAN Deutschland GmbH, Crailsheim, Germany) at 450 nm. The extinction values were transformed to DON content by a software package distributed by the manufacturer. For wheat, this immunoassay had been tested for performance by AOAC Research Institute (Gaithersburg, USA). For rye, similar results were recently obtained (Miedaner et al., 2003). We assumed, therefore, that the test is also suitable for triticale.
on an entry-mean basis (Fehr, 1987) as the relation of genotypic to phenotypic variance. Analyses were performed with the computer package PLABSTAT (Utz, 2000). All effects were assumed to be random variables. Results Moderate disease severity was observed in all environments for wheat and triticale as shown by FHB ratings (Table 1). Differences between environments were low with the exception of OLI 2000 where a high DON content connected with the highest ExAg value was reached in triticale despite a relatively low FHB rating. In FHB rating, genotypes ranged from 2.7 to 6.2 in wheat and from 2.8 to 5.3 in triticale on the 1–9 scale. Mean ExAg and DON content for environments were similar for both crops and generally showed the same tendency. The released varieties represented almost the entire range covered by the complete set of genotypes for all traits (Table 2). Traits showed similar tendencies with two exceptions: Ambras had a higher and Binova Table 1. Means of FHB and FDK rating, ExAg and DON content of 113 wheat and 55 triticale genotypes inoculated with an isolate of Fusarium culmorum in three (wheat) and six (triticale) environments Rating (1–9) FHB
FDK
ExAg (OD) DON (mg kg−1 )
HOH 2001
4.1
NDa
1.00
53.2
EWE 2001
3.7
ND
1.23
44.6
HOH 2003
4.2
ND
0.39
48.8
Mean
4.0
0.87
48.9
LSDb5%
0.2
0.04
Environment Wheat
Genotypic range 2.7–6.2 across environments
3.5
0.36–1.38
12.0–105.2
25.7
Triticale
Statistical analyses Analyses of variance were based on plot means and computed according to standard procedures (Cochran & Cox, 1957; Snedecor & Cochran, 1989). Residuals were independent and followed a normal distribution for FHB and FDK rating, and ExAg content, but not for DON content. Values of the latter trait were adjusted to normality by log transformation. Components of variance were expressed as coefficients of variation (CV%). Broad-sense heritabilities (h2 ) were estimated
Content
HOH 1999
3.4
3.8
0.70
OLI 1999
3.3
5.0
0.88
30.8
EWE 1999
4.0
4.5
0.84
29.4
HOH 2000
4.6
4.8
0.82
53.3
OLI 2000
2.6
6.8
1.55
91.8
EWE 2000
3.9
4.2
1.35
57.6 48.1
Mean
3.6
4.8
1.02
LSDb5%
0.3
0.4
0.14
Genotypic range 2.8–5.3 2.8–7.0 0.63–1.58 across environments a
7.1 24.2–74.0
ND, not determined. Least significant difference at probability P = 0.05 for the factor environment. b
127 Table 2. Means of FHB and FDK rating, ExAg and DON content of six wheat and seven triticale varieties inoculated with an isolate of Fusarium culmorum in three (wheat) and six (triticale) environments Rating (1–9)
Content
FHB
FDK
ExAg (OD)
DON (mg kg−1 )
Piko
3.1
NDa
0.67
28.1
Arina
3.3
ND
0.55
14.0
Pegassos
4.1
ND
0.96
57.8
Ambras
4.8
ND
0.96
74.4
Kontrast
4.8
ND
1.09
91.6
Genotype Wheat
Ronos
6.2
ND
1.29
83.0
LSDb5%
1.1
ND
0.24
21.4
Lasko
2.8
2.8
0.63
24.2
Moreno
3.3
3.2
0.78
33.3
Malno
3.8
5.1
0.96
36.7
Trimaran
3.9
4.7
1.32
47.5
Alamo
4.3
5.4
1.09
50.5
Modus
4.3
6.2
1.58
63.5
Binova
5.3
7.0
1.44
46.7
LSD b5%
0.4
1.1
0.42
18.1
Triticale
a lower DON content than expected from their FHB rating. Significant (P < 0.01) genotypic variances were found for all traits in wheat and triticale across environments (Table 3). Genotype-by-environment interaction was also significant throughout. This interaction was mostly smaller or similar to the genotypic value. In triticale, however, genotype-by-environment interaction for ExAg content considerably surpassed the coefficient of variation of the genotype. Heritabilities were moderate to high for all traits with the exception of ExAg content in triticale. Generally all associations between resistance traits were significant (P < 0.01, Table 4). The association between ExAg and DON content was high in wheat and moderate in triticale (Figure 1). Genotypic correlation coefficients surpassed the phenotypic coefficients. Head blight rating and DON content were also tightly associated in wheat (Figure 2A), but not in triticale. A tighter correlation, however, was shown between FDK rating and DON content (Figure 2B).
Discussion
a
ND, not determined. Least significant difference at probability P = 0.05 for the factor genotype. b
Wheat and triticale genotypes tested in this study are representative for FHB resistance of the presently
Table 3. Coefficients of variation of genotype, genotype-by-environment interaction, and error, and heritability for FHB and FDK rating, ExAg, and DON content (log transformed) for 113 wheat and 55 triticale genotypes inoculated with an isolate of Fusarium culmorum combined across three (wheat) and six (triticale) environments Rating (1–9)
Content
dfa
FHB
FDK
ExAg (OD)
DON (mg kg−1 )
Genotype (G)
112
13.95∗∗
NDb
19.84∗∗
9.38∗∗
G × environment
224
15.32∗∗
ND
11.78∗∗
4.47∗∗
Error
322
8.82
ND
12.00
4.90
0.65
ND
0.81
0.86
Parameter Wheat Coefficients of variation
Heritability Triticale Coefficients of variation 54
11.85∗∗
17.04∗∗
11.09∗
6.27∗∗
G × environment
270
7.39∗∗
11.31∗∗
31.80∗∗
6.42∗∗
Error
324
10.87
21.77
24.37
6.79
0.88
0.82
0.36
0.79
Genotype (G)
Heritability ∗ ,∗∗ Significant
at P = 0.05 and 0.01, respectively (F-test). of freedom. b ND, not determined. a Degrees
128 Table 4. Coefficients of phenotypic (rphen ) and genotypic (rgen ) correlation among FHB and FDK rating, ExAg and DON content (log transformed) for 113 wheat and 55 triticale genotypes inoculated with an isolate of Fusarium culmorum combined across three (wheat) and six (triticale) environments Wheat Correlation between FHB rating
FDK rating
Triticale
rphen
rgen
rphen
–FDK rating
NDa
ND
0.71∗∗ 0.93++
–ExAg content
0.76∗∗
0.93++
0.63∗∗ 1.01++
–DON content
0.80∗∗
0.98++
0.36∗∗ 0.43++
–ExAg content ND
ND
0.68∗∗ 1.09++
–DON content ND
ND
0.70∗∗ 0.86++
0.98++
0.60∗∗ 1.07++
ExAg content –DON content
0.86∗∗
rgen
∗∗ Significant
at P = 0.01. greater than twice its standard error. ND, not determined.
++ Estimate a
released varieties. The FHB resistant wheat varieties Arina and Piko (Miedaner et al., 2001a) and the currently most resistant triticale variety Lasko (Oettler & Wahle, 2001) were included. At the other end of the scale the highly susceptible varieties Ronos and Kontrast (wheat) and Binova (triticale) were tested. All other genotypes ranged within these extremes on a continuous scale. Significant (P < 0.01) variation among genotypes and moderate to high heritabilities offer good chances for resistance selection when tests are conducted in several environments. Only ExAg content in triticale had a considerably lower estimate due to the large genotypeby-environment interaction and error variances. The underlying reason for this is not obvious and needs further research. The high genotypic correlation coefficients between DON and ExAg content, which were corrected for the masking genotype-by-environment and error variances, show that both traits are tightly associated in inoculation experiments with a defined isolate. Measuring ExAg content by the newly developed ELISA is much cheaper than determining the DON content by a commercially available immunotest, because the prices for the test components of the latter are about 10-fold higher. An expensive purification and labelling of the antigen is not necessary in ExAg PTA ELISA. In contrast to the highly specific competitive DON ELISA, the PTA ELISA detects fungi at the species or genus level (Li et al., 2000). For both immunoassays, a similar laboratory equipment and well-instructed
technicians are necessary. Immunoassays provide indirect measures of grain infestation by Fusarium species that might be useful for resistance selection and phytopathological analyses. Both tests are ideally suited for all analyses with high numbers of samples. Recently developed quantitative PCR techniques are too slow and expensive, although tight correlations between fungal DNA and DON concentrations in wheat grain have been reported (Schnerr et al., 2002). More resistant wheat and triticale genotypes clearly had a lower ExAg content, i.e. they were less intensely colonized by F. culmorum and consequently showed much lower DON accumulation in their grains. Correlations with FHB rating were lower in triticale. Rating of FHB is more difficult than in wheat because of the widely varying colours of heads and awns and the differing shape of its heads. FDK rating had a higher correlation to DON content than FHB rating, probably because identical grain samples were used for both measurements. Similar correlations were reported for wheat genotypes (e.g. Mesterhazy et al., 1999), and Chelkowski (1989) even suggested to estimate DON content directly from percent of FDK for Polish conditions. FDK rating should be performed by the same person within an experiment and is only possible for grain samples from uniformly infected field plots. The high genotypic correlation between FHB rating and DON content in our population of adapted wheat genotypes illustrate that tolerance mechanisms (Mesterhazy, 1995), wilting phenomena, type III resistance (i.e. non-accumulation of DON; Mesterhazy, 1995) or associations with morphological traits which may interfere with the correlation (Miedaner & Perkowski, 1996) did not occur. Accordingly, DON/ExAg ratio did not significantly (P > 0.1) vary among wheat and triticale genotypes (data not shown). We found high DON contents in all experiments because of the artificial spray inoculation with a highly DON-producing isolate. This clearly represents an extreme situation, but low levels of FHB in resistance selection may lead to escapes which influence genotypic differences and genotype-by-environment interaction. It should be noted, therefore, that our conclusions might only be valid for inoculation experiments. The use of one isolate of F. culmorum results in a similar amount of DON per unit ExAg in all samples. In natural infections, however, several isolates are involved even on small-scaled fields (Miedaner et al., 2001b) that may produce widely differing amounts of DON on the same host variety (Gang et al., 1998). According to
129
Figure 1. Relationship between DON and ExAg content for (A) 113 wheat and (B) 55 triticale genotypes across three and six environments, respectively. Varieties are designated by their initials (Am, Ambras; Ar, Arina; Ko, Kontrast; Pe, Pegassos; Pi, Piko; Ro, Ronos; AL, Alamo; Mod, Modus; Tr, Trimaran; Bi, Binova; Ma, Malno; Mor, Moreno; La, Lasko). LSD5% = least significant difference at P = 0.05.
Snijders & Perkowski (1990), the correlation between FHB rating and DON content even varied for different isolates separately inoculated on 10 wheat genotypes. The highest correlation was obtained with the same isolate as used in our study. Moreover, in the humid climate of central Europe multiple infections by several Fusarium species are likely to occur (Parry et al., 1995; Lienemann, 2002). They might have different growing patterns within the host and produce different types and concentrations of mycotoxins. Likewise, the absence of DON in natural infections does not necessarily mean that no mycotoxins are present, because some isolates of F. graminearum and F. culmorum produce
nivalenol (Gang et al., 1998). Because the ExAg immunotest (PTA ELISA) detects all Fusarium species so far tested, correlations measured in this study have to be additionally analyzed in natural infections. For resistance selection, artificial inoculation is recommended to allow for a reasonable disease severity each year. When breeding populations of wheat are inoculated with the same isolate, it can be expected that the ExAg content will be a useful indicator for the DON concentration of the grain. According to our data, the wheat breeder can select for low visual FHB rating only. This trait is cheap and easy to assess and will result in a selected fraction with below-average ExAg
130
Figure 2. Relationship between DON content and (A) FHB rating for 113 wheat and (B) FDK rating for 55 triticale genotypes across three and six environments, respectively. See Figure 1 for abbreviations.
and DON content when genotypes are tested in several environments. The newly developed PTA ELISA was useful for genotypic differentiation, but did not yield higher correlations with DON content than symptom ratings. Reducing DON contamination in triticale can be based on FDK rating of a representative inoculated sample. In both crops, there is no real need to perform costly DON immunoassays in early generations, where large populations need to be screened. It might be recommendable to apply DON analysis at later stages to select genotypes which combine resistance to the Fusarium fungus and a very low mycotoxin content.
Acknowledgments The authors thank Dipl. Ing. agr. (FH) Andrea Bosch (Hohenheim), Mrs. Monika Wesemann, and Doreen Hentsch (Aschersleben) for their excellent technical assistance. The triticale research was financially supported by the Federal Ministry for Consumers’ Protection, Nutrition, and Agriculture (BMVEL), Bonn, Germany, and the Association for Promoting Private German Plant Breeding (GFP), Bonn. S.R. was supported by a grant from the Federal Ministry for Education and Research (BMBF), Bonn, Germany (InnoRegio FKZ: 03i0606A).
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