228 cereal grain (Gromadzka et al. 2008). Besides F. equiseti, ZEA is produced by other Fusarium species, including the most important pathogens of maize and wheat, i.e. F. cul- morum and F. graminearum. Also, glycosylated derivatives of ZEA were identified both in wheat and in maize grain. They can be regarded as potentially hazardous for human and animal health, as they can be converted to ZEA in the digestive tract (Berthiller et al. 2009; Sulyok et al. 2006). It can be accumulated in cereal grain at high levels, reaching several μg/g, and transferred with infected grain to cereal products, such as animal feed (Gromadzka et al. 2008). Isolates of F. equiseti species are highly variable in a number of morphological traits, including the size of apical cells, conidia shape, aerial mycelium texture, growth rate on standard media, pigmentation and sporodochia appearance (Kosiak et al. 2005). Also, a great diversity has been ob- served in metabolites composition and amounts among nat- ural F. equiseti populations, but ZEA, FUSCHR and FUS-X are the most commonly synthesised. To identify and assess the genetic diversity of the natural populations of pathogens, numerous molecular assays have been developed and applied. As far as the Fusarium genus is concerned, analysis of the highly polymorphic intron regions like the translation elongation factor 1α (tef-1α) appears as the most useful and versatile tool, as it allows the discrimination of the genotypes even on the sub-specific level (Kristensen et al. 2005; Stępień et al. 2011a). Therefore, a similar approach was used in this study in order to evaluate the variability of F. equiseti isolates from two populations (originating from Italy and Poland, plus an additional strain from Argentina), based on the partial sequences of the tef-1α. Additionally, we analysed the poly- morphism present in the coding regions of the two genes from the ZEA biosynthetic pathway (PKS13 and PKS4) and inside the TRI5 gene, which encodes a key enzyme of the trichothecene biosynthesis pathway—trichodiene synthase. We have examined the level of ZEA biosynthesis in rice cultures and the ability of all isolates to produce enniatins/ beauvericin and trichothecenes (from A and B groups) by the identification of the essential metabolic pathway genes esyn1, TRI13 and TRI4 using gene- and allele-specific polymerase chain reaction (PCR) assays. Materials and methods Fusarium strains and media Twenty-seven Fusarium strains were included in the study. Twenty-five of them represented two distinct populations of F. equiseti (originating from Italy and Poland). One strain originating from Argentina (ITEM 4323) was also analysed as a representative genotype from a distinct population. J Appl Genetics (2012) 53:227–236 Additionally, one F. langsethiae strain (KF 2640) was in- cluded in the study (Table 1). For the sequence analyses of TRI5 and PKS13 genes diversity, two strains of F. grami- nearum (KF 371 representing the nivalenol chemotype and KF 1413 representing the deoxynivalenol chemotype) were used as the outgroup. All isolates were identified using dedicated species-specific PCR-based markers (Table 2). Also, partial sequences of the tef-1α were amplified for molecular species identification and phylogenetic analysis. The providers of all strains were two culture collections: the KF Fusarium strain collection at the Institute of Plant Ge- netics, Polish Academy of Sciences, Poznań, Poland, and the ITEM collection at the Institute of Sciences of Food Production (ISPA-CNR), Bari, Italy. Growth rates The strains were examined for growth speed by plating on potato dextrose agar (PDA) medium. The growth speed of 25 isolates described as F. equiseti was mea- sured as the colony diameter on 90-mm Petri plates with solid PDA medium (Oxoid, Basingstoke, Hamp- shire, UK) after 4 days from inoculation. The cultures were grown in the incubator set at room temperature (20°C) with a 12-h photoperiod. The average was calculated from two replicates. DNA extraction, PCR primers, cycling profiles and DNA sequencing Genomic DNA of all isolates was extracted using a hexade- cyltrimethylammonium bromide (CTAB) method described in detail previously (Stępień et al. 2008). All primers used for the PCR-based identification of mycotoxin biosynthetic genes, along with references or accession numbers of sequences used for their design, are presented in Table 2. PCRs were done in 25-μl aliquots using PTC-200 and C- 1000 thermal cyclers (Bio-Rad, Hercules, CA, USA). Each reaction tube contained 1 unit of Platinum Hot- Start Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 2.5 μl of 10× PCR buffer, 12.5 pmol of forward/ reverse primers, 2.5 mM of each dNTP and about 10– 20 ng of fungal DNA. The PCR conditions were as follows: 15 min at 95°C, 35 cycles of (30–60 s at 94°C, 30– 60 s at 58-63°C, 1–2 min at 72°C) and 10 min at 72°C. Amplicons were electrophoresed in 1.5% agarose gels (Invitrogen) with ethidium bromide. For sequence analysis, PCR-amplified DNA fragments were purified with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following programme: 30 min at 37°C and 15 min at 80°C. Both strands were labelled using the BigDye Terminator 3.1 kit (Applied Biosystems, Foster