J Appl Genetics (2012) 53:227–236 Fig. 1 Most parsimonious tree for the isolates studied, created on the basis of the translation elongation factor 1α (tef-1α) sequences. The tree was obtained using the maximum parsimony approach and tested by bootstrapping (10,000 replicates) with a cut-off value of 50%. Abbreviations used for the origin and host species: IT – Italy, PL – Poland, AR – Argentina, Os – Oryza sativa, Zm – Zea mays, Ea – Equisetum arvense, Ac – Allium cepa, Ch – Chenopodium, Pi – Pinus root, Sl – Solanum lycopersicum, Or – Orobanche ramosa, Tr – Triticum sp., Le – Lycopersicon esculentum, Tc – triticale, Ta – Triticum aestivum 231 The highest toxin content was observed in Italian isolates: ITEM 5104 and ITEM 3190 (34,237 and 2273.2 ng/g re- spectively). The other Fusarium isolates were characterised by much lower abilities of the mycotoxin biosynthesis: 14 isolates produced less than 100 ng/g of the toxin and four isolates’ ZEA content only reached the range of 120– 460 ng/g. Sequence polymorphism in two genes from the zearalenone biosynthetic pathway Based on the sequences of two F. graminearum genes encod- ing polyketide synthases involved in ZEA biosynthesis (PKS4 and PKS13), we designed PCR primers to amplify portions of those genes using genomic DNAs from F. equiseti strains. Eight strains from the Italian population of F. equiseti did not yield any amplification using the PKS4-specific marker, while among the remaining strains (including F. avenaceum ITEM 2753), eight different amplicons were identified (Table 1). Concerning the PKS13 locus, most of the strains displayed the 532-bp allele and two (F. langsethiae KF 2640 and ITEM 6164) amplified a fragment of about 700 bp in length. Eleven strains gave no amplification at all (almost all were from the Italian group). Thus, the PKS4 gene seems to be more poly- morphic than PKS13, especially among the isolates from the Polish population. Sequence analysis of the amplified frag- ments should be performed to reveal the level of this poly- morphism, as some of the amplicons can be non-specific. We sequenced several chosen fragments of the PKS13 sequence to confirm the specificity of designed markers and to analyse their intra-specific diversity, as well as similarities between F. equiseti and F. graminearum. Based on the aligned sequences, a dendrogram was designed (Fig. 4). All the obtained PKS13 sequences were submitted to the NCBI GenBank and are publicly available under accession numbers JF966273–JF966279. Identification of enniatin and trichothecene biosynthetic genes To further examine the toxigenic abilities of the strains studied, we used gene-specific DNA markers to prove or exclude the capability of the strains to produce enniatins (by identifying the esyn1 gene) and trichothecenes (by identifying