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Fusarium head blight (FHB) is one of the most important diseases of wheat in many areas of the world. Besides causing yield loss, FHB often results in the accumulation of Fusarium-specific mycotoxins in wheat grain (Bottalico and Perrone 2002). Cultivation of susceptible wheat cultivars, the lack of FHB forecasting schemes implemented in practice and high disease pressure in regions of Croatia where wheat is grown in rotation with maize lead to frequent yield losses and increase the risk of mycotoxin accumulation in wheat grain, especially in years or conditions conducive to FHB development. Most of the Fusarium species reported to occur in wheat grain in Europe are potential producers of various mycotoxins, and their mycotoxigenic activity on wheat has been the object of numerous studies, with F. graminearum Schwabe and F. culmorum (W.G. Smith) Saccardo and their products deoxynivalenol (DON) and zearalenone (ZEN) being the most intensively studied. Limited data exist on the mycotoxigenic activity of fumonisin-producing Fusarium species in wheat (Marin et al. 1999) and wheat grain contamination with fumonisins. The most important producers of fumonisins are F. verticillioides (Saccardo) Nirenberg, F. proliferatum (Matsushima) Nirenberg, and F. nygamai Burgess & Trimboli (Leslie and Summerell 2006). The presence of F. verticillioides and F. proliferatum in wheat kernels implies that fumonisins could be present on wheat, among the other mycotoxins reported so far. Bottalico and Perrone (2002) suggested that the detection of fumonisin B1 (FB1) in wheat should be expected and pointed out that the relatively frequent occurrence of F. verticillioides and F. proliferatum in wheat in Europe could indicate the spread of these fumonisin-producing fungi in small-grain cereals. Presuming that the fungicides used for FHB management are active against all Fusarium species, they could be effective against the accumulation of various Fusarium mycotoxins by reducing fungal presence and activity in wheat spikes. Studies in which the relationship between fungicide treatments and mycotoxin accumulation in wheat has been investigated have shown that a positive correlation is not always present between fungicide efficacy, FHB severity and mycotoxin accumulation in wheat grain (Gareis and Ceynova 1994; Martin and Johnston 1982; Milus and Parsons 1994). The aim of the present study was to determine the presence of FB1 and ZEN in wheat grain in Croatia and to determine the relationship between fungicide effectiveness and FB1 and ZEN contamination of wheat kernels. This experiment also presented an opportunity to evaluate field performance of new products registered for FHB control in Croatia.

A field trial was conducted in 2006 near Kutjevo in northeastern Croatia (45°23’ N, 17°47’ E). Plots were arranged according to a randomized complete block design with four replicates, and the trial was exposed to natural FHB infections. All fungicides were applied twice, with the first application at GS 53 (1/4 of head emerged) and the second one at GS 60 (beginning of flowering) (Zadoks et al. 1974), using the rates recommended by the manufacturers diluted in 500 L ha-1 of water (Table 1). FHB severity assessment was done at GS 75 (medium milk) (Zadoks et al. 1974). It was assessed visually using a scale of 0 to 5 (0 = no symptoms visible; 1 = 1 to 5% of symptomatic spikelets; 2 = 6 to 10% of symptomatic spikelets; 3 = 11 to 25% of symptomatic spikelets; 4 = 26 to 50% of symptomatic spikelets; 5 = more than 50% of symptomatic spikelets). Both FHB severity and product efficacy were expressed as percentage.

Table 1

Fusarium head blight (FHB) severity, efficacy of fungicides, fumonisin B1 (FB1) and zearalenone (ZEN) contents in wheat grain

Fusarium head blight (FHB) severity, efficacy of fungicides, fumonisin B1 (FB1) and zearalenone (ZEN) contents in wheat grain

a Product efficacy was calculated using Abbot's formula where effectiveness (%) = (FHB severity in control - FHB severity in treatment) X 100/FHB severity in control.

b Means followed by the same letter are not significantly different (P = 0.05) according to Duncan's Multiple Range Test. Values in parentheses refer to the standard error of the mean.

c Means followed by the same letter are not significantly different (P = 0.05) according to ANOVA. Values in parentheses refer to the standard error of the mean.

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Grain samples from the fungicide efficacy trial were collected after harvest and stored at -20 °C prior to ZEN and FB1 contamination analysis, which was performed using HPLC with fluorescence detection. For ZEN analysis, the samples were cleaned up using C-18 solid phase extraction columns (Varian, Harbor City, CA, USA). ZEN bound to the column was eluted with 4 mL of methanol and evaporated to dryness. Before injection into the HPLC system, samples were redissolved in a methanol/water solution (80/20 v/v). The mobile phase for HPLC consisted of a methanol/water solution (80/20 v/v). The flow rate of the mobile phase was set to 0.5 mL min-1. Guard column LiChrospher RP-18 (Merck, Darmstadt, Germany) 4 x 4 mm and reversed-phase analytical column LiChrospher RP-18 (Merck, Darmstadt, Germany) 250 x 4 mm with 5 µm particles were used. Wavelengths of the detector were set at 274 nm for excitation and 440 nm for emission. Standard ZEN solutions were obtained by dissolution and dilution of the standard (Sigma Chemical Co., St. Louis, MO, USA) with methanol. The standard calibration curve was linear (R2 = 0.999) and the detection limit for ZEN was 0.39 µg kg-1. For FB1 analysis, the samples were cleaned up using the immunoaffinity column (Vicam, Watertown, MA, USA). FB1 bound to the column was eluted with 1.5 ml of methanol and evaporated to dryness. Before injection into the HPLC system, samples were redissolved in methanol and derivatized with 100 µL of o-phthaldehyde (OPA) solution (40 mg o-phthaldehyde dissolved in 1.0 mL of methanol and added to 5 mL of 0.1 M sodium tetraborate and 50 µL of mercaptoethanol). The mobile phase for HPLC consisted of methanol and 0.1 M NaH2PO4 (68/32 v/v) adjusted to pH 3.35 with o-H3PO4. The flow rate of the mobile phase was set to 0.8 mL min-1. Guard column LiChrospher RP-18 (Merck, Darmstadt, Germany) 4 x 4 mm and reversed-phase analytical column LiChrospher RP-18 (Merck, Darmstadt, Germany) 125 x 4 mm with 5 µm particles were used. Wavelength of the detector was set at 336 nm for excitation and 440 nm for emission. Standard FB1 solutions were obtained by dissolution and dilution of the standard (Sigma Chemical Co., St. Louis, MO, USA) with an acetonitrile/water solution (1/1 v/v). The standard calibration curve was linear (R2 = 0.997) and the detection limit for FB1 was 10 µg kg-1.

ZEN and FB1 content in grain samples from different fungicide treatments were determined by analysis of variance at P = 0.05 with the ANOVA procedure of SAS 9.1 (SAS Institute, Cary, NC, USA). Linear correlations were generated and Pearson correlation coefficients were calculated with the CORR procedure of SAS 9.1 using untransformed data of ZEN content, FB1 content, fungicide efficacy and FHB severity from all replicates.

FB1 and ZEN contents were determined in all wheat grain samples (Table 1). Mean FB1 and ZEN contents in all tested samples were 308.1 µg kg-1 and 3.59 µg kg-1, respectively. Mean FB1 content ranged from 446.6 µg kg-1 in samples treated with prothioconazole + tebuconazole (Prosaro 421 SC®) to 182.0 µg kg-1 in samples treated with tebuconazole + triadimefon (Folicur BT 225 EC®). Mean ZEN content ranged from 5.33 µg kg-1 in samples treated with fluoxastrobin + prothioconazole (Fandango®) to 2.33 µg kg-1 in samples treated with epoxiconazole + kresoxim-methyl (Controlan®). Mean FB1 and ZEN contents in untreated control samples were 220.7 µg kg-1 and 4.21 µg kg-1, respectively.

The detection of FB1 in wheat grain samples confirmed the hypothesis of Bottalico and Perrone (2002) that these mycotoxins are likely to occur in wheat. Marin et al. (1999) hypothesized that the low incidence of fumonisin-producing Fusarium spp. in wheat grain could be the reason for the absence of fumonisins in wheat. Research on Fusarium spp. populations in wheat grain in Croatia revealed the common presence of F. verticillioides in kernels (Cosic et al. 2004). Among 846 isolates of Fusarium isolated from wheat kernels in eastern Croatia from 1996 to 2002, 166 (20%) were even identified as F. verticillioides; only F. graminearum (53%) was found in higher percentage (Cosic et al. 2004). Considering the obvious common presence of fumonisin-producing F. verticillioides in wheat grain in Croatia, the presence of FB1 in the samples analyzed was not unexpected, although such results are in contrast with the study of Marin et al. (1999). These authors reported that two isolates of F. verticillioides and two of F. proliferatum produced minimal quantities of FB1 in irradiated wheat grain (maximum amount of 3.5 µg g-1) compared with irradiated maize grain (maximum amount of 2019.2 µg g-1). This would mean that wheat grain is an unsuitable substrate for FB1 production, which was not confirmed by the results of the present study.

On the other hand, ZEN is a toxin frequently found in wheat in European countries (Bottalico and Perrone 2002). The mean ZEN content found in the present study (3.59 µg kg-1) was higher than those found in maize samples in Croatia, which ranged from 0.2 to 1.2 µg kg-1 (Mitak et al. 2005) and from 0.62 to 3.22 µg kg-1 (Domijan et al. 2005). ZEN concentrations detected in the present study are generally low compared with those found in some other European countries (De Nijis et al. 1996; Lepschy-von Gleissenthal et al. 1989; Perkowski et al. 1990; Vrabcheva et al. 1996).

No significant differences between treatments were found among FB1 and ZEN levels in the grain samples analyzed. No significant correlation was found between FB1 content in wheat grain and the efficacy of the fungicides tested or between FB1 content in wheat grain and FHB severity. Correlation coefficients were 0.056 and -0.097, respectively. Similarly, no correlation was found between ZEN content in grain and fungicide efficacy or between ZEN content and FHB severity, with correlation coefficients of 0.172 and -0.215, respectively. One of the possible reasons for the lack of correlation between fungicide efficacy and FB1 or ZEN content in wheat grain in the present study could be the period during which mycotoxin-producing Fusarium spp. colonize wheat grain. If such colonization occurs at late growth stages, from the hard dough to the end of ripening, it is likely that fungicides applied earlier lose their effect on fungi that develop on spikes and grain late in the growing season. Regardless of such hypothesis, studies have shown that the interaction between mycotoxin production, the environment and fungicidal activity of active ingredients is complex (D’Mello et al. 1998; Pirgozliev et al. 2003). Most of these studies have focused on DON, and it is obvious that all three cases can occur. Fungicides can reduce, increase, or have no effect on mycotoxin content in wheat grain, and it is hard to predict their effect on mycotoxin production (Gareis and Ceynova 1994; Martin and Johnston 1982; Menniti et al. 2003; Milus and Parsons 1994; Pirgozliev et al. 2002; Simpson et al. 2001). It should be emphasized that the present study included only one year and location, and more research is needed to make hypothetical conclusions.

The efficacy of all products tested on FHB severity was higher than 70% (Table 1). The tebuconazole + triadimefon mixture (Folicur BT 225 EC®) was the most efficient one with 85.7% efficacy, followed by prothioconazole + tebuconazole (Prosaro 421 SC®) with 82.5% efficacy, and propiconazole + cyproconazole (Artea 330 EC®) with 80.2% efficacy, while carbendazim (Bavistin SL®) was the least efficient product (72.1%). FHB severity ranged from 7.6% on plots treated with the tebuconazole + triadimefon mixture to 53.0% on the untreated control (Table 1). Results from the present study confirm the fact that even after two fungicide treatments during the growing season, some damage resulting from FHB is likely to occur in conditions conductive to FHB development. Fungicide efficacy in this study was higher than 70%, which can be considered a good result in conditions of high disease pressure. Jones (2000) reported that complete control of FHB with less than six to eight fungicide treatments cannot be expected. All fungicides evaluated in the present study, except carbendazim, had one or both active ingredients from the triazole group, so their effectiveness is not unexpected. Various triazole fungicides are effective in FHB management (Hutcheon and Jordan 1990; Menniti et al. 2003; Mesterházy and Bartók 1997; Pirgozliev et al. 2002; Simpson et al. 2001). The same has been proven for carbendazim in field trials (Ellner 1997; Mesterházy and Bartók 1997). In the matter of efficacy, results from the present study are in accordance with other field studies in which tebuconazole- containing fungicides showed the highest efficacy for controlling FHB among all products tested (Cromey et al. 2001; Ellner 1997; Matthies and Buchenauer 2000; Mesterházy and Bartók 1997; Simpson et al. 2001).