Introduction

Food security and quality are important issues for a rapidly growing human population. The European Union (EU) is one of the world’s major cereal producers and consumers, with wheat being the most important cereal consumed. The five biggest EU wheat producers are currently France, Germany, the United Kingdom, Poland and Romania (EC 2015). Fungal pathogens produce an extraordinary diversity of biologically active secondary metabolites. Health risks associated with the consumption of cereal products contaminated with Fusarium mycotoxins are of major concern worldwide (Groves et al. 1999; Desjardins 2006). Such dangerous compounds should be minimized in wheat, a primary source of carbohydrates for both humans and domesticated animals. In the last decade, human population growth, land degradation and global climate change have placed pressure on food production systems, resulting in a greater demand for cereals (FAO 2013). As a result, the principles of integrated pest management (IPM) have been incorporated into the European Union’s (EU) Framework Directive on the sustainable use of pesticides and crop rotations (Council Directives 79/117/EEC and 91/414/EEC). IPM systems comprise many components, including soil tillage, crop rotation, resistance elicitors, cultivars and deployment of crops in mixtures (Park et al. 2009).

Species belonging to the genus Fusarium are ubiquitous in soil, air and water, and on plants and animals (Burgess 1981; Marasas et al. 1984; Nelson et al. 1994; Elvers et al. 1998; Bennett and Klich 2003; Nicholson et al. 2004). Depending on the ecological context, soil-borne Fusarium species may be parasites, endophytes or pathogens of healthy host plants; they are known for their ability to survive in soil in the form of spores or as saprotrophs (Leslie et al. 2006; Aoki et al. 2014). The most common pathogens occurring on winter and spring wheat in Poland and Northern Europe are Fusarium culmorum (Wm.G. Sm) Sacc., Gibberella zeae (Schwein.) Petch (anamorph of Fusarium graminearum Schwabe), Gibberella avenacea R.J. Cook (anamorph of Fusarium avenaceum (Fr.) Sacc.), and Fusarium poae (Peck) Wollenw. Other Fusarium species (e.g. F. sporotrichioides (Sherb.)) are less important due to their reduced incidences and aggressiveness in the Kujavia-Pomerania region of the northern plains of Central Europe (Wakuliński and Chełkowski 1993; Bai and Shaner 1994; Parry et al. 1995; Chełkowski 1998; Arseniuk et al. 1999; Bottalico and Perrone 2002; Edwards 2004; Logrieco and Visconti 2004; Abdullah and Atroshi 2016; Basler 2016; Tralamazza et al. 2016; Weber et al. 2016). These pathogens cause various diseases, such as root rot, foot rot, stem base rot, crown rot, Fusarium seedling blight (FSB) and Fusarium head blight (FHB-scab). FHB reduces wheat grain quantity and quality (Champeil et al. 2004a; Xu et al. 2005; Schmidt et al. 2016), and this disease has re-emerged in many cereal-growing regions worldwide (MacMullen et al. 1997; Jones and Mirocha 1999). Several fungal secondary metabolites, including deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), moniliformin (MON) and their derivatives may contaminate wheat grains as a result of FHB (Bottalico 1998; Chełkowski 1998; Snijders 2004; Schollenberger et al. 2005; Wiśniewska 2005; Tralamazza et al. 2016). In Poland, DON and a derivative, 3-acetyldeoxynivalenol (ADON), were identified in 1985 (Visconti et al. 1986), while ZEN was found in 1980 (Chełkowski et al. 1984) and has been recorded only in low amounts (Perkowski et al. 1990, 1991, 1997).

Thus far, breeders have not been able to produce a wheat cultivar fully resistant to FHB (Korbas and Horoszkiewicz-Janka 2007), although breeding for resistance is a necessary long-term strategy. Planting Fusarium-infected seed for cereal crops results in reduced plant density due to seedling blight (Timmermans et al. 2009). Burlakoti et al. (2010) showed that combining resistance to initial infection (type I) and resistance to spread within infected spikes (type II) remarkably enhanced resistance to FHB caused by G. zeae (F. graminearum) in spring wheat. Thus, varieties should be chosen for planting based on a combination of data, including level and type of FHB resistance and tolerance to seedling blight.

Many reports state that the most effective method to reduce losses caused by FHB of wheat is to plant resistant crop varieties in combination with appropriate agronomical practices and chemical protections (Clark et al. 2009; Blandino et al. 2012; Willyerd et al. 2012). 25 spring wheat cultivars from the Polish National List and 35 resistant cultivars/lines from Canada, Germany, Japan, Mexico and China were investigated by Góral and Walentyn-Góral (2014)) in 2010–2012 for their resistance to FHB following inoculation with F. culmorum. In Polish cultivars, the FHBI index (FHBi) was 28.1% (range of 15.8–45.6%), while cultivars and lines from the resistant collection were mostly highly resistant to FHB (mean index was 5.5%, at a range 0–26.0%). Lines ‘CJ 9306’ and ‘CJ 9311’ and cultivar Sumai 3 (all from China) were very highly resistant and showed no disease symptoms (Góral and Walentyn-Góral 2014).

FHB incidence has been described in the context of various factors, such as the type of farming system (Champeil et al. 2004b; Lenc 2015a), agronomic practices (Czaban et al. 2011; Horoszkiewicz-Janka et al. 2012) and environmental conditions (Doohan et al. 2003). Agronomic practices, such as crop rotation, mineral fertilization, organic matter soil inputs, tilling and others, play crucial roles in the management of Fusarium diseases (Wegulo et al. 2015). Non-inversion tillage may increase wheat grain infection by G. zeae as compared with inversion tillage, whereby residues are buried in the soil (Leplat et al. 2013). FHB’s occurrence is strongly dependent on specific weather conditions, predominantly rainfall. Some studies suggest that tillage protects wheat against Fusarium infestation (Dill-Macky and Jones 2000), while others state that weather conditions are much more important (Schaafsma et al. 2001). According to Duveiller (2008) and the International Maize and Wheat Improvement Center (CIMMYT), two causative agents of FHB, G. zeae and F. culmorum, are strongly influenced by climatic changes. FHB outbreaks may result from an early rainy season combined with farming system changes, especially expansion of corn crop growing area and the presence of corn stubble residues (Zhang et al. 2012; Steinmüller et al. 2004). Airborne Fusariums are known to be transported long distances (Keller et al. 2014) and G. zeae aerobiological characteristics have been thoroughly investigated (Goswami and Kistler 2004). Still, the relationships between FHB and grain contamination by Fusarium mycotoxins with respect to agronomic factors need to be fully understood because wheat cultivation has been simplified, with wheat monocultures becoming more popular, especially in their winter forms.

The purpose of this work was to investigate the impact of four experimental factors, including crop sequence, sowing date, fungicide use and cultivar, on FHB of wheat to identify conditions that minimize the risk of grain contamination by mycotoxins via integrated pest management methods.

Materials and methods

Field trials

Three field experiments were conducted from 2010 to 2013 at the Experimental Station of Cultivar Testing in Chrząstowo (53o11’N 17 o35’E), Poland, Kujavia-Pomerania region, Naklo vicinity, using two wheat (Triticum aestivum L. emend. Fiori et Paol.) cultivars the bread group cv. Cytra and the qualitative group cv. Monsun. Neither cultivar requires vernalization and both can be sown in late fall (facultative term) or spring. Crops were planted in the Alfisols typical of the region, which were classified as “wheat good complex” according to the Polish Soil Classification. A cultivator with a roller was used for ploughing and tilling of 72 total plots (16.5 m2). Seeds were placed at 3 cm depth with 12 cm row spacing; the target density (TD) of wheat was 500 seedlings m−2. According to the standard practices recommended by the Centre for Cultivar Testing (COBORU 2013), nitrogen fertilizer was applied with the following schedule: 40 kg of N per hectare before sowing, 50 kg per hectare at the 31–32 stage of the BBCH scale (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) (Witzenberger et al. 1989; Lancashire et al. 1991) and 40 kg per hectare at the BBCH 45–47 stage. Phosphorus and potassium were applied in doses of 60 kg P2O5 and 75 kg K2O per hectare, respectively, before sowing. Seeds were coated with Baytan Universal 094 FS (triadimenol 75 g/L, imazalil 10 g/L, fuberidazol 9 g/L) at 400 mL per 100 kg seeds. The dates of wheat sowing, fungicide applications and disease assessments during the three study years are presented in Table 1.

Table 1 The dates of wheat sowing, anthesis, fungicide applications and disease assessments during the three study years
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Factors and treatments

Experimental factors and treatments included pre-crop type (sugar beet, corn, wheat), sowing date (late autumn, spring), cultivar type (Cytra, Monsun) and fungicide application. These cultivars exhibit differential susceptibilities to Fusarium. The popular ‘Monsun’ cultivar, which produces a greater seed yield than ‘Cytra’, displays unstable, moderate FHB susceptibility, while ‘Cytra’ is stably susceptibility to FHB (Góral and Walentyn-Góral 2014). Fungicide applications included a control group designated “untreated” (without foliar and head treatments), and a treatment program designated “fungicide”, based on two treatments, made at T1 (BBCH 30–32) and T2 (BBCH 65), with the first treatment a mixture of prothioconazole and spiroxamine (commercial product Input 460 EC at dose 1 L/ha) and the second a mixture of fluoxastrobin and prothioconazole (Fandango 200 EC at dose 1 L/ha). A total of 24 combinations of 4 experimental factors in 3 replications in the split-plot-block layout were studied.

Disease assay

Wheat heads at BBCH 77 (late milk stage) were selected from 100 random plants on each wheat plot, and per cent index of Fusarium head blight (FHBi) was assessed according to the procedures established in OEPP/EPPO PP 1/26(4) (2012)). The number of husks showing FHB symptoms per head and the number of diseased heads were used to calculate FHBi, designating the percentage of all heads with disease symptoms. Tissue samples were collected and examined using a Leica DM 2500 microscope and Leica M125 stereomicroscope. Fungal isolation in artificial media was required for the identification of pathogenic species.

Identification techniques

One hundred 5 mm2 fragments were cut out from the borders of both healthy and infected ear husks. Prepared material was rinsed for 45 min in running water, disinfected in 1% AgNO3 solution for 15 s, rinsed three times for one min each time in sterile distilled water and placed on Potato Dextrose Agar (PDA; Difco) with streptomycin (50 mg/L) in Petri plates. Fungi were incubated for 7–10 days at 20 °C in a day-night cycle. The colonies on each plate were then examined macro- and microscopically and distinguished on the basis of colour, hyphal characteristics, growth rates, and sporulation rates. Colonies of each species were counted and representative fungi were identified by morphotyping on PDA and Synthetic Nutrient Agar (SNA; KH2PO4 1 g/L, KNO3 1 g/L, MgSO4∙7H2O 0.5 g/L, KCl 0.5 g/L, glucose 0.2 g/L, and sucrose 0.2 g/L) using the protocols of Booth (1971) and Kwaśna et al. (1991).

To confirm the species classification of Fusarium isolates, additionally polymerase chain reactions (PCR) were performed. The PCR assay was performed on selected isolates that, using traditional methods, were determined as G. avenacea (F. avenaceum), F. culmorum and G. zeae. Every year we examined 20 isolates from each species. Species-specific SCAR (Sequence Characterized Amplified Region) primers, JIAF/R for G. avenacea (Turner et al. 1998), Fc01F/R for F. culmorum and Fg16NF/Fg16NR for G. zeae (Nicholson et al. 1998), were used. DNA was extracted according to the modified Doyle and Doyle (1990) method. The amplification reactions were carried out in a thermocycler (Eppendorf Mastercycler ep gradient, Germany) using a Taq PCR Core Kit (QIAGEN Inc., USA). The PCR products were separated in TBE buffer by electrophoresis in 1.4% agarose gels stained with ethidium bromide and visualized under UV light.

Mycological analysis

Harvested material from the experimental plots (grain at 14% moisture with a minimum weight of 500 g) was thoroughly mixed and milled under sterile conditions. Twenty g of each sample was placed individually in a sterile bag of a Stomacher-type homogenizer, suspended in 180 mL of sterile liquid prepared according to PN EN ISO 6887–1 and homogenized for 90 s. Determination of the total number of fungal species was carried out according to PN ISO 7954 with one alteration (spread plate method of 1 mL and 0.1 mL, in triplicate). From the initial homogenized mixture (material diluted 1:10) a series of dilutions were performed. The inoculation was performed according to Koch on Yeast Extract Glucose Chloramphenicol Agar (YGC). Samples were incubated for 5–7 days at 25 ± 1 °C. Following incubation, colonies were counted and the results were expressed as the number of colony forming units (CFU) per 1 g of sample [cfu/g].

Mycotoxin analysis

12.5 g of harvested grain was homogenized with 50 mL of ACN:H2O (80:20) for 3 min and the extract was filtered using Fluted Filter Paper (Vicam). 40 μl of zearalanone (ZAN, internal standard for ZEN) solution was added to 4 ml of the extract, and the mixture was applied to a BondElut Mycotoxin® column (Agilent). Then, 50 μl of internal standard solutions (13C DON, 13C T-2 toxin, 13C HT-2 toxin) were added to 2 ml of the purified extract, and the mixture was evaporated to dryness under nitrogen. Then, 495 μl of MeOH:H2O 1:4 was added to the vial and the sample was vortexed. The analytical method of liquid chromatography tandem mass spectrometry (LCMS/MS) used mycotoxin standards for the analysis of the major metabolites of Fusarium. Mycotoxin presence and concentration (deoxynivalenol = DON, nivalenol = NIV, 3-Acetyldeoxynivalenol = ADON, T-2 toxin = T2, HT-2 toxin = HT-2 and zearalenone = ZEN) was assessed using HPLC with MS/MS detection. HPLC: Nexera (Shimadzu, USA); mass spectrometer: API 4000 (AB Sciex, Foster City, CA, USA); chromatographic column: Gemini C18 (150 × 4.6 mm, 5 μm) (Phenomenex Inc., Torrance, CA, USA); mobile phase: A: H2O + 5 mM CH3COONH4 + 1% CH3COOH, B: MeOH +5 mM CH3COONH4 + 1% CH3COOH; flow rate: 0.7 ml/min; injection volume: 10 μl. Limits of detection (LODs) and limits of quantification (LOQs) were 1 and 3 μg/kg, respectively, for DON, NIV and ADON; 0.2 and 0.6 μg/kg for T-2, 0.7 and 2 μg/kg for HT-2 and 0.06 and 0.2 μg/kg for ZEN. For statistical analyses, samples below LOQ were given at the LOQ value.

Statistical analyses

The arcsine angular transformation according to the Bliss (1938)) was used to obtain a normal distribution for FHBi (%) and Fusarium ratio (%). The Bliss transformation is typically applied to data having original binomial distributions expressed in percentage, most frequently assuming values within the range of 0–100%. Mould (cfu/g), DON (μg/kg) and ZEN (μg/kg) concentrations in grain were log-transformed due to high right skewing. The other mycotoxins were not statistically analysed due to levels of low detection or lack of repeatability within and between years and treatments. Shapiro-Wilk’s test was used to assess normality. The variables, FHBi, overall moulds concentration, Fusarium ratio, DON and ZEN concentration, were analysed by five-way mixed model (years was a random effect); pre-crop type, sowing date, fungicide application and wheat cultivar were the fixed effects. Analyses of variance (ANOVA) were carried out to determine the effects of year and all fixed factors, as well as the 2nd order interactions between fixed factors, estimated by the GLM for split-plot-block design with 4 residuals. The higher order (3rd, 4th) interactions between effects and the interactions of random (years) with fixed factors were omitted to avoid the confounding of main effects and the low-order interactions with higher order interactions (Hinkelmann and Kempthorne 2005). The means were separated according to Tukey’s HSD test with p ≤ 0.05 designating a significant difference. Mean data from the two cultivars x three pre-crop types x fungal treatment type x three years (n = 36) were used to assess correlations between total mycotoxin concentration and 5 determinant variables: development duration at BBCH 71–89, FHBi, G. zeae and F. culmorum ratio and total Fusarium ratio (FR) in wheat grain separately for two dates of sowing, according to the r-Pearsons’ coefficient. These variables were chosen following the ANOVA and means separations, as we noticed that in spring sowings the mean values were higher and the ranges were wider than in the late autumn sowings (Table 6). We therefore calculated correlations separately for the 36 cases of late autumn sowing and 36 cases of spring sowing. For this value of n, p = 0.05 corresponds to r = 0.33, and p = 0.01 corresponds to r = 0.42. Analyses were performed using Statistica 12.0, StatSoft software.

Results

Head and grain infestation

The Monsun cultivar grew and developed faster than cv. Cytra, and late-autumn-sown crops grew and developed faster than spring-sown crops (Table 1). In 2011 plants reached BBCH 65 (50% of anthers mature) between June 7–16, when rainfall was low (decade sum 6.9–15.6 mm) and air temperature was high (average 20 °C) (Table 2). Average FHBi was 6.96% overall, and was highest for wheat sown in spring (11.5%,Table 3). In 2012, half anthesis occurred in the 2nd and 3rd week of June (Table 1), with abundant rain (monthly sum 113.9 mm) and relatively low air temperature (monthly average 15.5 °C). The highest FHBi was noted in untreated wheat (25.1%) and was significantly higher in wheat sown after a pre-crop of wheat or corn as compared to sugar beet (Table 4). The cold spring in the third year caused slower crop growth, and wheat plants reached BBCH 65 just after mid-June, with little precipitation (decade sum 0.5 mm) and moderate temperature 18.6 °C. This season did not favour the Fusarium infection, and FHBi was 4.64%. Over all study years, three of the four agronomic factors had significant effects on FHBi. When wheat followed after wheat or corn (pre-crop, F (2;4) = 10.6), FHBi was higher compared with a pre-crop of sugar beet. A double application of fungicides at BBCH 30–32 and BBCH 65 (fungicidal factor, F (1;2) = 24.5) resulted in significantly reduced FHBi (2.6%) compared with untreated wheat (14.5%). The Cytra cultivar (cultivar effect, F (1;2) = 19.8) had an FHBi twice that of cv. Monsun (Tables 3 and 4).

Table 2 Rainfall (mm) and air temperature (°C) in June–July in the years of study (2010–2013) and for the past five decades (1965–2014)
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Table 3 Mean squares from analysis of variance with wheat characteristics
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Table 4 Fusarium head blight index (FHBi) on wheat for different pre-crop types, sowing dates, fungicide treatments and cultivars. Data represent means ± se
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Results are means from 2011 to 2013. Fungicide treatments are untreated and a program based on two treatments at T1 (BBCH 30–32) and T2 (BBCH 65), where the first treatment is an application of a mixture of prothioconazole and spiroxamine and the second is a mixture of fluoxastrobin and prothioconazole.

The dominant Fusarium species, identified based on morphology, that occurred on wheat heads was F. culmorum (21.1%), followed by G. avenacea (F. avenaceum) (17.2%), while G. zeae (F. graminearum) isolations were sparse (7.1%) over all treatments (Fig. 1). Other species were present in up to 54.5% of isolations, although most were identified as non-pathogenic. Wheat grown after corn revealed the highest prevalence of FHB species (over 60%). However, the three Fusarium species were identified twice as frequently when wheat was grown in monoculture than after sugar beets (Fig. 1). Furthermore, in untreated wheat, 60% of the fungal samples isolated were recognized as FHB-causative species. Fusarium species composed 60% of isolated fungi for wheat sown in spring, and only 27% in autumn (Fig. 1). The identification of G. avenacea, F. culmorum and (G. zeae) F. graminearum was additionally carried out using the PCR assay with species-specific SCAR primers. The JIAF/R primers verified the previous microscopic identification of G. avenacea and confirmed that all of the selected isolates belonged to this species. For the G. avenacea isolates, the amplification product of 220 bp was obtained. The identifications of F. culmorum and G. zeae were confirmed using the Fc01F/R and Fg16NF/Fg16NR primer sets, respectively. The F. culmorum isolates’ product of 570 bp was amplified, while for G. zeae the product was 280 bp.

Fig. 1
figure 1

Fusarium species isolations on wheat heads for different pre-crop types (wheat, corn, beet), sowing seasons (late autumn, spring,), fungicide treatments and cultivars (Cytra, Monsun)

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The number of types of moulds found on wheat grains depended on three experimental factors: pre-crop (F (2;4) = 10.3), sowing date (F (1;2) = 27.4), and fungicide application (F (1;2) = 18.9), while the cultivar type was insignificant (Table 3, Fig. 2a-d). Measured moulds on grains (cfu/g) was similar for wheat grown after corn or after wheat, with the highest representation of Fusarium species in the case of wheat followed by wheat (18.8%,Fig. 3a). The spring date of sowing favoured the number of moulds on grain (12,759 CFU/g) and the ratio of Fusarium species (18.4%). These numbers were two- and three-folds higher for wheat planted in spring than in late autumn (Fig. 2b, 3b). A significant interaction was found between cultivar and sowing season (F (1;2) = 19.1), both for the number of moulds and Fusarium ratio (Fig. 2e, 3e). Cultivar Cytra was more susceptible (22.7%) than Monsun (13.8%) to grain infestation by Fusarium species (Fig. 3d) and was much more susceptible in spring (22.7%) than in late autumn sowing (9.00%) – Fig. 3e. A similar pattern was observed in the number of moulds found on both cultivars planted after the different pre-crop types (Fig. 2f, 3f). However, the ratio of Fusarium isolations to other moulds found on the two cultivars differed with respect to pre-crop type. Cytra planted after wheat was more susceptible to Fusarium (20.5%) than Monsun (17.1%). Moreover, the Fusarium ratio in the moulds from the grain was significantly higher in case of Cytra than in case of Monsun planted after corn or sugar beet (Fig. 3f). The number of mould units from untreated wheat grain was almost three times as high, and Fusarium isolations three times higher, as that from grain of fungicide-treated wheat (Fig. 2d, 3d). The fungicide program, including prothioconazole and spiroxamine at T1 and a mixture of fluoxastrobin and prothioconazole at T2, effectively reduced both total moulds on grain (cfu/g) and Fusarium isolations.

Fig. 2
figure 2

The amount of mould (CFU/g) on wheat grains for different pre-crop types (a), sowing dates (b), fungicide treatments (c), cultivars (d) and interactions between cultivars / sowing date (e) and cultivars / pre-crop type (f). Data represent means + se. $ the same letters indicate the homogenous group according to HSD Tukey’s test at p = 0.05 for the individual treatment effects overall the years, # small letters indicate the homogenous group according to HSD Tukey’s test at p = 0.05 for within the interactions overall the years

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Fig. 3
figure 3

The proportion of Fusarium on wheat grains for different pre-crop types (a), sowing dates (b), fungicide treatments (c), cultivars (d) and interactions between cultivars / sowing date (e) and cultivars / pre-crop type (f). Data represent means + se. Explanations – see Fig. 2

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Mycotoxin profile and quantities depend on pre-crop type, fungicide control, sowing date and wheat cultivar

The concentrations of six mycotoxins were recorded: DON, NIV, ADON, ZEN, HT-2 and T-2. Data for DON and ZEN were analysed by ANOVA as only these mycotoxins were present in more than 80% of samples (excluding samples below LOQ). In 2012, the highest DON concentration (1250 μg/kg) was recorded (data not shown) for the grain of cv. Cytra sown in the spring after corn, without fungicidal control. DON concentration was affected by the main agronomic factors and their interactions: pre-crop of wheat (F (2;4) = 7.51), sowing season (F (1;2) = 20.3), interaction between pre-crop and wheat sowing season (F (1;2) = 19.7), fungicidal treatment (F (1;2) = 25.0), cultivar (F (1;2) = 18.5), the cultivar and pre-crop interaction (F (1;2) = 18.6), and the cultivar and sowing season interaction (F (1;2) = 20.2) (Table 3). ZEN concentration was affected by fungicidal treatment (F (1;2) = 20.1), cultivar (F (1;2) = 41.9), the cultivar and pre-crop interaction (F (1;2) = 27.7), and the cultivar and sowing date interaction (F (1;2) = 24.9) (Table 3). The mean values for all mycotoxins from all study years, except the mean for T-2 toxin, which was below LOQ, are presented in Table 5. Generally, mycotoxin concentrations were higher for cv. Cytra than cv. Monsun, and were favoured by spring season sowing and pre-crop of wheat or corn. Grain from untreated Cytra grown after corn in the spring had the highest concentrations of DON (605 μg/kg), ADON (103 μg/kg), ZEN (17.5 μg/kg) and HT-2 (5.53 μg/kg). Monsun under the same conditions produced grain with much lower toxin concentrations: DON, 154 μg/kg; ADON, 11.6 μg/kg; ZEN, 1.07 μg/kg; and HT-2 < 2 μg/kg. Fungicidal treatment did not suppressed DON in Cytra grain when this wheat was grown after corn. Reduced toxin concentrations were observed in grain harvested from crops sown in late autumn as compared to spring crops (Table 5).

Table 5 Toxic fungal metabolites in grains for different cultivars, dates of sowing, pre-crop types and fungicide treatments. Results are means from 2011 to 2013
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The relationships between mycotoxin concentration in wheat grain and five determinant variables were calculated using r–Pearson coefficient of correlation (Fig. 4). These determinants included: duration of the period between the end of flowering (BBCH 69) and the beginning of kernel abscission (fully ripe stage, BBCH 89), FHBi, F. culmorum isolations, G. zeae isolations and Fusarium ratio (FR) as a % of total isolations. Table 6 display the results of the mean and ranges of six characteristics where the tendency to the higher means and wider ranges refer to the spring sowings. We therefore calculated correlations separately for the 36 cases of late autumn sowing and 36 cases of spring sowing. Stronger positive correlations (p < 0.01) between six mycotoxin concentration and the agronomic determinants were obtained for spring-sown wheat. Longer development times between BBCH 69 and 89 correlated with higher mycotoxin levels (r = 0.85), as shown by increasing FR (r = 0.86) and increasing incidences of both G. zeae (r = 0.70) and F. culmorum (r = 0.65) on wheat heads (Fig. 4). For late autumn wheat, mycotoxin levels correlated only with FHBi (r = 0.51), G. zeae (r = 0.67) and F. culmorum isolations (r = 0.88), and not with BBCH 69–89 duration and FR in grain. G. zeae isolations correlated with mycotoxin concentrations in grain at the same level in both sowing seasons (Fig. 4).

Fig. 4
figure 4

Correlations between total mycotoxin content and development duration at BBCH 69–89, incidence of Fusarium head blight and the proportions of G. zeae. F. culmorum and total Fusarium (FR) in wheat grains for different sowing dates

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Table 6 Means and ranges of wheat characteristics correlated with total mycotoxin concentration in grain
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Discussion and conclusions

In the present study, four agronomic fixed factors (pre-crop, sowing date, fungicide treatment and cultivar) were studied in relation to FHBi, FR, Fusarium species present on grain and the concentrations of six FHB-associated mycotoxins in planted wheat crops. F. culmorum was the dominant species occurring on wheat heads (21.1%). This pathogen is one of the predominant Fusarium spp. in cooler climates such as northern, central and western Europe (Wagacha and Muthomi 2007), as well as in Poland (Golinski et al. 2002; Korbas and Horoszkiewicz-Janka 2007; Lenc 2015a; Weber et al. 2016). G. zeae dominates in areas with warmer climates, especially where summer is warm (Magan et al. 2002; Hofgaard et al. 2016). This fungus is relatively rare in the spring wheat cultivars although it is increasingly contributing to FHB (Czaban et al. 2011; Lenc et al. 2015b). In our study, this pathogen was identified in 7.1% of all isolations. Both species were molecularly identified in PCR assays with species-specific primers that proved to be reliable for isolates from Poland by Baturo-Ciesniewska and Suchorzynska (2011). FHBi was affected independently by the pre-crop type, fungicidal treatment and cultivar type, but not sowing date. FHBi was also dependent on seasonal weather conditions during the three years of our study. FHBi ranged from 4.64% in 2013 to 13.84% in 2012, with mean 8.48%, which indicates a low level of disease. However, 25% of untreated wheat heads were diseased in 2012, which was higher than in other study years. Such high incidences in 2012 can be explained by weather conditions favourable to FHB, including high rainfall (114 mm) and cool air temperatures (15.5 °C) during the critical phase, BBCH 65. Rain intensity, duration, and frequency, and the size and velocity of falling drops, affect the splash dispersal of spores and the success of infection (Shin et al. 2014). However, intense rain may have a decreased effective dispersal effect by washing off newly-dispersed spores, which, therefore, did not contribute to increased Fusarium infections (Lenc et al. 2015b). Moreover, wind can increase the primary rain-dispersal distance in a downwind direction and decrease it upwind. Generally, wind-dispersal distances are longer than rain dispersal distances alone, and the pure splash dispersal is mostly local (Sache 2000).

The two wheat cultivars tested in our trials exhibited different FHB susceptibilities. Cv. Cytra had an FHBi twice that of cv. Monsun, which was expected after Góral and Walentyn-Góral (2014)) findings. Thus, more CFU/g on grain and a higher Fusarium ratio was an attribute of cv. Cytra grain. Consequently, higher mycotoxin levels were found in Cytra grain, especially for DON, ADON, HT-2 and ZEN. Moreover, the interaction of sowing season and pre-crop type was significant in term of Fusarium ratio (%) in both wheat cultivars. Grain from more-susceptible Cytra had a higher Fusarium ratio when planted in spring or after wheat. This same pattern was not seen in the FHBi; because FHBi was assessed in samples collected directly from the field. Mycelia and conidia were probably not present on the grain surface, and consequently, they were not present in the liquid that was used as the inoculum on the YGC medium.

Our results showed that FHB was observed twice as frequently when wheat was grown in monoculture as compared to after sugar beets. Wheat sown after sugar beets had very low incidences of head diseases, as well as lower mycotoxin concentrations. Czaban et al. (2011) reported that intensive wheat cropping, high nitrogen inputs and incorporation of straw into the soil are more conducive to grain infection by FHB than IPM practices. Interactions of factors including host genetics, pathogen aggressiveness and toxin production capacity, and environmental conditions, particularly moisture, influence FHB development and the accumulation of mycotoxins in FHB-infested wheat (Gautam and Dill-Macky 2012). In seasons with rainfall levels, relative humidities and temperatures favouring FHB infestation, tillage and fertilization treatments have little to no impact on disease development. During seasons with moderate weather conditions, FHB may occur more frequently in no-till plots compared to conventional tillage plots, with subsequent increases in mycotoxin concentrations in grain (Lori et al. 2009). We tilled the plots used in this study using a uniform pattern for all pre-crop types and wheat cultivars. Pre-crop type impacted mould CFUs in grain as independent factor with higher amounts after wheat or corn, while Fusarium ratio (%) after wheat. We found that DON and ZEN concentrations were higher in cv. Cytra and illustrated the interactions between cultivar and pre-crop and cultivar and sowing date in the same meaning as Fusarium isolations from moulds. In current study, wheat grown after corn exhibited the highest FHBi (11.17%) and the highest FR (over 60%). This is in agreement with the findings of Nitzsche et al. (2002), which described the risk of FHB in the Saxonia region for wheat following various pre-crop types, and concluded that corn as a pre-crop increased the risk of Fusarium infestation. A 10-year survey of randomly collected winter wheat samples within the Czech Republic also revealed the significant adverse effect of corn as the pre-crop on the accumulation of DON in wheat grain (Chrpová et al. 2015). Moreover, DON production by both of the DON-producing species F. culmorum and G. zeae was significantly higher in mixed infections with other species. The presence of G. avenacea (F. avenaceum) among these species appeared to markedly promote the production of DON and NIV. Because G. avenacea was isolated in 17.2% of our samples, the same effect could be influencing the levels of mycotoxins produced by F. culmorum and G. zeae that we detected.

Sowing in late autumn results in earlier wheat development and ripening as compared with spring sowing (Wenda-Piesik et al. 2016). In current study, sowing season also impacted both of these disease characteristics, with higher intensity for spring sowings. We observed higher means and wider ranges of FHBi, higher Fusarium ratios in grains, higher F. culmorum and G. zeae percentages in isolations, and increased duration of the phases between the end of flowering and the beginning of kernel abscission in spring sowings as compared to late autumn sowings. A longer developmental time for plants between BBCH 69–89 correlated with increased mycotoxin levels, as evidenced by the increasing FHBi and incidence levels of both G. zeae and F. culmorum on heads. Usually, wet weather results in the prolongation of this stage (from the end of flowering to the beginning of the fully ripe stage) in spring wheat infected by Fusarium spp., and favours the presence of the inocula. For late autumn-sown wheat, the total mycotoxin concentration was correlated with FHBi, and G. zeae and F. culmorum incidences on heads, but not with the duration of BBCH 69–89 or FR in grain. Due to the advanced development and faster kernel maturation, wheat sown in late autumn exhibited reduced grain contamination levels and lower mycotoxin concentrations. Correlations and regressions among FHB symptoms caused by F. poae and F. avenaceum, fungal incidences, and toxin accumulation in wheat grains were studied on Swiss varieties under controlled inoculation conditions (Vogelsgang et al. 2007). The NIV content and F. poae incidence in grain was highly correlated, with an r 2 of 0.90, while the correlation between disease symptoms was less strong (r 2 = 0.56). Conversely, the correlation between the F. avenaceum incidence in grain and the MON content was rather low (r 2 = 0.59), while with the correlation between disease symptoms and MON was greater (r 2 = 0.77). In that study, the distribution of data had a high overall infection rate by F. avenaceum, starting at 70% infected grains, and its narrow range (70–90%) explained the lower correlation with MON. Toxin concentrations in grains of plants that were artificially inoculated might not reflect the situation in commercial grain fields (Vogelsgang et al. 2007). Here, correlations exist in data collected under natural infection conditions. There were wide ranges for the features determining the mycotoxin concentrations, and their variations were related to the weather conditions and four agronomic factors. Their strongest correlations were: Fusarium ratio (r = 0.86), G. zeae isolations (r = 0.70) and F. culmorum isolations (r = 0.65), which explained the toxin content in wheat grain from spring term sowing. The mycotoxin levels on wheat grain coming from late autumn sowing were correlated with FHBi (r = 0.51), G. zeae (r = 0.67) and F. culmorum isolations (r = 0.88). This significantly corroborates the theory of a relationship between FHB and mycotoxins in grain, which was described previously by Browne (2007) regarding the F. graminearum incidence and DON accumulation. However, the associations between disease symptoms, fungal incidence on grain and toxin content have not always been shown. Horoszkiewicz-Janka et al. (2012) found no correlation between different pre-crop types (rapeseed, corn and wheat) or tillage system (reduced vs. traditional) and DON or ZEN concentrations in wheat sown from 2009 to 2011 in Central Poland.

Fungicide applications to suppress FHB may be challenging, in part due to uneven flowering times within a single field, which may necessitate multiple costly applications. Due to the short application window (the period during, or shortly after, anthesis), unforeseen events such as unfavourable weather can prevent optimal application timing (Wegulo et al. 2015). Our analyses showed that fungicide treatment independently affected mould CFUs in grain as well as Fusarium isolations. The program including applications of the fungicides prothioconazole and spiroxamine at T1 and a mixture of fluoxastrobin and prothioconazole at T2 effectively reduced head disease and FR, as well as total moulds on grain and Fusarium incidence. However, when cv. Cytra was grown after corn, DON levels were relatively high. This indicated that cultivars susceptible to FHB should also be protected at the end of the flowering stage. Proper timing and application of fungicides is critical. If the window of opportunity is missed and infection occurs, later applications of fungicide will not prevent formation of DON (Yuen and Schoneweis 2007). Thus, a third treatment at stage T3 (BBCH 69) with, for example, a mixture of prothioconazole and tebuconazole (Paul et al. 2005), could be justified in wet June–July. Under moderate conditions, IPM practices such as crop rotations and proper cultivar choices should be employed to maintain low inoculum levels, with fungicides used only when necessary.

Co-occurrence of Fusarium mycotoxins, with the potential for synergistic or antagonistic toxin interactions, is a recurring problem in wheat production systems. European regulations set maximum tolerated limits and guidance values for several mycotoxins in food and feed, and designate methods for mycotoxin sampling and analysis (European Commission (EC) 2006a, 2006b, 2013).

In our investigation, the mean DON, NIV, ADON, ZEA, HT-2 and T-2 levels in grain did not exceed the maximum permissible values for unprocessed wheat. However, our study suggests that it is most important to monitor and mitigate FHB risk for susceptible cultivars, in years with high June rainfalls, when wheat sowing follows corn or wheat and when no fungicidal treatment is used.