Fusarium species and Fusarium mycotoxins in grain of barley in Poland in 2009 and 2010. Short communication

Próby ziarna jęczmienia jarego ze zbiorów w 2009 i 2010r. zostały przeanalizowane pod kątem zawartości DNA gatunków Fusarium i toksyn fuzaryjnych (trichotecenów B). Próbki pochodziły z różnych pól z Radzikowa, w środkowej Polsce. Jakościowe i ilościowe oznaczanie gatunków Fusarium w ziarnie przeprowadzono techniką real-time PCR. Toksyny fuzaryjne w ziarnie analizowano metodą chromatografii gazowej. W ziarnie jęczmienia wykryto siedem gatunków Fusarium. Dominujące gatunki to F. avenaceum, F. graminearum i F. poae. Wykryto również występowanie F. culmorum, F. langsethiae, F. sporotrichioides i F. tricinctum. Stężenie trichotecenów B (deoksyniwalenolu, niwalenolu) w ziarnie było niskie. Najwyższy współczynnik korelacji deoksyniwalenol vs. DNA Fusarium stwierdzono dla F. graminearum. Jeśli chodzi o niwalenol, najwyższy był współczynnik korelacji z DNA F. poae.


Introduction
Fusarium head blight (FHB) is a disease of cereals (including barley) 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. avenaceummoniliformin, enniatins and beauvericin (BEA) producer; F. poae -NIV, BEA producer. F. langsethiae and F. sporotrichioides -T-2 and HT-2 toxin producers, are also prevalent (Bottalico, 1998;Bottalico and Perrone, 2002;Jestoi et al., 2008;Vogelgsang et al., 2008;Somma et al., 2010). Because of the diversity of Fusarium species causing Fusarium head blight, monitoring of changes in the Fusarium population on wheat is important. The frequency of species infecting wheat is not stable and changes depending on the weather in a particular year. Large differences are also observed between different regions of wheat production in Europe. For example, other species are dominant in northeastern Europe, as well as in the southwestern part of the continent (Bottalico, 1998;Bottalico and Perrone, 2002). Species compositions change over time, which is the results of global warming and changes in acreage of major cereal crops, i.e. an increase of maize area.
Barley is less infected by FHB compared to durum wheat or bread wheat (Langevin et al., 2009). However, its grain can also be contaminated with Fusarium toxins (Edwards, 2009;Malachova et al., 2010). Their presence (as well as the presence of Fusarium mycelium) is particularly important for malt barley, as it has a negative impact on beer quality (Havlova et al., 2006;Sarlin et al., 2007).
Data on barley contamination with Fusarium toxins or the frequency of Fusarium species infecting this cereal are much less available than for bread wheat. Hence, it would be interesting to find what the current situation in this field is. The aim of the present study was to determine the presence of Fusarium species and the content BIULETYN IHAR Nr 288 / 2020 Tomasz Góral, Piotr Ochodzki, Linda Kaergaard Nielsen, Dorota Walentyn-Góral of trichothecene type B mycotoxins in barley grain to compare species frequency with earlier reported data.

Material and methods
Five samples of spring barley grain from 2009 (2) and 2010 (3) were analysed. Samples were collected from two cultivars: 'Rufus' and 'Rubinek'. Barley was grown in five commercial fields near Radzików, Central Poland. Barley was harvested using a combine harvester. Ten subsamples weighing 1 kg were taken from the harvested grain and mixed thoroughly. Afterwards, a 1 kg grain sample was taken for further analysis. The collected samples were stored at -20°C before DNA and mycotoxin extraction. Qualitative and quantitative determinations of eight Fusarium species in the grain were performed by real-time PCR. The 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. poae, F. graminearum, F. langsethiae, F. sporotrichioides and F. tricinctum. The detailed methodology of DNA extraction and real-time PCR was described by Góral et. al (2019). The trichothecenes of group B -deoxynivalenol (DON), nivalenol (NIV) were quantified using gas chromatography techniques. The detailed methodology was described by Góral et. al (2019).
The original Fusarium DNA amount and toxin concentrations were transformed to logarithmic values in order to obtain a normal distribution for the variables. The relationships between the results for Fusarium DNA and

Results and discussion
Five samples of grain of spring barley collected from fields in Radzików, Central Poland, were analysed (Tab. 1). All samples contained DNA of Fusarium species at an average value of 11,287 pg of DNA per g of wheat DNA (Tab. 1). The samples from 2010 were more contaminated with Fusarium than the samples from 2009 (17, Seven Fusarium species were detected in the barley grain. Dominating species were F. avenaceum (4,336 pg/g), F. graminearum (3,464 pg/g) and F. poae (2,153 pg/g) (Tab. 1, Fig. 1). These species were found in all samples. Fusarium sporotrichioides was found in four samples at an average DNA concentration of 618 pg/g. Fusarium culmorum was present in three samples, but at a low concentration of 175 pg/g. Fusarium langsethiae was found only in one sample ('Rufus' 10), but was the dominating species in this sample, and the DNA concentration amounted to 2,444 pg/g. Fusarium tricinctum was also found in one sample ('Rubinek' 09) at 266 pg/g.
The concentration of Fusarium DNA in barley grain in 2010 was higher than that in wheat grain in 2010 (Góral et al., 2019). The composition of Fusarium species infecting barley grain was similar to that of wheat, with F. graminearum prevailing over F. culmorum (Tomczak et al., 2002;Stępień and Chełkowski, 2010;Góral et al., 2019). According to Nielsen et al. (2014) avenaceum, F. langsethiae, F. culmorum, F. poae, and F. graminearum, which were found in >85% of the samples (Nielsen et al., 2011). F. tricinctum was found in 67% of the samples, F. sporotrichioides in 15%, and F. equiseti in 2%. In wheat, the most frequent were F. avenaceum, F. graminearum and F. culmorum. Species composition in the above three countries seems to be similar. More species were involved in Fusarium head blight in barley than in wheat. Several species were also found in barley grain in northern USA (Salas et al., 1999). However, other than in Europe, Fusarium graminearum was the primary pathogen causing FHB epidemics and comprised from 62% to 64% of all Fusarium species isolated from infected kernels from 1994 to 1996. The authors also isolated F. sporotrichioides, F. poae, and F. avenaceum and stated that these species were involved in FHB infection, but to a limited extent. The above results show the effect of climatic conditions between northern Europe and the continental USA on Fusarium species in barley.
Fusarium langsethiae was found primarily in northern Europe on oat and barley (Yli-Mattila BIULETYN IHAR Nr 288 / 2020 Tomasz Góral, Piotr Ochodzki, Linda Kaergaard Nielsen, Dorota Walentyn-Góral et al., 2008;Edwards et al., 2012). The occurrence of F. langsethiae on wheat in Poland was confirmed in 2008 (Łukanowski et al., 2008). This species was found mainly in northern Poland; 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 a low level (8% of the samples) (van der Fels-Klerx et al., 2012). Czaban et al. (2015) detected the presence of F. langsethiae in the years 2008-2010 in south-eastern Poland. However, this is the first report on the presence of F. langsethiae on barley in Poland. The concentration of trichothecene toxins (DON, NIV) was low (Tab. 1) and was similar to that detected in naturally infected barley grain samples in the United Kingdom in -2005(Edwards, 2009-2011(Nielsen et al. 2014. Edwards (2010) found only one sample, which exceeded the legal limit for DON. Mycotoxin levels were also similar to that detected in barley in Poland in 1997 (Perkowski et al., 2003) and in the Czech Republic in he years 2001 and 2005 (Hajslova et al., 2007), but higher than that detected in the years 2005(Malachova et al., 2010.
The highest amount of DON was found in the sample 'Rubinek 10', which was the most Fusarium contaminated sample. In addition, this sample contained the highest amount of NIV and F. poae DNA, which is a producer of NIV (Stenglein, 2009