Species of the genus Fusarium and Fusarium toxins in the grain of winter and spring wheat in Poland

The aim of the study was to determine the presence of Fusarium species and mycotoxins in wheat grain from harvest in 2009 and 2010 in Poland. Samples from different locations were analyzed for the content of DNA of Fusarium species and mycotoxins. In 2009, DNA of F. graminearum and F. poae was present in all samples, F. culmorum in 82% of samples, and F. avenaceum in 55% of samples. In 2010, the highest content of DNA was found for F. graminearum followed by F. avenaceum, F. poae and F. langsethiae. The amount of F. culmorum DNA was very low. The most frequently occurring species were F. poae and F. graminearum, however, the amount of F. poae DNA was lower. In 2009, deoxynivalenol was detected in all samples. In 2010, the average content of deoxynivalenol was lower than in 2009. Nivalenol was detected at very low concentration in both years. Significant correlations between content of F. graminearum DNA and deoxynivalenol concentration in the grain and between content of F. poae DNA and nivalenol concentration in the grain in 2009 were found.


Introduction
Fusarium head blight (FHB) is a disease of wheat caused by a complex of toxicogenic fungi of the genus Fusarium (Parry et al. 1995). The main species of this complex in Europe are F. graminearum and F. culmorum identified as deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) producers. However, other Fusarium species producing mycotoxins are also prevalent: F. avenaceum -moniliformin, enniatins and beauvericin (BEA) producer; F. poae -NIV, BEA producer. Fusarium langsethiae and F. sporotrichioides -T-2 and HT-2 toxins producers are also prevalent (Bottalico and Perrone 2002;Jestoi et al. 2008;Vogelgsang et al. 2008;Somma et al. 2010;Imathiu et al. 2013). Fusarium graminearum and F. culmorum are highly pathogenic species, which can cause severe epidemics of FHB. The other species are medium or weakly pathogenic, however, due to the wide prevalence, they may also cause mycotoxin contamination of grain (Uhlig et al. 2007;Yli-Mattila et al. 2008;Nielsen et al. 2011;Dinolfio and Stenglein 2014).
Because of the diversity of Fusarium species causing FHB, monitoring of changes in the Fusarium population on wheat is important. Frequency of species infecting wheat Tomasz Góral, Piotr Ochodzki, Linda Kaergaard Nielsen... is not stable and changes depending on the weather in particular year (Xue et al. 2019). Differences are also observed between regions of wheat production in Europe. For example, other species dominate in North-Eastern Europe (equal share of three species F. avenaceum, F. culmorum, F. graminearum) than in south-western part of the continent (mainly F. graminearum) (Bottalico and Perrone 2002). Species compositions changes over time, which is the results of climate warming, and changes in the acreage of major cereal crops -particularly increase of the maize area (Sundheim et al. 2013;Hofgaard et al. 2016;Maiorano et al. 2008;Vaughan et al. 2016). The main reported effect of the above factors is increase in F. graminearum occurrence and decrease in F. culmorum (Parikka et al. 2012;Miller 2008;Scherm et al. 2013;Hofgaard et al. 2016;Bilska et al. 2018). Chandelier et al. (2011) analysed winter wheat samples from Belgium over 2003-2009 period. They found that main species were F. avenaceum and F. graminearum; however, their frequency changed depending on year from 20 to 100%. The frequency of F. poae was relatively constant over the years (about 70%). The overall incidence of F. culmorum decreased during the study, from 80% in 2003 to 10% over the final three years. Similarly, Isebaert et al. (2009) observed that F. graminearum and F. culmorum were the most important species in Northern Belgium in [2002][2003][2004][2005]. They found correlation between crop prevalence and both species frequency. F. graminearum dominated in areas of maize cultivation, F. culmorum in areas small grain cereals cultivation. In Luxemburg, the most common species isolated from wheat heads were F. graminearum, F. avenaceum and F. poae. Increase of frequency of F. graminearum and decrease in F. culmorum were observed (Giraud et al. 2010). Winter wheat cultivated in the Netherlands in 2009 was studied for Fusarium species and toxins (van der Fels-Klerx et al. 2012). In samples collected on harvest, authors found dominance of F. graminearum. F. avenaceum and Microdochium nivale were also frequent. However, in the pre-harvest samples, only F. graminearum and M. nivale were present. Waalwijk et al. (2004) analysed wheat heads and grain collected in the Netherlands in 2001. In 2001 in samples collected at late milk stage, F. graminearum was predominant; however, some samples contained also F. avenaceum and/or F. culmorum. At harvest, F. graminearum dominated almost completely. In 2002 the weather conditions were more favorable for FHB, and they found relative dominance of F. graminearum in grain from the Netherlands and almost complete in samples from France. According to Birzele et al. (2002) in 1997 and 1998 the dominating species in Germany in wheat grain were F. avenaceum, F. poae, F. culmorum and F. graminearum. Frequencies of two last species were similar, however percentage of F. graminearum increased in 1998. In Germany in 2008, F. graminearum sensu stricto was the predominant species followed by F. culmorum. Other species (F. poae, F. tricinctum, M. nivale etc.) were identified in small amounts (Talas et al. 2011). Similar results were obtained by Birr et al. (2020) who analyzed winter wheat grain samples from seven locations in Germany from 2013 to 2017. In Hungary, in year 2010, which was very favorable for FHB development, predominantly F. graminearum was isolated from wheat grain (Laszlo et al. 2011). Waalwijk et al. (2003) analyzed wheat ears with FHB symptoms collected in Netherlands in 2000 and 2001. They found that F. graminearum was the dominating Fusarium species in both years. As they stated, this was significant change comparing results from the 1980s and 1990s, which showed that F. culmorum was the predominant species in the Netherlands. They presume that this shift could be connected with an increase in maize acreage. F. graminearum, unlike F. culmorum, is a major pathogen on maize and, can survive on maize debris (Xu and Nicholson 2009;Maiorano et al. 2008). The other factor could be climate warming which favors F. graminearum as it has higher optimal temperature of development (Vaughan et al. 2016). The good example of this shift can be first detection of F. graminearum in wheat grain collected in 2017 in West Siberia, Russia (Gagkaeva et al. 2019) as well as absence of F. graminearum until 2012 in FHB infected cereal (Supronienė et al. 2010(Supronienė et al. , 2016 The prevalence of FHB pathogens differed significantly between studied countries in 2001 and 2002 (UK, Ireland, Italy and Hungary) (Xu et al. 2005). Overall, all pathogens (F. graminearum, F. culmorum, F. avenaceum and F. poae) were commonly detected in Ireland and to a lesser extent in the UK. In contrast, only two species, F. graminearum and F. poae, were regularly detected in Italy and Hungary. Fusarium culmorum was rarely detected except in Ireland. The latter country has the coolest summer weather among four studied countries. Authors stated that the increase in F. graminearum, especially in the UK, appears to have been at the expense of F. culmorum. The replacement of F. culmorum by F. graminearum as the predominant FHB pathogen was also reported in Bavaria (Obst et al. 1997) where the change was linked with increased maize production in Poland in years 2016 and 2017 more than 80% of isolates collected from symptomatic wheat heads were F. graminearum, and less than 4% were F. culmorum (Bilska et al. 2018). F. graminearum dominated in wheat grain in 2012, and was replaced by F. poae in 2013 (Wolny- Koładka et al. 2015). It is worth to notice that in Poland grain maize acreage increased considerably from 1990 (59 000 ha) to 2017 (above 1 215 500 ha).
The Fusarium species can be isolated from cereal kernels and identified using classical and/or molecular methods (Wiśniewska et al. 2014). The molecular method widely used for identification and quantification of Fusarium DNA concentration in samples is real time PCR (Niessen 2007;Nicolaisen et al. 2009;Nielsen et al. 2011Nielsen et al. , 2013Horevaj et al. 2011).
The aim of the present study was to determine the presence Fusarium species and content of mycotoxins in wheat grain in Poland, Samples were collected in 2009 and 2010. Results were compared with Fusarium species frequency reported earlier and the results obtained after 2010.

Cereal grain samples
Fifty samples of wheat grain were collected during the harvesting season 2010. They originated from 25 experimental stations of COBORU (the Research Centre for Cultivar Testing) located in different regions of Poland ( Figure 1; marked with numbers). Two winter wheat cultivars 'Bogatka' (medium resistant to FHB) and 'Muszelka' (susceptible) were included. The winter wheat was grown with a moderate nitrogen input (avg. 90 kg/ha of N) and without chemical control of diseases. The grain was harvested using combine harvester. and 2010 (circles). Location numbers correspond to these in Table 2 Rysunek 1 Additionally, 11 samples of wheat grain from 2009 (5 locations) and 8 samples from 2010 (2 locations) were analyzed. Samples were collected from different locations/fields and cultivars of spring and winter wheat ( Figure 1, Table 1, Table 4).
Wheat grain samples were stored in a freezer at -20°C before DNA and mycotoxins extraction.

DNA extraction and analysis
Grain samples of 300g were initially ground with a laboratory grinder and 5 g was powdered in liquid N 2 with eight steel balls using Geno/Grinder 2000 (OPS Diagnostics, Bridgewater, NJ). DNA was extracted from 100 mg of that powdered sample using a modified CTAB method (http://gmo-crl.jrc. ec.europa.eu/summaries/NK603-WEB-Protocol-Validation.pdf) as described by Nicolaisen et al. (2009). DNA extracted from the wheat samples was further purified using a DNeasy kit (Qiagen) according to the manufacturer's instructions.
The Fusarium isolates: F. avenaceum 9605, F. culmorum 9560, F. equiseti 8752, F. graminearum 1955, F. langsethiae 8051, F. poae 8452, F. sporotrichioides 1926, and F. tricinctum 8048 were grown and extracted as described in Nielsen et al. (2011). They were grown on potato dextrose agar (PDA) medium at 22°C under 12 h of light and 12 h of darkness for 1-2 weeks prior to DNA extraction. PDA plates before inoculation were covered with sterile cellophane membranes (Horevaj et al. 2011). Mycelium was scraped off the cellophane membrane using a spatula and ground in liquid N 2 with eight steel balls using a Geno/ Grinder 2000 (OPS Diagnostics, Bridgewater, NJ). Powdered mycelium (100 mg) was used for DNA extraction, using the same method as for grain samples. The concentration of DNA from Fusarium isolates was determined using NanoDrop 1000 (Thermo Fisher Scientific, MA).
Qualitative and quantitative determinations of eight Fusarium species in grain were performed by real time-PCR. Primers used were based on fungal TEF-1α gene sequences, designed by Nicolaisen et al. (2009), specific for the different Fusarium species: F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. langsethiae, F. poae, F. sporotrichioides, and F. tricinctum. Real-time PCR was carried out in 12.5 μl consisting of 6.25 μl of 2× SYBR Green PCR Master Mix (Applied Biosystems), 250 nM each primer, bovine serum albumin at 0.5 μg/μl, and 2.5 μl of template DNA. PCR reactions were performed in duplicate on all samples. Genomic DNA from grain samples and pure cultures was diluted 1:10 before PCR.
PCR was performed on a 7900HT Sequence Detection System (Applied Biosystems) using the following cycling protocol: 2 min at 50°C; 95°C for 10 min; 40 cycles of 95°C for 15 s and 62°C for 1 min; followed by dissociation analysis at 60 to 95°C. For the plant assay annealing and extension was performed at 60 °C. Standard curves for Fusarium species and wheat were made of five-fold dilution series using pure fungal DNA and wheat DNA. The amount of fungal DNA was calculated from the cycle threshold (Ct) values using the standard curve. The result of each individual sample from each species-specific assay were evaluated by studying the dissociation curve and Ct value, as SYBR Green binds to all double stranded DNA and might create false positives. The plant EF1α assay was used to provide a normalized measurement for Fusarium DNA in each sample, which was calculated as picograms of fungal DNA per micrograms of plant DNA according to Nicolaisen et al. (2009).

Analysis of Fusarium toxins
The type B trichothecenes -deoxynivalenol (DON), nivalenol (NIV) were quantified using gas chromatography with electron capture detection (GC-ECD) technique. Mycotoxins were extracted from 5 g of ground grains using 25 ml of an aqueous solution of acetonitrile (acetonitrile: water 84:16) in a shaker 90 min, 300 r.p.m.. Samples were centrifuged (3000 rpm min -1 , 5 min.), and the extract was purified with MycoSep® 227 Trich+ columns (Romer Labs Inc., Union, MO). One milliliter of the internal standard solution (chloralose) was added to 4 ml of purified extract. The solvent was evaporated to dryness in the stream of air. Mycotoxins were derivatized to the trimethylsilyl derivatives using a derivatizing agent Sylon BTZ (BSA + TMCS + TMSI, 3: 2: 3, Supelco). After dissolution of sample in isooctane, excess of derivatizing agent was decomposed and removed with water. The organic layer was transferred to autosampler vial and analyzed chromatographically with gas chromatograph SRI 8610C, with BGB-5MS column of 30 m in length, and an internal diameter of 0.25 mm.
The carrier gas was hydrogen, adjusted to pressure 12 psi, with nitrogen as a make-up gas at 60 mL/min. Elution was carried out in the temperature gradient: Initial temperature was 170 °C, increased to 250 °C at 5 °C/min., and increased from 250 °C to 300 °C at 10 °C/min., followed by a holding time of 5 min., and decreased to 170 °C. Mycotoxin detection was carried out using electron capture detector Tomasz Góral, Piotr Ochodzki, Linda Kaergaard Nielsen... (ECD). Identification of individual compounds was made by comparing the retention times of the pure standards of mycotoxins. The concentration of mycotoxins was established based on the calibration curve, using chloralose as the internal standard. Results were corrected for recoveries, ranged from 73% (NIV) to 85% (DON). The limits of detection (LOD) was on average 5 µg/kg, and limit of quantification 10 µg/kg.

Statistical analysis
The original Fusarium DNA and toxin concentrations were transformed to logarithmic values to obtain a normal distribution for the variables. The relationships between the results for Fusarium DNA and Fusarium toxins were investigated by Pearson correlation tests. Principal component analysis was used to analyze relationship between concentrations of DNA of F. avenaceum, F. culmorum, F. graminearum, F. langsethiae and F. poae in grain samples from 25 locations. Next, PCA was applied to analyze relationship between concentrations of Fusarium toxins (DON, NIV, ZEN) and DNA of producing species F. culmorum, F. graminearum and F. poae in grain samples from 25 locations. The correlation and PCA analyses were performed using Microsoft® Excel 2010/ XLSTAT©-Pro (Version 2013.4.07, Addinsoft, Inc., Brooklyn, NY, USA).

Concentration of Fusarium species DNA and DON, NIV and ZEN mycotoxins levels in samples of grain of spring and winter wheat collected in 2009
Zawartość DNA gatunków Fusarium oraz mykotoksyn DON, NIV i ZEN w próbach ziarna pszenicy jarej i ozimej zebranych w 2009 r.

Sample name Próba
Despite large differences in Fusarium DNA content in the grain samples, amount of F. graminearum DNA was the highest in nine samples ( Figure  2). F. culmorum dominated only in a sample from Nagradowice and in sample from Trzebnica concentrations of F. avenaceum and F. graminearum DNA were similar.  (Table 1). The most contaminated were the grain samples of winter wheat 'Nagradowice' and 'Debina 2' and spring wheat 'Radzików 1'. Levels of NIV were much lower. On average, it was 68 μg/kg. NIV was detected in seven samples. The highest concentration was in the grain of winter wheat from 'Debina 2' and winter wheat 'Radzików 4'. ZEN was detected in six samples at the average level of 45 μg/ kg. Considerable amounts of ZEN were found in samples from Nagradowice and in samples of spring wheat 'Radzików 1' and winter wheat 'Dębina1'.

Samples from 2010
In 2010, average concentration of Fusarium DNA was 1970 pg/μg (1430 pg/μg in 'Bogatka' grain and 3770 pg/μg in 'Muszelka' grain) ( Table  2). The difference in Fusarium DNA concentration between cultivars was statistically significant according to paired samples t-test. The highest concentration of DNA was detected in the grain from Zadąbrowie, South-Eastern Poland ( Figure  1). The DNA amount was five-six times lower in the grain from Czesławice (South-Eastern Poland), Rychliki, Radostowo (Northern PL) and Głubczyce (Southern PL). Very low concentration of DNA was found in the grain from Naroczyce, Nowa Wieś Ujska (Western Poland), Kawęczyn (Central Poland), and Rarwino (North-Western Poland). At a regional scale, the highest Fusarium DNA concentration was observed in the grain from South-Eastern and North-Eastern Poland and the lowest concentrations was observed in the grain from Western, North-Western and Central Poland (Figure 1). Of the eight Fusarium species tested, five were detected in wheat grain. DNA of F. equiseti, F. sporotrichioides and F. tricinctum was not detected in any sample. The highest was the content of F. graminearum DNA (1252 pg/μg), then F. avenaceum (259 pg/ μg), F. langsethiae (237 pg/μg) and F. poae (168 pg/μg) (Figure 3). The content of F. culmorum DNA (55 pg/μg) was very low.
The most frequently occurring species were F. poae (detected in 74% of samples) and F. graminearum (detected in 52% of samples) (Figure 3). In 18% of samples F. poae was the only species found. F. langsethiae was detected in six samples (five from three locations in Northern Poland -Wyczechy, Radostowo, Rychliki). The concentration of F. langsethiae in these samples was relatively high (1972 pg/μg) as compared with an average for samples containing F. graminearum DNA (2235 pg/μg). F. poae was detected in all samples of medium resistant cultivar 'Bogatka' but only in 48% of samples of susceptible 'Muszelka'. Another species F. avenaceum was also found more frequently in the grain of 'Bogatka' (32%) than 'Muszelka' (20%). Three other species (F. culmorum, F. graminearum, F. langsethiae) were detected in the grain of both cultivars with similar frequency.
Amounts of DNA of Fusarium species weakly correlated with each other. Only coefficient of correlation of F. graminearum with. F. culmorum was statistically significant (r = 0.461, p = 0.02). Positive relationship was found between F. avenaceum and F. culmorum or F. graminearum (r = 0.306, r = 0.162) as DNA of the first species was mostly detected in the same locations as the other two species -1, 4, 14, 19, 22 (only F. graminearum), and 24. DNA of F. langsethiae did not correlate with other species, as it was found only in six samples. Otherwise, F. poae DNA did not correlate with other species because the species was present in the most of samples (74%) and in the most samples (except two) amounts of F. poae DNA were similar.
Biplot produced by PCA analysis on DNA concentration of five Fusarium species showed uneven distribution of these species in different locations (Figure 4). F. culmorum was present mostly in the same locations as F. graminearum (except 12). F. avenaceum was present in the same six locations as F. culmorum and F. graminearum (except 22, where only the second species was detected). In three locations (7, 8, 13) this species was accompanied only by F. poae. As it was mentioned earlier, F. langsethiae was found in four locations (12,15,17,23). In Słupia (17)   Average content of DON was low and amounted to 96.2 μg/kg, at a range from.49.3 to 552.0 μg/ kg (Table 3). The content of NIV was very low -55.2 μg/kg, at a range 49.2 -70.8 μg/kg. The average content of DON for 'Bogatka' was 78.2 μg/kg, and 114.2 μg/kg for 'Muszelka'. Difference of DON content between cultivars was not statistically significant. The highest concentration of DON was found in samples of both cultivars from Zadąbrowie and Czesławice, South-Eastern Poland ( Figure  1). High concentration of this toxin was also found in the samples of 'Muszelka' from Wrócikowo, Lućmierz and Głubczyce.
ZEN was detected in 12% of samples of 'Bogatka' and in 60% of samples of 'Muszelka' cultivars. Average content was 23.9 μg/kg and was 3 times higher in the grain of 'Muszelka' than in 'Bogatka'. The difference in ZEN content between cultivars was statistically significant according to paired samples t-test. High concentration of ZEN was present in samples of 'Muszelka' and 'Bogatka' grain from Zadąbrowie (248 and 206 μg/kg, respectively) and in 'Muszelka' sample from Czesławice (128 μg/kg).
Six samples of the grain containing DNA of F. langsethiae were analyzed for T-2/HT-2 toxins. In all the samples, the total concentration of both mycotoxins was below detection limit of 35 μg/kg. Amount of Fusarium DNA in grain correlated significantly with concentration of Fusarium toxins (DON, NIV, ZEN) (Table 3). F. graminearum DNA correlated significantly with DON and ZEN concentrations, whereas F. culmorum DNA with ZEN concentration only. DNA of F. poae did not correlate with DON and ZEN -toxins not produced by this species. There was some positive relationship between F. poae and NIV concentration. Summarized amount of F. culmorum and F. graminearum DNA did not improve the strength of correlation with the toxins. Correlation of NIV with F. graminearum + F. poae DNA (possible NIV producers) was statistically significant (r = 0.511). Table 3 Tabela 3 Biplot produced by PCA analysis distinguished some locations based on concentrations of DNA of three Fusarium species and Fusarium toxins ( Figure 5). In Zadąbrowie (24), we found the highest amount of DON and ZEN as well as amount of F. graminearum DNA. Grain from Czesławice (2) were characterized by the highest amounts of F. poae DNA and NIV but also have high concentrations of the others toxins/DNA. On the other hand, in Słupia (17) concentration of F. poae DNA and NIV was low, but analysis showed high concentration of F. culmorum accompanied by moderate concentration of F. graminearum and DON. Tomasz Góral, Piotr Ochodzki, Linda Kaergaard Nielsen... In the four locations (5, 10, 11, 13) the concentration of DNA of three Fusarium species as well as the concentration of toxins were low. In another eight locations ( Figure 5, solid line), concentration of toxins was low, but amount of Fusarium DNA varied from low (23) to high (6). In Ruska Wieś (14) we found the highest concentration of F. culmorum DNA (511.8 pg/μg). Five locations (2, 3, 7, 21, and 22) could be characterized by above average concentration of NIV and moderate to high concentration of F. poae DNA. This species was present at considerable amounts also in samples from other locations (1,8,9,12,20) but NIV concentration was low.

Coefficients of correlation between concentration of DNA (pg/μg) of three Fusarium species and concentration (μg/kg) of mycotoxins DON, NIV and ZEN in grain of winter wheat cultivars 'Bogatka' and 'Muszelka' from 2010 harvest in 25 locations
In samples of the grain of spring and winter wheat collected from Radzików and neighboring Młochów we found more Fusarium DNA than in most samples of 'Bogatka' and 'Muszelka' ( Table 4). The highest amount of DNA was present in samples of winter wheat 'Tonacja' and 'Zawisza' (6998 pg/μg and 5738 pg/μg, respectively). In spring wheat, it was lower, except for the sample of 'Raweta' from Radzików (Raweta R1) (5513 pg/μg). Four Fusarium species were detected in grain. F. langsethiae and F. sporotrichioides were not present. F. avenaceum dominated in three samples (on average 1797 pg/μg of DNA) and F. graminearum in three (1432 pg/μg). In one sample (Tonacja R) amounts of DNA of these species were similar. F. poae was present in all samples of winter wheat (270 pg/μg). In the grain of spring, wheat 'Raweta' from Młochów only this species was present. The concentration of F. culmorum DNA was generally the lowest of all species (67 pg/μg).
The concentration of trichothecene toxins was low (Table 4). ZEN amount was below limit of detection. The highest concentration of DON was found in the samples with high concentration of F. graminearum and F. culmorum DNA -Zawisza R and Raweta R1. The same was true for NIV concentration in grain. No relation was found between F. poae and NIV; however, total concentration of F. graminearum and F. poae correlated the best with NIV amount.

Discussion
Presence and concentration of Fusarium DNA in naturally infected wheat in two years of the study was generally in accordance with data on occurrence of Fusarium species on wheat in Poland. According to the published data, dominant species on wheat spikes and kernels were F. culmorum, F. graminearum, F avenaceum and F. poae (Perkowski et al. 1990;Goliński et al. 1996;Bottalico and Perrone 2002;Stępień et al. 2008;Chełkowski et al. 2012;Wiśniewska et al. 2014;Kuzdraliński et al. 2017;Bilska et al. 2018;Iwaniuk et al. 2018). Proportions Species of the genus Fusarium and Fusarium toxins in the grain of winter and spring wheat in Poland of these four species changed depending on year and study as well as region of sampling. Other species were also detected were not present in all published results, for example F. langsethiae (Łukanowski & Sadowski 2008), F. sporotrichioides (Kuzdraliński et al. 2017), F. tricinctum (Wiśniewska et al. 2014).
Weather in 2009 was more favorable for FHB development than in 2010, which is also reflected in the difference in amount of Fusarium DNA and mycotoxins (Table 5).  In some regions (e.g., Radzików) in 2010, the drought conditions occurred in June and July with high temperatures and infrequent, heavy rainfalls. Despite differences in weather and limited number of samples in 2009, F. graminearum was occurring more frequently than F. culmorum. Amount of DNA of the first species was also higher in both years. While F. culmorum DNA was very low in 2010, we can conclude that dry weather is affecting to a large extent occurrence of this species (Scherm et al. 2013). In the Netherlands in 2009 incidence and amount of F. culmorum DNA was similarly low as in our study (van der Fels-Klerx et al. 2012). Authors found this species only in 2% of samples and DNA concentration was 80-times lower than for F. graminearum. Tomczak et al. (2002) analyzed Fusarium species causing FHB epidemics in 1998 and 1999 in two regions of Poland. In 1998 in northern and central regions F. avenaceum dominated, being followed by Fusarium graminearum and F. culmorum with similar frequency. In 1999, ranking of species was the same; however, frequency of F. graminearum was 3-5 times higher than F. culmorum. Authors reminds that no F. graminearum was detected in the previous decade (1980's) in wheat grown in Northern Poland. Kuzdraliński et al. (2018) found dominance of F. graminearum in samples of wheat grain from South-Eastern Poland collected in 2013. F. culmorum was fifth species as regards frequency. Wiśniewska et al. (2014) found that F. culmorum was the most common species on strong infected heads of wheat in 2009. They analyzed samples from six locations, and only in two from Southern Poland F. graminearum prevailed over F. culmorum. Iwaniuk et al. (2018) observed variability in F. culmorum and F. graminearum frequency in grain of spring wheat collected in 2017 in North-Eastern Poland. First species dominated in two cultivars and the second in two others. Stępień and Chełkowski (2010) summarized frequencies of Fusarium species infecting wheat heads in Poland from 1985to 2009. In 1985 and Microdochium nivale dominated, F. culmorum being the third species. In 2009, F. graminearum dominated and F. culmorum was the second species with about half frequency of first species. Increase in F. graminearum was obvious; however, differences between years were substantial. F. culmorum predominated in some localities in several studies. It may be explained by the influence of local weather conditions on the frequency of species. Variability of Fusarium species can be high even at the single field level (Xu et al. 2008b) Sexual stage of Fusarium graminearum is Gibberella zeae, which produces sexual spores (ascospores) in perithecia (Desjardins 2003). For F. culmorum perfect stage is not known and fungus produces only asexual spores -macroconidia (Scherm et al. 2013). Thus F. graminearum can disperse and infect host plants with ascospores and macroconidia, whereas F. culmorum only with macroconidia. Nature of F. graminearum is the homothallic which allows the production of large masses of ascospores and effectively compete against F. culmorum (Waalwijk et al. 2003). In a German study, the important contribution of ascospores to inoculum pressure was emphasized (Obst et al. 2002). Ascospores required a relative humidity below 53%, whereas macroconidia required relative humidity of above 80% for germination, as was observed by Beyer et al. (Beyer et al. 2005). It can be another factor favoring F. graminearum over F. culmorum under dry conditions.
Fusarium poae was the most frequently species detected in grain (100% of samples in 2009 and 74% of samples in 2010). In 2010 in 9 samples out of 50 it was the only Fusarium species present. However, amount of F. poae DNA was about 10-times lower than F. graminearum DNA in dry 2010 year and up to 200 times lower in year 2009 of weather favorable for FHB. According to other reports F. poae was frequently isolated from wheat spikes and kernels in Poland (Goliński et al. 1996;Lenc et al. 2015;Kuzdraliński et al. 2017;Iwaniuk et al. 2018). This is a weak pathogen of cereals, however, is widespread on wheat in Europe (Vogelgsang et al. 2008(Vogelgsang et al. , 2019Isebaert et al. 2009;Xu et al. 2003;Lindblad et al. 2013;Polišenská et al. 2021). Vogelgsang et al. (2019) in eight-year survey found similar pattern. The highest frequency of F. graminearum and F. poae in winter wheat, but 3-times higher amount of F. graminearum. Audenaert et al. (2009) observed dominance of F. poae in Flanders in 2007 and in 2008 it was isolated with lower frequency. In 2007, the infection pressure was very high as compared with 2008. The authors suggested that this is because F. poae was a secondary pathogen infecting the weakened heads. Additionally, high frequency of occurrence of F. poae was explained by its sporulation strategy. This species produces very large amounts of microconidia in a dry powdery form that can easily invade cereal heads. It could be true for dry conditions and wind dispersal, because for splash dispersal Hörberg (2002) did not find any difference in patterns between F. poae microconidia and much larger macroconidia of F. culmorum. It is to add that F. poae as a weak pathogen was rarely isolated when only FHB symptomatic wheat kernels were analyzed (Bilska et al. 2018). Xu et al. (2008a) associated F. poae with dry and warm weather conditions, whereas F. graminearum with warm/humid conditions. F. avenaceum and F. culmorum were both associated with niches of cooler/wet/humid conditions. This was confirmed for F. poae by Covarelli et al. (2013) but they observed that in dry season of 2009 F. graminearum was replaced by F. poae and also by F. avenaceum. Parikka et al. (2012)  Results showed that this favored F. poae spread on wheat. Only in the South/South-Eastern Poland weather was warm and humid, and F. graminearum dominated in the grain samples from this region.
Low F. poae DNA in the grain observed in our study could be explained by lower aggressiveness of this species as compared to F. graminearum (Vogelgsang et al. 2008;Stenglein 2009). It was also found that F. poae that predominated in wheat glumes was not detected in grain, which was infected by F. culmorum, F. avenaceum and M. nivale (Doohan et al. 1998). Authors did not detect F. graminearum in wheat samples (collected in England, UK in 1994) which is good example of later Fusarium species shift in Europe. Polley and Turner (Polley and Turner 1995) found that F. poae was associated with distinct glume spot lesions and was the most frequently isolated from glumes. Doohan (1998) supposed that the infection process and colonization by F. poae differs from that of other Fusarium species causing FHB.
Fusarium poae is known as NIV producer (Thrane et al. 2004;Schollenberger et al. 2006). Consequently, we detected NIV in most samples but at very low quantities. In Poland, NIV was found primarily in oats infected by F. poae (Perkowski et al. 1997). Edwards et al. (Edwards et al. 2012) found that correlation of nivalenol concentration Species of the genus Fusarium and Fusarium toxins in the grain of winter and spring wheat in Poland in oat grain and F. poae DNA was highly significant but only accounted for 9% of the variance. It showed that other species such as F. graminearum and F. culmorum were involved in NIV production. NIV chemotypes of these species are not frequent in Poland. Stępień et al. (2008) found that only 12% of F. graminearum isolates in Poland displayed the NIV chemotype.
Besides NIV, F. poae isolates were found to produce wide range of toxins including type A and B trichothecenes, beauvericin, enniatins, moniliformin, and others (Bottalico and Perrone 2002;Thrane et al. 2004;Uhlig et al. 2006;Stenglein 2009;Somma et al. 2010). The surveys of wheat harvested in Poland in 2006 and 2007 as well as in 2013 showed that increased importance of F. poae in the FHB complex in Poland (Kulik and Jestoi 2009;Wolny-Koładka et al. 2015).
In 1994 Norwegian researchers found "powdery F. poae" strains which were the most abundant potential producer of HT-2 and T-2 toxins in cereals (Kosiak et al. 2003). In 1999 these F. poae strains were proved to produce T-2 toxin (Torp and Langseth 1999). Strains originated mainly from Norwegian oats but were found also on wheat in Austria and the Netherlands. Further these strains were described as a new species F. langsethiae by Torp and Nierenberg (2004). The species was being found primarily in Northern Europe on oats and barley Edwards et al. 2012).
Occurrence of F. langsethiae on wheat in Poland was confirmed in 2008 ). This species was found mainly in Northern Poland (including Radostowo mentioned in present study), however it was present in some samples of wheat grain from Central Poland . In 2009 F. langsethiae was found on wheat grain in the Netherlands but at low level (8% of samples) (van der Fels-Klerx et al. 2012). Presence of F. langsethiae was detected by Czaban et al. (2015) in years 2008 -2010 in South-Eastern Poland. Percentages of winter wheat kernels colonized by this species was low. It ranged from 0 to 2.9% in susceptible cultivar 'Kris' in 2010. In our research, we did not detect F. langsethiae in 2009, however limited number of samples was analyzed. In 2010, DNA of this species was found mainly in samples from Northern Poland and in only one from southern region at low concentration.
F. langsethiae and F. poae are favored by dry conditions (Supronienė et al. 2010;Parikka et al. 2012;Czaban et al. 2015), however it seems that the first species prefer lower temperatures than the former. Kokkonen et al. (2012) found that F. langsethiae produced the highest amount of the type A trichothecenes at 15 o C, whereas F. poae could produce beauvericin at both cool and warm conditions. Deoxynivalenol (DON) was the toxin which amount was the highest in the analysed grain samples. In their review, Perkowski et al. (2004) summarized results of several papers on mycotoxins in cereal grain in Poland. Amounts of DON detected in wheat grain were similar to these in present work in 2010, but lower than in 2009. DON concentration in 2009 and 2010 was similar to that detected by Czaban et al. (2015) in four winter wheat cultivars in the same years. Authors found DON mainly in the grain from 2009 and in 2010, DON was present only in small concentrations. In 2017 (moist season) and 2018 (dry season), Bryła et al. (2019) observed similar pattern of DON concentration: low in 2018 and high in 2017. Lindblad et al. (2013) found similar amounts of DON in grain of winter wheat collected in Sweden in 2009 and 2011. We detected higher amounts of DON, especially in 2009. In 2010, it was also higher, however did not exceeded the legislative limit of 1250 μg/kg like in Swedish samples in 2011.
DON accumulation was closely associated with the presence of F. graminearum (Bryła et al. 2015;Lindblad et al. 2013). Coefficient was very high in 2009, because of high DON accumulation and high F. graminearum DNA amount in grain.
In this year DON concentration correlated strongly also with F. culmorum DNA despite its low concentration in the most of samples. In 2010, coefficients were lower and significant only for F. graminearum.
Nivalenol (NIV) accumulation was much lower than DON and amounts was comparable to detected by Bryła et al. (2015) in 2017 and 2018. Its concentration was significantly associated with the presence of F. graminearum and F. poae in 2009. In 2010, coefficients were insignificant but positive for all three possible NIV producers: F. culmorum, F. graminearum and F. poae. Xu et al. (2003) studied wheat grain samples harvested in 2001 from UK, Ireland, Italy, and Hungary. They did not find quantitative relationships between amount of Fusarium DNA and the concentration of the mycotoxins in the grain. However, for total F. graminearum and F. culmorum DNA and DON concentration linear model was nearly significant. In the next survey (Xu et al. 2008a) they studied Fusarium species frequency and mycotoxin content in wheat samples from the same countries over two years (2003)(2004). They found DON being the most frequently detected toxin. DON amount correlated strongly with F. graminearum DNA. NIV was related significantly only to the amount of F. culmorum DNA. As regards ZEN, authors found strong association with both F. culmorum and F. graminearum. In 2005 in Poland, the highest amount of ZEN was found in wheat grain infected by F. graminearum (Gromadzka et al. 2008). In grain were F. culmorum was the main pathogen, ZEN content was 10-times lower. We found higher amounts of ZEN in both 2009 and 2010 comparing with results obtained by Czaban et al. (2015) for the same years (all values below LOD = 10 μg/kg). However, ZEN content was very diverse and high in individual samples (above 100 μg/kg).