Fusarium Species in the Grain of Winter Wheat in Poland in 2014–2017 and 2019

1. Introduction

Fusarium head blight (FHB), also known as scab, is a severe wheat disease (Triticum aestivum) in Europe and other regions caused by fungi of the genus Fusarium [1]. Several species of Fusarium can cause FHB, including Fusarium graminearum and Fusarium culmorum. These fungi infect the developing wheat spikes, causing the grain to become discolored and shriveled, thereby reducing the yield and quality of the crop. The presence of mycotoxins produced by Fusarium species in infected grains can also pose a risk to human and animal health. The two species mentioned above are identified as producers of deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN). Other Fusarium species that cause FHB include F. avenaceum, a producer of moniliformin, enniatins, and beauvericin (BEA); F. poae, a producer of diacetoxyscirpenol (DAS), NIV, and BEA; F. langsethiae and F. sporotrichioides, producers of T-2 toxin, HT-2 toxin, and DAS, also occur [2,3,4,5,6,7]. Among these species, Fusarium graminearum and F. culmorum are highly pathogenic to wheat, causing severe epidemics of FHB. The other species are moderately or weakly pathogenic; however, they may also contaminate wheat grain with mycotoxins [8,9,10,11].

Currently, F. graminearum is considered the most important species causing FHB in Europe and other regions, as it is the most virulent and capable of producing high levels of mycotoxins. Phylogenetic studies of the F. graminearum strain collection revealed that F. graminearum is a species complex comprising 11 distinct species [12,13,14]. The most common species worldwide within the F. graminearum species complex (FGSC) is F. graminearum sensu stricto. In Europe, F. graminearum s.s. was isolated almost exclusively [15,16,17]. Only single isolates of other species (F. boothi, F. cortaderiae) were found, as shown in the analysis of the influence of climate on the distribution of FGSC species by Backhouse [18] and in the bibliographic analysis by Del Ponte et al. [19].

The composition of Fusarium species infecting wheat varies depending on the climate of the wheat-growing region. In Europe, other species are more common in the Northeast, where F. avenaceum, F. culmorum, and F. graminearum s.s. each make up about the same share, compared to the Southwest of the continent, where F. graminearum s.s. is mostly found [20,21].

The composition of the species changes with time, which is the result of climate warming and changes in the acreage of the main cereal crops, such as an increase in the area of maize [22,23,24,25]. The primary reported effect of these factors is the increase in the frequency of F. graminearum and the decrease in F. culmorum [26,27,28]. Climate warming could favor F. graminearum s.s., as it has a higher optimal temperature for development than F. culmorum. F. graminearum s.s., unlike F. culmorum, is a significant pathogen on maize and can survive on maize debris [24,29]. It is worth noting that in Poland, the area used for maize cultivation has expanded significantly, from 59,000 hectares in 1990 to 1,814,974 hectares in 2024 (https://kukurydza.info.pl/powierzchnia-uprawy-kukurydzy-w-polsce-w-2024-r/).

The frequencies of Fusarium species infecting wheat heads in Poland from 1985 to 2009 were summarized by Stępień and Chełkowski [30]. In 1985, F. avenaceum and Microdochium nivale were the dominant species, with F. culmorum as the third most common. By 2009, F. graminearum became dominant, and F. culmorum ranked second, with its frequency about half that of F. graminearum. The rise of F. graminearum was evident; however, the differences between certain years were statistically significant. According to various studies conducted in Poland after 2010, F. graminearum is now the most prevalent species of Fusarium [31,32,33]. A higher frequency of F. culmorum was observed only in some studies [34,35]. Other species commonly detected include F. avenaceum and F. poae [33,36].

There has been a recent increase in FHB cases in Poland, raising concerns among farmers and researchers about its impact on food safety and security [37,38,39]. This disease presents a major risk in areas with high humidity and rainfall, as these conditions foster the growth and spread of Fusarium fungi. [33,36,40,41]. In Poland, farmers and researchers use various strategies to reduce the impact of FHB. These include planting resistant cultivars, adopting integrated disease management practices, and developing new technologies to detect and lower mycotoxin contamination [42,43]. However, FHB still presents a significant challenge for the Polish cereal crop industry, which must meet the stricter limits on Fusarium toxin levels in cereal grain coming into effect in 2024 [44]. Ongoing research and development efforts are essential to address this problem.

The study aimed to evaluate the diversity of Fusarium species infecting wheat kernels across various regions of Poland from 2014 to 2019.

2. Materials and Methods

2.1. Wheat Grain Samples

Wheat grain samples were collected during the harvest seasons of 2014–2017 and 2019. They came from 26 experimental stations of COBORU (the Research Centre for Cultivar Testing; https://coboru.gov.pl/), situated in different regions of Poland (Figure 1). Two winter wheat cultivars, moderately susceptible to FHB—‘Arkadia’ and ‘Bamberka’ (which was replaced in 2017 by ‘Hondia,’ a moderately resistant variety to FHB)—were included [45]. According to COBORU data (https://coboru.gov.pl/), these three cultivars were among the most widely grown in Poland during the study years. Winter wheat was cultivated in 10 m² plots with moderate nitrogen input (average 90 kg/ha of N) and no chemical control of diseases, except for seed treatment with fungicides. Herbicides and insecticides were applied as needed. The grain was harvested with a combine harvester.

The number of samples collected and analyzed varied from year to year.

2.2. Sample Preparation

Pure isolates from five Fusarium species (F. culmorum, F. avenaceum, F. graminearum s.s., F. poae, and F. langsethiae) were cultivated on PDA medium. The surface of the PDA in the Petri dishes was covered with sterilized cellophane discs. Mycelial cultures were incubated in darkness at 22°C for one week. Between 1 and 2 grams of mycelium from each species were harvested for DNA extraction. The samples were ground in liquid nitrogen using a mortar and pestle.

Wheat grain samples (300 g) were finely ground using Ultra Centrifugal Mill ZM 300 (Retsch Polska, Verder Polska Sp. z o.o., Katowice, Poland). Next, 5 g of ground grain was powdered in liquid N₂ using Geno/Grinder 2000 (OPS Diagnostics, Bridgewater, NJ). Two replicates (200 mg) of subsamples were taken from each powdered sample for DNA isolation.

2.3. DNA Isolation

DNA extraction from pure mycelia and plant samples was conducted using a CTAB method. 750 µl of CTAB extraction buffer (20 g/l CTAB, 1.4 M NaCl, 0.1 M Tris-HCl, 20 mM Na₂EDTA) with 20 µl of RNase A (10 mg/ml) was added to each subsample. Buffer volumes, together with the volumes of chemicals used in subsequent steps, were increased proportionally, if needed. Samples were incubated at 65°C for 15 min. Next, 20 µl of Proteinase K (20 mg/ml) was added, and samples were incubated at 65°C for 60 min. Subsequently, samples were centrifuged for 10 min at 16000 x g. The supernatant was transferred into a new tube, and 500 µl of chloroform was added. Then, samples were shaken for 30 s and centrifuged for 10 min at 16000 x g. The upper layer was transferred to a new tube and mixed with another 500 µl of chloroform. After 5 min of centrifugation at 16000 x g, the upper layer was combined with two volumes of CTAB precipitation solution (5 g/l CTAB, 0.04 M NaCl). The mix was incubated for 60 min at room temperature and centrifuged for 5 min at 16000 x g. After removing the supernatant, the pellet was dissolved in 350 µl of 1.2 M NaCl. Next, 350 µl of chloroform was added, and the mix was shaken for 30 s. After 10 min centrifugation at 16000 x g, the upper phase was transferred to a new tube and mixed with 180 µl of isopropanol. The sample was centrifuged for 10 min at 16000 x g. The supernatant was removed, and the pellet was washed in 500 µl of 70% ethanol and centrifuged for 10 min at 16000 x g. The DNA pellet was dried and then dissolved in 100 µL of deionized, sterile water. DNA concentration was measured using the Nanophotometer Pearl (Implen GmbH, Munich, Germany).

2.4. Real-Time PCR

Real-time PCR analyses with SYBR Green intercalating dye were performed on a 7500 Fast instrument (Life Technologies). Primer sequences developed by Nicolaisen et al. [46] are listed in Table 1. The authors state that F. graminearum primers (Fgram F, Fgram R) detect all species in the F. graminearum species complex. Exceptions are two species from South America—F. cortaderiae and F. brasillicum.

PCR reactions were conducted in a 12.5 µl total volume. Standard curves for Fusarium species were prepared using subsequent dilutions of DNA derived from pure cultures: 0.001, 0.01, 0.1, 1, and 10 ng/µL. The standard curve for the EF1α reference gene was prepared from wheat DNA in concentrations of 0.5, 1, 2, 5, and 10 ng/µl. Samples for the real-time PCR reaction were diluted to 5 ng/µl. The PCR mix consisted of 1x SYBR Green PCR Mastermix (Life Technologies), 250 nM primer F and R (Sigma), and 0.5 µg/µl BSA (Thermo Scientific). The temperature profile of the PCR reaction was as follows:50°C, 2 min; 95°C, 10 min; (95°C, 15 s; 62°C, 1 min) x 40. For the EF1α gene, the annealing temperature was 60°C. Following the PCR reaction, melt curve analysis was conducted in the temperature range of 60-95°C to check the specificity of the obtained products.

The concentration of each Fusarium species was measured using a standard curve. The results are expressed as picograms of fungal DNA per microgram of plant DNA, following Nicolaisen et al. [46].

2.5. Statistical Analysis

Statistical analysis was performed using Microsoft® Excel 2016 and XLSTAT© (Version 2020.4.1.1027, Addinsoft, Paris, France) software.

The relationships between DNA levels of five Fusarium species were analysed using Pearson’s correlation tests (XLSTAT procedure: Correlation tests). Before analysis, all variables that were not normally distributed were log10-transformed.

The data on DNA levels of five Fusarium species in 111 wheat grain samples, collected over five years from 26 locations, were then analysed using multivariate statistical methods. Principal component analysis (XLSTAT procedure: Principal Component Analysis PCA) was used to demonstrate how grain samples from different locations and years are distributed in relation to the variation explained by the first two principal components and how DNA levels of Fusarium species influence these components. PCA results also reveal relationships among variables based on the angles between variable vectors.

To identify a potential link between Fusarium DNA levels in grain and the summarized weather variables across 92 data windows, Spearman’s rank correlation coefficient was employed. Weather summaries with a significant Spearman correlation (p < 0.01) were identified as key weather factors that could influence Fusarium colonization of wheat grains. A phenological stage was assigned to these significant weather summaries.

3. Results

3.1. Weather Conditions

The weather conditions at the 26 grain sampling locations varied (Figure 2). The average temperature in May fluctuated over the five years, ranging from 10.2°C to 16.3°C. The most considerable temperature variation was recorded in 2019, while the smallest was in 2014. In June, the average temperature fluctuated between 13.9°C and 22.8°C over the five years. The most tremendous variation was observed in 2017, with the least in 2014. July’s average temperature ranged from 15.9°C to 22.0°C during these years, with the highest variation in 2015 and the lowest in 2014.

When comparing the five years, the lowest average temperature in May was recorded in 2019 and 2015, while the highest was in 2016. We noticed a slow decrease in the average temperature starting from 2016. In June, the lowest average temperature was recorded in 2014 and the highest in 2019. The average temperature in June increased each year from 2014 and was particularly high in 2019. The lowest average temperature in July was recorded in 2017 and 2019, with the highest in 2014. We observed a gradual decline in the average temperature in this month from 2014 onward.

Regarding precipitation, the monthly total rainfall in May ranged from 40.7 to 91.8 mm over five years. The greatest variation occurred in 2019 and 2014, while the least was in 2016. In June, the annual total rainfall ranged from 36.6 to 76.4 mm across five years. The largest variation was in 2019, and the smallest in 2015. In July, the total rainfall over five years varied from 45.9 to 141.2 mm. The highest variation was in 2016, and the lowest was in 2015.

Over five years, the lowest precipitation in May was observed in 2016 and 2017, while the highest occurred in 2014 and 2019. We saw a gradual decline in rainfall from 2014 to 2016, followed by an increase until 2019. In June, the lowest precipitation was recorded in 2019 and 2015, and the highest in 2017. The June rainfall was fairly consistent across the five years. The lowest precipitation in July was recorded in 2019, and the highest in 2016. We observed a gradual rise in rainfall in July from 2014 to 2016, followed by a subsequent decrease until 2019.

3.2. Fusarium Species

DNA from five Fusarium species was detected in most samples over a five-year period. Trace amounts of Fusarium DNA were detected in 40% of samples in 2014, 35% in 2015, 36% in 2016, 8% in 2017, and 28% in 2019. The highest total of Fusarium DNA was identified in 2017, and it was notably higher than in other years (Figure 3).

In 2014, F. graminearum s.s. dominated, and the amount of DNA from this species was significantly higher than that of the other species. Only traces of F. avenaceum DNA were detected. In 2015, F. poae was the dominant species, and its DNA amount was greater than that of the others. DNA from F. avenaceum and F. culmorum was detected only in trace amounts. In 2016, the DNA levels of the five species did not differ significantly; however, the highest amounts were seen for F. avenaceum and F. graminearum s.s., while F. langsethiae had the lowest amount. In 2017, F. graminearum s.s. again dominated, and its DNA amount was significantly higher than that of the other species. In 2019, the DNA levels of the five species did not differ significantly, with F. avenaceum having the lowest amount. No DNA from F. culmorum was detected.

The highest concentrations of total Fusarium DNA during the five-year study were found in southeastern Poland, particularly in the Podkarpackie province, as well as in the northwestern regions, including Zachodniopomorskie and Pomorskie provinces (Figure 4). Elevated levels of Fusarium DNA were also detected in wheat grain from the southern Opolskie province. Additionally, the Małopolskie province in the south showed higher concentrations of Fusarium DNA compared to other regions. The lowest levels were observed in Kujawsko-Pomorskie (north-central Poland), Podlaskie (eastern Poland), and Dolnośląskie (southwestern Poland).

Over five years of experimentation, the regional distribution of Fusarium DNA showed significant variation (Figure S1). In 2014, Fusarium DNA was mainly found in grain samples from southeastern Poland (Podkarpackie, Lubelskie, and Małopolskie provinces), with particularly high levels in Podkarpackie (2804 pg/μg). In 2015, a large amount of Fusarium DNA was detected in samples from southern Poland, especially in Małopolskie. Unlike in 2014, high concentrations also appeared in samples from Mazowieckie. In 2016, Fusarium DNA was primarily found in samples from northwestern Poland (Zachodniopomorskie), with a concentration of 1757 pg/μg, while other regions showed lower levels. An exception was Śląskie in southern Poland, which exceeded the average for 2016 at 501 pg/mg (Figure 3). In 2017, the highest Fusarium DNA levels were in samples from northwest and north Poland (Zachodniopomorskie, Pomorskie, and Warmińsko-Mazurskie). Levels were also high in parts of central Poland (Wielkopolskie and Łódzkie) and southern Poland (Opolskie). Unfortunately, samples from Podkarpackie were not available this year. In 2019, Fusarium DNA was mainly found in grain samples from northern (Pomorskie and Warmińsko-Mazurskie) and southeastern Poland (Podkarpackie, Małopolskie, and Świętokrzyskie), with high levels also in western Poland (Lubuskie).

We analyzed the frequency of Fusarium species in Polish regions over five years (Figure S2). DNA from F. aveanceum was found in significant amounts only in two northern regions—the Zachodniopomorskie and Pomorskie provinces. In other regions, it was present in very low or trace amounts. The DNA concentration of F. culmorum in wheat grain was generally low, mainly detected in samples from the northwestern Zachodniopomorskie province and the southern Śląskie province. Only traces of F. culmorum DNA were found in most parts of eastern Poland, as well as in the south and west. DNA from F. graminearum s.s. was detected in the highest amounts, mostly in southeastern and southern Poland (particularly in the Podkarpackie province), as well as in northern and northwestern Poland (mainly Pomorskie). It was also present in central and west-central regions. Only trace levels of this species’ DNA were found in the Mazowieckie and Lubuskie provinces. The presence of F. langsethiae DNA was confirmed in significant quantities in two southern regions (Opolskie and Podkarpackie) and one in western Poland (Lubuskie). In five of the sixteen provinces, F. langsethiae was either not detected or found only in trace amounts. The DNA of F. poae was detected in most provinces, with the highest concentrations in the southern Małopolskie province and the east-central Mazowieckie province. Only low or trace amounts of this species’ DNA were observed in two regions.

Table 2. Pearson correlation matrix of DNA concentrations from five Fusarium species.

Variables (n=111)	F. avenaceum	F. culmorum	F. graminearum s.s.	F. langsethiae
F. culmorum p-value	0.469 0.000
F. graminearum s.s. p-value	0.246 0.009	0.249 0.008
F. langsethiae p-value	-0.012 0.899	0.055 0.567	0.067 0.488
F. poae p-value	-0.050 0.599	0.037 0.703	0.031 0.745	-0.037 0.0698

Table 2. Pearson correlation matrix of DNA concentrations from five Fusarium species.

Variables (n=111)	F. avenaceum	F. culmorum	F. graminearum s.s.	F. langsethiae
F. culmorum p-value	0.469 0.000
F. graminearum s.s. p-value	0.246 0.009	0.249 0.008
F. langsethiae p-value	-0.012 0.899	0.055 0.567	0.067 0.488
F. poae p-value	-0.050 0.599	0.037 0.703	0.031 0.745	-0.037 0.0698

The DNA amounts of three Fusarium species—F. avenaceum, F. culmorum, and F. graminearum s.s.—were significantly correlated. The highest correlation was observed between F. avenaceum and F. culmorum. In samples with high levels of Fusarium DNA, these two species were often found together. The correlation coefficients between F. graminearum and either F. avenaceum or F. culmorum were significant but lower. F. graminearum s.s. was present in fewer samples, along with one of these species, in higher amounts. However, some grain samples were infected equally by all three species. No correlation was found between the DNA of F. langsethiae or F. poae and that of the other species.

The relationships between species were further analyzed using Principal Component Analysis (PCA). A strong connection was found among the DNA concentrations of F. avenaceum, F. culmorum, and F. graminearum s.s. (Figure 5). All three species showed high positive loadings on factor F1. These species were often detected together in the same grain samples. High DNA levels were mainly observed in samples from 2017 and 2016, with some notable amounts also found in samples from 2014 and 2019. No significant link was observed between these species and F. langsethiae or F. poae. For factor F2, the loadings were highly positive for F. poae and highly negative for F. langsethiae. F. langsethiae DNA was only detected in larger amounts in certain samples, mainly from 2016, 2017, and 2019. F. poae was consistently found in many samples across all years, but in similar moderate quantities. The loadings for factor F3 showed that in some samples—mainly from 2019—both species were present in significant amounts (Figure S3). Again, there was no relationship between these species and F. avenaceum, F. culmorum, or F. graminearum s.s.

Factor loadings for factor F4 were strongly positive for F. graminearum and slightly negative for the other species (Figure S4). This indicates the dominance of F. graminearum as a key pathogen in the FHB complex of wheat.

The relationships between two weather variables (Tmean, PREC), estimated over 14-day windows during the growing season, and the total Fusarium DNA content in winter wheat grain at harvest were analyzed using Spearman’s rank correlation coefficient (Figure 6). A positive correlation was observed between DNA content and precipitation during flowering (BBCH 61-69) and ripening (BBCH 87-89). Conversely, Tmean during stem elongation (BBCH 30-39) and milk-dough (BBCH 75-83) development was negatively correlated with DNA content in the harvested grain. Tmean was positively correlated with DNA content at booting-heading (BCH 45-51).

4. Discussion

This study highlights the significant effect of weather conditions on the presence and distribution of Fusarium species in winter wheat grain. The annual fluctuations in average temperatures during May, June, and July—particularly the extreme differences in 2019 and 2014—and the more stable conditions in 2017 show that temperature is a key factor in Fusarium development. This is further supported by the negative correlation between temperature during the stem elongation and milk-dough stages and Fusarium DNA content. Previous research has also highlighted the role of temperature in influencing the dynamics of Fusarium species [47,48,49].

Precipitation levels showed significant yearly variation, with notable differences between the dry 2015 and the wet years of 2016 and 2017. The positive correlation between Fusarium DNA content and precipitation during the flowering and ripening stages indicates that increased moisture may encourage the growth and spread of Fusarium species. This aligns with previous research highlighting the importance of wet conditions in Fusarium development [50,51,52].

The detection of Fusarium DNA in most samples collected in the late 2010s, with the highest concentrations in southeastern and northwestern Poland, indicates that regional climate factors influence the prevalence of Fusarium. We found that F. graminearum s.s. was the primary cause of FHB. The proportion of the previously dominant species, F. culmorum, decreased significantly. It was found in higher amounts only in the cooler northwestern region. Duba et al. [53], investigating wheat samples from northeastern and southeastern Poland (collected in 2104 and 2015), found F. culmorum dominance only in grain from the cooler northern region.

Several studies examining changes in Fusarium populations, mainly in Western Europe, have shown similar trends. According to Birzele et al. [54] in 1997 and 1998, the dominant species in wheat grain in Germany were F. avenaceum, F. poae, F. culmorum, and F. graminearum. The frequencies of the last two species were similar; however, the percentage of F. graminearum increased in 1998. Waalwijk et al. [55] analyzed wheat heads and grain collected in the Netherlands in 2001 and 2002. In 2001, samples collected during the late milk stage mainly contained F. graminearum; however, some samples also had F. avenaceum and/or F. culmorum. At harvest, F. graminearum was almost exclusively dominant. In 2002, weather conditions were more favorable for FHB, leading to a relative dominance of F. graminearum in grain from the Netherlands and nearly complete dominance in samples from France. Isebaert et al. [56] observed that F. graminearum and F. culmorum were the most prevalent species in Northern Belgium from 2002 to 2005. They found an interesting correlation between crops and the frequency of both species (maize – F. graminearum, small grain cereals – F. culmorum). In Luxembourg, the most common species found on wheat heads in 2008 were F. graminearum, F. avenaceum, and F. poae. An increase in the frequency of F. graminearum and a decrease in F. culmorum were observed [57]. Chandelier et al. [58] analyzed winter wheat samples from Belgium over the 2003-2009 period. They found that the main species were F. avenaceum and F. graminearum; however, their frequency varied depending on the year, ranging from 20% to 100%. The frequency of F. poae remained relatively constant over the years (about 70%). The overall incidence of F. culmorum decreased during the study, from 80% in 2003 to 10% in the final three years. In Germany in 2008, F. graminearum s.s. was the main species, followed by F. culmorum. Other species (F. poae, F. tricinctum, M. nivale, etc.) were found in small quantities [59]. In Hungary, in 2010, which was a very favorable year for FHB development, F. graminearum was primarily isolated from wheat grain [60]. Winter wheat grown in the Netherlands in 2009 was examined for the presence of Fusarium species and toxins [61]. In samples collected at harvest, the authors observed a dominance of F. graminearum. F. avenaceum and Microdochium nivale were also common. However, in the preharvest samples, only F. graminearum and M. nivale were found.

Fusarium poae was the most commonly detected species in grain. It appeared in 98% of samples, compared to 91% for F. graminearum. The amount of F. poae DNA was approximately three times lower than that of F. graminearum over a five-year period. However, during dry years like 2015, F. poae dominated, with its DNA levels being eight times higher than F. graminearum. In similarly dry conditions in 2019, the DNA amounts for both species were similar.

According to published studies, F. poae was frequently isolated from wheat spikes and kernels in Poland in 1993, as well as in 2006-2007 [62,63]. It is a weak pathogen of cereal spikes; however, it is widespread on wheat across Europe [7,56,64,65]. In 2001 and 2002, Xu et al. [64] discovered that F. poae was the most common species infecting grain at harvest in three studied countries (UK, Italy, Hungary). Only in cooler Ireland was F. poae less frequent. Audenaert et al. [66] observed that this species was dominant in Flanders in 2007, although it was isolated less frequently in 2008. The higher infection pressure in 2007 compared to 2008 was attributed to F. poae’s nature as a secondary pathogen. Additionally, the frequent occurrence of F. poae is connected to its sporulation strategy. This species produces large amounts of tiny microconidia in a dry, powdery form that can easily invade cereal heads. This process may be especially effective under dry conditions and wind dispersal, as Hörberg [67] found no difference between the dispersal patterns of F. poae microconidia and the much larger macroconidia of F. culmorum during splash dispersal.

Xu et al. [68] linked F. poae with relatively dry and warm conditions, while F. graminearum was associated with warm and humid environments. F. avenaceum and F. culmorum were both linked to cooler, wet, and humid environments. Parikka et al. [69], who predicted an increase in the importance of F. poae (along with F. langsethiae) under drier conditions in Scandinavia, also confirmed this.

The strong correlation among F. avenaceum, F. culmorum, and F. graminearum s.s. suggests they may share ecological niches or respond to similar environmental triggers. The PCA analysis supports these findings, indicating that these species frequently coexist, especially in samples from 2017 and 2016. However, the lack of correlation with F. langsethiae or F. poae suggests that these species may respond to different environmental conditions. Xu et al. [64] stated that when weather conditions are unfavorable (e.g., drought) for F. graminearum, it can be replaced by secondary FHB species, such as F. poae, which was the most common. The negative correlation between temperature during the stem elongation and milk-dough stages and Fusarium DNA content suggests that higher temperatures at these stages may not promote Fusarium infection, possibly due to faster plant growth or changes in plant susceptibility. Conversely, the positive correlation during booting and heading suggests that the temperature at this stage could lead to increased infection rates.

Overall, this study emphasizes the complex interactions between weather conditions and the presence of Fusarium species in winter wheat grain. Understanding these relationships is essential for developing effective management strategies to reduce the impact of Fusarium on wheat production. The composition of FHB-causing Fusarium species is key to understanding toxin profiles in wheat grain. Future research should focus on exploring how temperature and precipitation specifically influence Fusarium growth and identifying potential methods to lower Fusarium contamination in wheat.