Journal of the Science of Food and Agriculture J Sci Food Agric 85: (2005) DOI: /jsfa.1965

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1 Journal of the Science of Food and Agriculture J Sci Food Agric 85: (2005) DOI: /jsfa.1965 Effect of fungicides on the development of Fusarium head blight, yield and deoxynivalenol accumulation in wheat inoculated under field conditions with Fusarium graminearum and Fusarium culmorum Miriam Haidukowski, 1 Michelangelo Pascale, 1 Giancarlo Perrone, 1 Davide Pancaldi, 2 Claudio Campagna 3 and Angelo Visconti 1 1 Institute of Sciences of Food Production, CNR, Via G. Amendola 122/o, Bari, Italy 2 Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi di Bologna, Viale Fanin 46, Bologna, Italy 3 Syngenta Crop Protection SpA, Via S. Sofia 21, Milan, Italy Abstract: The effect of different fungicide treatments on Fusarium head blight (FHB) development, grain yields and deoxynivalenol (DON) accumulation in wheat was evaluated after artificial inoculation under field conditions with a mixture of Fusarium graminearum and Fculmorum. The trials were carried out using commercially available products on five different cultivars of soft and durum wheat (Serio, Genio, Bracco, Duilio and Orobel) in two separate experimental fields located in the North of Italy. Treatments with Tiptor S (cyproconazole plus prochloraz) and a mixture of Horizon 250 EW (tebuconazole) plus Amistar 250 SC (azoxystrobin) significantly reduced the FHB disease severity (by between 25 and 77%) and DON content (by between 32 and 89%) in the grain as compared with the inoculated control. Yields (tonne ha 1 ) and thousand grain weight (TGW) were higher in plots subjected to fungicide treatments. Tetraconazole (Eminent 40 EW) showed a markedly reduced effectiveness compared with the other treatments. Regression analysis showed a strong correlation of disease severity with DON levels (positive correlation)and with yields ortgw (negative correlation) for individual cultivars and locations. Fusarium graminearum, Fculmorumand Fpoaewere the species most commonly isolated from all trials, including inoculated and non-inoculated control plots Society of Chemical Industry Keywords: conazole fungicides; deoxynivalenol; Fusarium head blight; azoxystrobin; wheat INTRODUCTION Fusarium head blight (FHB) is a worldwide disease of wheat and other small-grain cereals caused by the combination of various fungi including Microdochium nivale and different Fusarium species (mainly F graminearum and F culmorum). The disease can cause total or partial ear premature senescence with consequent reduction of both crop yields and grain quality. In the case of strong fungal attacks, heavy yield losses (up to 70%) of crop have been observed. Moreover, the infected kernels represent one of the principal sources of fungal inoculation when used as seed. 1,2 Most of the Fusarium species causing FHB, under favourable environmental conditions, can produce various toxic secondary metabolites, ie trichothecenes, zearalenone and moniliformin. The occurrence of such natural contaminants (mycotoxins) in cereals is of great concern because their presence in feeds and foods is often associated with chronic or acute mycotoxicoses in livestock and could threaten human health. 3,4 Deoxynivalenol (DON) is a trichothecene mycotoxin primarily produced by F graminearum and F culmorum and it has been shown to be the most common mycotoxin contaminant associated with FHB infected grains. 5,6 Deoxynivalenol can cause haematic and anorexic syndromes and neurotoxic and immunotoxic effects in mammals. Surveys on the occurrence of DON showed it to be a common contaminant of wheat worldwide. 7 Legal limits for DON in wheat and/or cereals and derived products intended for human consumption have been recently Correspondence to: Michelangelo Pascale, Institute of Sciences of Food Production, CNR, Via G. Amendola 122/o, Bari, Italy michelangelo.pascale@ispa.cnr.it (Received 12 January 2004; revised version received 10 May 2004; accepted 13 May 2004) Published online 11 October Society of Chemical Industry. J Sci Food Agric /2004/$

2 M Haidukowski et al fixed by several European countries including Austria, Germany and The Netherlands. 8 The US Food and Drug Administration (FDA) established the advisory level of 1.0mgkg 1 for DON in all finished wheat products. 9 Advisory levels for Fusarium toxins (including DON) are currently under discussion by the European Commission. The association of diseases caused by toxigenic Fusarium spp with grain contamination by the relevant mycotoxins is well-known. 3,6,10 Several studies have shown that a reduction in FHB severity leads to a decrease in mycotoxin content in grains both naturally or artificially infected. Potential measures of control to reduce the severity of the disease and the consequent accumulation of mycotoxins in kernels include the use of varieties resistant to FHB, the use of appropriate farming practices (such as crop rotation, tillage, nitrogen fertilizer, irrigation and seed treatment) and 2,5,10 17 the use of fungicides or biological antagonists. The Scientific Committee on Plants (SCP) of the European Commission, after evaluation of the influence of pesticides on the mycotoxin accumulation in food, concluded that there is not sufficient evidence that pesticides play a prominent and consistent role in preventing or inhibiting the production of mycotoxins by toxigenic fungi and recommended that more research should be carried out with respect to the effects of plant protection products against mycotoxigenic fungi responsible for major plant diseases, such as FHB, and to evaluate their efficacy against mycotoxin production. 18 Conflicting evidence on the role of fungicides in controlling mycotoxin production by Fusarium species have been recently reported. For example, several studies have shown that some triazole fungicides, alone or in combination, reduce FHB incidence and DON accumulation, whereas the novel azoxystrobin based fungicides can even increase DON production, 11,13 15,17,19 although they can partially control FHB. In recent years, FHB has been severe in Italy, particularly in the Northern and Central regions. The principal causative pathogens were shown to be Fusarium graminearum and Fculmorumfollowed by F avenaceum, Fpoaeand Microdochium nivale In addition, in several monitoring programs carried out in Italy, DON was frequently found in wheat kernels, sometime at levels higher than 1 mg kg The purpose of this study was to evaluate the preventive effect of some commercial fungicides against the development of FHB, grain parameters and accumulation of DON in cultivars of durum and soft wheat grown in Northern Italy, inoculated under field conditions with a mixture of Fusarium graminearum and Fculmorum. The possibility of using visual disease parameters as indicators of wheat contamination by DON in conditions of severe FHB epidemic was also evaluated. EXPERIMENTAL Fungal inoculum The strains of Fusarium graminearum and F culmorum (ITEM 126 and ITEM 6273, respectively) used to inoculate wheat were obtained from the culture collection of the CNR Institute of Sciences of Food Production (Bari, Italy). Both the strains were isolated from wheat in Northern Italy and were able to produce DON in vitro when grown on autoclaved wheat. Spores were obtained by growing the fungal strains in shaking cultures in 1 l Erlenmeyer flasks containing 400 ml of V8 juice (Campbell Grocery Products Ltd, King s Lynn, Norfolk, UK) liquid medium (V8 juice 200 ml + CaCO 3 3 g brought to 1 l with distilled water) previously sterilized by autoclaving for 15 min at 121 C. After 7 days incubation in the dark at 25 C and 150 rpm, the flask contents were filtered through two layers of cheesecloth to obtain a spore suspension. The concentration of the inoculum, consisting mainly of conidia, was measured with a Thoma camera (HBG Henneberg-Sander GmbH, Lutzellinden, Germany) at the light-microscope. Fungicides Four commercially available products Tiptor S (cyproconazole plus prochloraz) and Amistar 250 SC (azoxystrobin), manufactured by Syngenta Crop Protection (Milan, Italy), Horizon 250 EW (tebuconazole), manufactured by Bayer CropScience (Milan, Italy), and Eminent 40 EW (tetraconazole), manufactured by Isagro Italia (Milan, Italy) were used in the field experiments (Table 1). Field trials During the growing season, two separate field trials were carried out in Bologna and Ravenna province (northern Italy), respectively. Wheat cultivars Genio and Serio (Triticum aestivum L) were used in both localities and the cultivars Bracco and Duilio or Bracco and Orobel (Triticum durum Desf) were used in Bologna and Ravenna, respectively. Cultivars were selected based on their high susceptibility to Fusarium Table 1. Active ingredients, product name and application rate of fungicides used in the field trials Trademark a Active ingredient (a.i.) Application rate (g a.i. ha 1 ) Eminent 40 EW tetraconazole 120 Tiptor S cyproconazole + prochloraz Horizon 250 EW + Amistar 250 SC tebuconazole + azoxystrobin b b a Eminent 40 EW manufactured by Isagro Italia, (Milan, Italy); Tiptor SandAmistar 250 SC manufactured by Syngenta Crop Protection (Milan, Italy); Horizon 250 EW manufactured by Bayer CropScience (Milan, Italy): b half manufacturer s recommended dose rate for azoxystrobin. 192 J Sci Food Agric 85: (2005)

3 Effect of fungicides on Fusarium-inoculated wheat head blight and their widespread cultivation in the region where the experiments were performed. All plots were cultivated according to normal agronomic practices which were standardized across both sites. The experimental design was a randomized block with four replicate plots in both localities for each trial (three fungicide treatments, one inoculated and one non-inoculated control). Each plot had a surface area of 18 m 2 (2 9m). Plots were artificially inoculated by spraying 700 ml of spore suspension containing a mixture of conidia of Fculmorum( conidia ml 1 )andf graminearum ( conidia ml 1 ) with an Echo motorized pump (Model SHR 4100, Kioritz Corporation, Tokyo, Japan). Delivery pressure at the nozzle (Teejet 8002 VS, TeeJet-LH Agro South Europe, Oilvet Orleans, France) was 2.5 atm, and the distance of the nozzle from the ears was 4 5 cm in order to avoid spore dispersion. Fungicide treatments were performed within 24 h before inoculation by using the same Echo pump with a delivery pressure at the nozzle (Teejet 8003 VS, TeeJet-LH Agro South Europe, Oilvet Orleans, France) of 3 atm and a rate of 500 l ha 1. Both inoculation and treatments were performed with a relative humidity above 75% and in the absence of wind. Initially it was planned to spray fungicides and fungal inoculum at the start of anthesis (Zadoks Growth Stage 60; GS 60). 26 Owing to adverse weather conditions in that period, some wheat cultivars (cv Genio in Bologna and cv Serio and cv Genio in Ravenna) were sprayed at mid-anthesis (GS 65). The effectiveness of fungicides in disease control, their influence on yield parameters and DON content in kernels were determined by comparison with artificially inoculated untreated plots (untreated control). In each trial, non-inoculated control plots were left in order to check the presence and consistency of natural inoculum. Visual disease assessment Visual disease assessment and on-field activity of the fungicides were evaluated at late milk stage (GS 77) by examining about 100 ears collected at random from each plot. The percentage of ears infected with FHB (incidence) and the percentage of infected area of the ear (severity) were calculated as a mean value of the experimental plots (n = 4). Disease severity was assessed by using a scale similar to that of Parry et al, 27 with eight evaluation classes (0, 2, 5, 10, 25, 50, 75 and 90% area infected) and applying the following formula: (number of ears per evaluation class evaluation class)/total number of scored ears Grain yields Ears were mechanically harvested at about 13% kernel relative humidity (RH) (GS 90 92) by using a smallplot combine harvester and the grain yields (tonne ha 1 ) were determined. Subsequently, a homogeneous 2 kg sample of grain was taken from each plot to determine the weight (g) of 1000 kernels (TGW). RH was measured by using a moisture meter Mod. WILE- 35 (Farmcomp Agroelectronics, Tuusula, Finland). DON analysis DON concentrations were determined following the method reported by Pascale et al, 25 based on immunoaffinity column clean-up of extracts and toxin determination by HPLC/UV. Briefly, 25 g of ground samples were added to polyethylene glycol (PEG- 8000) and extracted with water by blending. Extracts were filtered through filter paper (Whatman no. 4) and glass microfibre filter (Whatman GF/A) and cleanedup by DONTest immunoaffinity column (VICAM, Watertown, MA, USA). The toxin was determined by reversed-phase HPLC (Waters 625LC, Milford, MA, USA) with a diode-array UV detector (Hewlett Packard, Palo Alto, CA, USA) set at 220 nm. The column was a Waters C 18 Symmetry Shield, mm, 5 µm (Waters, Milford, MA, USA) preceded by a 0.5 µm Rheodyne guard filter. The mobile phase consisted of a mixture of acetonitrile:water (10:90) eluted at a flow rate of 1.0ml min 1. Recovery experiments were performed in triplicate using DONfree wheat samples spiked at levels of 0.1, 0.5, 1.0 and 2.0mgkg 1. Recoveries were higher than 80% in the range mgkg 1 of DON with relative standard deviations less than 10%. DON standard used for recovery experiments and HPLC calibration curves was purchased from Sigma-Aldrich s.r.l. (Milan, Italy). Appropriate dilutions of sample extracts before loading on immunoaffinity columns were necessary to avoid saturation of the DON-antibody binding sites. The detection limit of the method was 0.05 mg kg 1 (signal-to-noise ratio 3:1). Mycological examination Mycological examination was performed as reported by Menniti et al. 14 Briefly, glume, rachis, sub-glume and chaff of 10 ears per plot were washed in sterile water, disinfected in a 1% sodium hypochlorite solution for 2 min, rinsed twice in sterile water to eliminate any hypochlorite residue and dried on sterile filter paper, then placed in Petri dishes containing agar water. Plates were incubated in the dark. After 7 days, the developed mycelium was placed in Petri dishes containing potato dextrose agar (PDA) (Oxoid S.p.A., Milan, Italy) with the addition of 0.5gl 1 of PCNB (pentachloronitrobenzene) and 0.05 g l 1 of streptomycin sulphate. Plates were incubated in the dark at 22 C and after 5 days were placed under near UV alternating light and dark (12 h photoperiod) for a further 10 days to favour sporulation. Ear sampling was carried out at late milk maturity stage (GS 77) on noninoculated plots (natural inoculum). The Fusarium species were identified according to Nelson and Burgess. 28,29 Isolation frequency of Microdochium J Sci Food Agric 85: (2005) 193

4 M Haidukowski et al nivale and Fusarium species was calculated as number of isolates of the species divided by the total number of Fusarium spp + Microdochium isolates. Statistical analysis Data were processed by variance analysis (ANOVA) at p = 0.05 to indicate statistically significant differences between means (Tukey s test). Percentage data were arcsine transformed prior to statistical analysis to normalize and homogenize the variance. Regression analysis was performed for each combination of the investigated parameters (disease severity, DON content, yield and TGW) to assess the relevant correlation. All data were processed using the GraphPad InStat (Sigma, St Louis, MO, USA) statistical software. RESULTS Fusarium graminearum ( %), F culmorum ( %) and F poae ( %) were the species most commonly isolated from the ears of non-inoculated wheat cultivars (natural inoculum). In addition, F avenaceum (up to 10.0%), F equiseti (up to 2%) and Microdochium nivale (up to 12.1%) were also isolated. F graminearum and F culmorum were the predominant species accounting for 58 96% of the total fungal contamination in the different tested cultivars and locations. Results relevant to Fusarium head blight, yield parameters and DON contamination in the two experimental fields are summarized in Tables 2 and 3. The four replicate plots for each treatment showed a general agreement and reproducibility of the effect of the fungicides. Visual assessment of FHB Fusarium head blight incidence and disease severity in untreated control (artificial inoculum) were very high in both localities with values of % and 42 96%, respectively. High values of Fusarium head blight incidence (72 100%) and disease severity (26 80%) were also observed in untreated noninoculated plots. Higher values of disease severity were observed in the Ravenna trials compared with those in Bologna, for the three cultivars tested in both locations (Serio, Genio and Bracco). The artificial inoculation with F culmorum and F graminearum caused an increase of the disease severity on all cultivars and locations (average % increase = 51 ± 29) relative to non-inoculated control at both early (GS 60) and mid-anthesis (GS 65). No significant difference was observed between the effect of inoculations performed at beginning of anthesis (GS 60, average percentage increase = 55 ± 30) or mid-anthesis (GS 65, average percentage increase = 44 ± 31). A higher FHB disease severity, ranging from 80 to 96%, was observed in plots inoculated at GS 65 as compared with those inoculated at GS 60 (42 78%). Tebuconazole plus azoxystrobin and cyproconazole plus prochloraz treatments significantly (p < 0.05) reduced both the incidence and the severity of FHB compared with the untreated control. The fungicide effect on FHB incidence was less evident than that on FHB severity. The use of tetraconazole significantly reduced the severity of the disease in the tested cultivars (except for cvs Duilio and Serio in Bologna), although it produced effects lesser than those obtained with the other fungicides. Table 2. Effect of fungicides on FHB, yield, TGW and DON content in wheat Bologna trial Fusarium head blight Cultivar GS a Fungicide treatment I (%) b DS (%) c Yield (t ha 1 ) TGW (g) DON (mg kg 1 ) Serio d Non-inoculated control 89.7 a f 26.0 a 5.3 b 31.6 a 1.85 a 60 Inoculated control 99.6 c 41.9 b 4.6 a 29.0 a 9.80 c 60 Tetraconazole 97.8 bc 41.6 b 5.3 b 31.5 a 8.02 b 60 Cyproconazole + prochloraz 93.0 ab 21.4 a 6.5 c 35.9 b 6.70 b 60 Tebuconazole + azoxystrobin 89.8 a 22.9 a 6.7 c 36.9 b 6.65 b Genio d Non-inoculated control 92.1 b 47.5 b 5.2 b 29.6 ab 8.81 a 65 Inoculated control b 80.0 d 3.8 a 26.2 a c 65 Tetraconazole 96.7 b 60.0 c 4.8 b 28.0 ab b 65 Cyproconazole + prochloraz 82.7 a 38.9 a 5.9 c 31.9 b 9.91 ab 65 Tebuconazole + azoxystrobin 76.5 a 32.2 a 6.2 c 36.9 b 8.72 a Bracco e Non-inoculated control 98.6 b 29.3 b 5.1 b 43.5 a 1.91 a 60 Inoculated control b 59.0 d 3.5 a 41.5 a 9.87 c 60 Tetraconazole 98.5 b 45.2 c 4.5 b 45.4 a 5.84 b 60 Cyproconazole + prochloraz 75.1 a 13.5 a 6.5 c 51.6 b 1.40 a 60 Tebuconazole + azoxystrobin 79.9 a 14.9 a 6.3 c 50.3 b 1.10 a Duilio e Non-inoculated control 99.0 ab 39.6 a 4.9 bc 40.3 ab a 60 Inoculated control b 58.7 b 4.0 a 39.1 a b 60 Tetraconazole 94.4 b 55.7 b 4.5 ab 42.9 ab b 60 Cyproconazole + prochloraz 97.3 ab 31.2 a 5.6 cd 44.1 b a 60 Tebuconazole + azoxystrobin 95.7 a 25.9 a 6.1 d 50.9 c a a Zadoks growth stage for fungal inoculation and fungicide treatment; b incidence; c disease severity; d soft wheat; e durum wheat; f values followed by the same letter are not significantly different at p = 0.05 according to Tukey s test. 194 J Sci Food Agric 85: (2005)

5 Effect of fungicides on Fusarium-inoculated wheat Table 3. Effect of fungicides on FHB, yield, TGW and DON content in wheat Ravenna trial Fusarium head blight Cultivar GS a Fungicide treatment I (%) b DS (%) c Yield (t ha 1 ) TGW (g) DON (mg kg 1 ) Serio d Non-inoculated control 95.8 bc f 64.4 bc 6.9 a 32.1 a 1.78 b 65 Inoculated control c 85.5 d 7.0 a 33.6 ab 4.92 c 65 Tetraconazole 95.8 bc 71.3 c 9.3 b 36.5 b 1.94 b 65 Cyproconazole + prochloraz 92.3 b 63.8 b 9.7 b 39.8 c 1.67 b 65 Tebuconazole + azoxystrobin 84.5 a 53.8 a 10.0 b 40.3 c 0.92 a Genio d Non-inoculated control c 80.0 c 6.0 b 36.6 b 0.80 a 65 Inoculated control c 96.0 d 2.4 a 27.6 a d 65 Tetraconazole 97.3 bc 82.0 c 5.3 c 35.1 b c 65 Cyproconazole + prochloraz 93.5 ab 64.4 bc 6.1 c 36.3 b 9.32 b 65 Tebuconazole + azoxystrobin 92.5 a 52.8 a 6.3 c 36.6 b 5.40 ab Bracco e Non-inoculated control 92.4 b 50.6 b 5.5 b 33.3 a 0.05 a 60 Inoculated control c 78.0 c 4.0 a 32.3 a 4.37 d 60 Tetraconazole 94.8 b 51.0 b 5.8 b 41.1 b 1.96 c 60 Cyproconazole + prochloraz 91.3 ab 36.3 a 7.4 c 43.4 b 1.04 bc 60 Tebuconazole + azoxystrobin 88.1 a 29.3 a 7.5 c 42.6 b 0.67 b Orobel e Non-inoculated control 72.5 a 60.5 c 6.8 c 43.1 c 0.13 a 60 Inoculated control 90.0 b 71.3 d 4.7 a 40.9 a 3.85 c 60 Tetraconazole 88.3 b 61.9 c 5.5 b 42.1 b 3.70 c 60 Cyproconazole + prochloraz 72.8 a 49.6 b 7.0 c 46.0 d 2.16 b 60 Tebuconazole + azoxystrobin 66.3 a 27.5 a 7.0 c 45.8 d 2.73 bc a Zadoks growth stage for fungal inoculation and fungicide treatment; b incidence; c disease severity; d soft wheat; e durum wheat; f values followed by the same letter are not significantly different at p = 0.05 according to Tukey s test. Grain yields and TGW Yield and TGW values in untreated controls (artificial inoculum) ranged from 2.4 to 7.0tha 1 and from 26.2 to 41.5 g, respectively. An increase in both yield and TWG values was observed in both localities when cultivars were treated with tebuconazole plus azoxystrobin and cyproconazole plus prochloraz. No significant difference was observed between the two treatments (with the exception of the cv Duilio in Bologna for TWG value). The highest yields were obtained for the cv Serio after tebuconazole plus azoxystrobin treatment (6.7tha 1 in Bologna and 10.0tha 1 in Ravenna). The highest TWG value was found for cvs Bracco (51.6 g, Bologna) and Orobel (46.0 g, Ravenna) when the cyproconazole plus prochloraz treatment was used. The use of tetraconazole also showed yields and TWG values higher than those observed for the untreated control, although they were not always statistically significant (p = 0.05). Nevertheless, these values were always inferior to those obtained with the other fungicide treatments. Yield and TWG values in untreated noninoculated plots ranged from 4.9 to 6.9tha 1 and from 29.6 to 43.5 g, respectively. DON contamination DON levels in artificially inoculated wheat cultivars (untreated control) ranged from 3.9 (cv Orobel, Ravenna) to 46.7mgkg 1 (cv Genio, Ravenna). Cyproconazole plus prochloraz and tebuconazole plus azoxystrobin treatments significantly reduced, between 32 and 89%, DON content in kernels of the examined cultivars as compared with levels found in inoculated untreated control samples. No significant difference was observed between the two treatments, with the exception of cv Serio in Ravenna, where the reduction was greater for the latter treatment. Also, the treatment with tetraconazole significantly reduced the DON content (between 18 and 62%, with the exception of cv Duilio in Bologna and cv Orobel in Ravenna), although it was less effective than the two above-mentioned treatments (cyproconazole plus prochloraz and tebuconazole plus azoxystrobin). DON was detected in all the non-inoculated samples (natural inoculum) with high levels in two cultivars of wheat from Bologna trials (8.8mgkg 1, cv Genio and 13.6mgkg 1, cv Duilio). Correlation between disease severity, yield, TGW and DON levels In Table 4 are reported the coefficients of correlation between disease severity (DS), grain parameters (yield and TGW) and DON levels of wheat cultivars from the field trials. In the inoculation experiments (treated and untreated with fungicides), DS, yield, TGW and DON levels were highly correlated with each other when the regression analysis was performed with data from individual cultivars and locations. When naturally contaminated plots were also included for the statistical analysis of individual cultivars and locations, the average correlation coefficients dropped. No or limited correlation was found between DS, yields, TGW and DON levels when data from all cultivars and locations were analysed together. J Sci Food Agric 85: (2005) 195

6 M Haidukowski et al Table 4. Correlation coefficients between DS, grain parameters (yield and TGW) and DON levels of wheat cultivars treated with fungicides All data a Serio b Genio b Bracco b Duilio b Serio c Genio c Bracco c Orobel c (n = 8) Average ± SD All experiments d DS vs DON ± 0.28 DS vs yield ± 0.14 DS vs TGW ± 0.10 Yield vs TGW ± 0.05 Yield vs DON ± 0.23 TGW vs DON ± 0.23 Inoculation experiments e DS vs DON ± 0.10 DS vs yield ± 0.05 DS vs TGW ± 0.06 Yield vs TGW ± 0.04 Yield vs DON ± 0.04 TGW vs DON ± 0.09 a Data from all cultivars and locations analyzed together; b Bologna trial; c Ravenna trial; d three fungicide treatments, one inoculated control and one non-inoculated control; e three fungicide treatments and one inoculated control. DISCUSSION The environmental conditions during the experimental season were particularly favourable to the development of FHB disease in wheat grown in both locations examined herein and in several other regions in Italy. 30 Recent surveys on the occurrence of DON in cereals collected in Italy have indicated widespread DON contamination in wheat samples grown in Northern and Central Italy with high incidence, and levels of contamination up to 15 mg kg ,30. In the present study, high levels of DON were found in wheat samples from all inoculated plots and two naturally infected plots, which may be of some concern for human and animal health. Inoculation of the ears with Fculmorumand F graminearum significantly increased the DS as compared with the non-inoculated control (natural inoculum), with consequent decrease in grain yield in all cultivars, with the exception of cv Serio (Ravenna). A significant increase in DON levels was observed in all the inoculated plots, in agreement with findings of previous reports for similar experiments of Fusarium inoculation carried out on wheat both in glasshouse and in the field. 2,31 Despite the well-known susceptibility of most durum wheat varieties, the two soft wheat cultivars used in this study, ie Serio and Genio, were highly susceptible to FHB in both locations, with the cv Genio being the most susceptible. These cultivars were previously shown to be particularly susceptible to FHB in previous experiments (D Pancaldi, personal communication). Milus and Parsons 32 reported that, in the presence of a strong infective pressure (ie artificial inoculum), treatments with fungicides (including several conazole fungicides) do not affect FHB incidence, DON levels, yield and test weight. In our experiments, all fungicide treatments decreased both the incidence and the severity of the disease and increased both grain yield and TGW as compared with the inoculated controls. Decrease in incidence of the disease was less evident than decrease in FHB severity. Deoxynivalenol levels were significantly reduced (up to 89%) by the fungicide treatments as compared with the inoculated control. Our finding are in agreement with other investigations, proving the effectiveness of some conazole fungicides, including prochloraz and tebuconazole, both in glasshouse and in field trials producing a reduction of FHB in wheat (up to 91%) and DON accumulation. 13,14,17,33 38 Prochloraz and tebuconazole have been shown to improve TGW and reduce FHB in wheat inoculated with F culmorum in the glasshouse. 39 They also produced similar effects in field experiments after single inoculation of four cultivars of durum wheat with F graminearum and F culmorum. 14 These data were confirmed by our findings after treatments with prochloraz or tebuconazole present in Tiptor S formulation and in Horizon 250 EW, respectively. Azoxystrobin is reported to increase grain yield, dry matter and protein and delay senescence, 40 and has been used in this study in combination with tebuconazole to increase the efficacy of the treatment. Results to date indicate that azoxystrobin, when applied at full rate to inoculated wheat in field trials or in glasshouse, either has no effect, or can significantly increase the DON concentration in harvested grain, even though FHB symptoms are reduced. 17,19 However, when applied as preventive treatment, it may also reduce DON concentration. 15 This result is unlikely to be a direct effect on the toxin-producing Fusarium but, indirect, via the activity of azoxystrobin on other fungi present within the wheat ear environment. 11,15 Our findings with the azoxystrobin tebuconazole combination did not show a significant increase in grain parameters (yield and TGW) with respect to the treatment with Tiptor S (cyproconazole plus prochloraz). Moreover, the DON-stimulating effect of azoxystrobin, if any, could not be observed under our experimental conditions 196 J Sci Food Agric 85: (2005)

7 Effect of fungicides on Fusarium-inoculated wheat because either the treatment was performed as a preventive one or the DON-reducing effect of tebuconazole overwhelmed the hypothetical DON increase by azoxystrobin. Results obtained by using tetraconazole (Eminent 40 EW) showed, for the first time, the efficacy of this fungicide for FHB control. The field experiments showed an increase in production (between 0.5 and 2.9tha 1 ) on all cultivars, compared with inoculated control plots, mainly due to its capacity to reduce the severity of the disease in most of the cultivars. The TGW was also significantly higher for some cultivars. Finally, a significant decrease of DON content was observed for most of the tested cultivars. The possibility of using visual assessment of the disease to predict mycotoxin contamination in cereals has been reported by several authors. 5,41,42 A good correlation between visual FHB symptom ratings and DON levels in wheat was observed in greenhouse (r = 0.65) and field (r = 0.82) experiments after inoculation with F graminearum, suggesting that disease severity can be used as a good indicator of DON content in severe FHB epidemics. 5 No correlation was found in other studies between FHB severity and DON in harvested grain, despite the good correlation between the amount of trichotheceneproducing Fusarium and DON present within the grain. 11,15 We indeed found a strong correlation between disease severity and DON content for each experiment (average r = 0.93 ± 0.10, n = 8) when only the inoculated plots (treated and untreated with fungicides) were considered, in which almost all the fungal mass was attributable to the DON-producing strains of F graminearum and F culmorum used for inoculation. The average correlation coefficient dropped drastically to 0.75 (±0.28) when data relevant to naturally contaminated plots were also included. The contribution of non-toxigenic species/strains to FHB disease could be assumed as the major factor responsible for such correlation decrease that was particularly evident for the cultivar Orobel (r = 0.13). Moreover disease severity, yields, TGW and DON levels in our inoculation experiments were significantly correlated with each other when the regression analysis was performed with data from individual cultivars and locations, whereas no significant correlation was observed when all data obtained within the study were considered together. Our data also agree with previous investigations with respect to the relationship between FHB disease and yield, showing a strong negative correlation, strictly related to the inoculation with fungal species/strains known to cause wheat FHB. 5,14,17,39 Despite the forced conditions, ie artificial inoculation of highly pathogenic and toxigenic species of Fusarium, these findings show that the application of a combination of fungicides containing tebuconazole or prochloraz, either at the beginning of the flowering stage (GS60) or at a later stage (GS 65), provides a strong reduction of the FHB disease caused by F graminearum and F culmorum, allowing both an increase in grain yields and a considerable reduction of DON content in the wheat kernels. Our results, obtained under field conditions, strongly support similar studies on the efficacy of tebuconazole and prochloraz and provide useful information for wheat protection programs against toxigenic fungi responsible for FHB disease and the consequent mycotoxin accumulation in grains. The high correlation between visual disease severity and DON content in wheat kernels supports previous findings suggesting that the visual disease rating can be used as an indicator of mycotoxin contamination, particularly in case of severe epidemics. ACKNOWLEDGEMENTS The authors would like to thank Dr V Lippolis for his technical help in the deoxynivalenol analysis and the Consorzio Agrario di Bologna e Modena (Bologna, Italy), the Consorzio Agrario di Ravenna (Ravenna, Italy) and the Società Italiana Sementi SIS (S. Lazzaro di Savena, Bologna, Italy) for providing wheat seeds and contributing to field experiments. 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