J Appl Genetics (2012) 53:227–236 233 Fig. 4 Most parsimonious tree for six chosen isolates based on the sequences of the polyketide synthase (PKS13) gene from the ZEA metabolic pathway. The tree was obtained using the maximum parsi- mony approach and tested by bootstrapping (10,000 replicates) with a cut-off value of 70%. The F. graminearum KF 371 strain was used as the reference. Abbreviations used for the origin and host species: IT – Italy, PL – Poland, Or – Orobanche ramosa, Tc – triticale, Ta – Triticum aestivum polymorphism, which can explain our lack of amplification and identification problems. However, after analysing the tef-1α sequence, it was proven that eight of nine isolates still represent F. equiseti, despite the lack of the amplification of the FeF/FeR marker. Similarly, the analysis of tef-1α has proven a considerably high level of polymorphism, as it was already postulated before for other members of the Fusa- rium genus (Kristensen et al. 2005; Stępień et al. 2011b). The only exception was ITEM 6164 strain. We only were able to amplify a fragment of the tef-1α gene using primers designed for the Clonostachys genus and amplifying a re- gion adjacent to the studied one (results not shown). It is rather unusual concerning the considerable level of conserva- tion present inside this gene (Geiser et al. 2004; Kristensen et al. 2005). The latest findings suggest that sequences which are unique at a species (or group of species) level (e.g. mycotoxin biosynthetic genes) serve as better targets for designing species-specific markers (Alexander et al. Fig. 5 Most parsimonious tree for the 14 strains tested, based on the trichodiene synthase (TRI5) gene sequences. The maximum parsimony approach was used and tested by bootstrapping (10,000 replicates) with a cut-off value of 50%. Two strains of F. graminearum (DON chemotype, KF 1413, and NIV chemotype, KF 371) were used as references. Abbreviations used for origin and host species: IT – Italy, PL – Poland, Os – Oryza sativa, Zm – Zea mays, Or – Orobanche ramosa, Le – Lycopersicon esculentum, Tc – triticale, Ta – Triticum aestivum 2011; Fernández-Ortuño et al. 2010; Niessen et al. 2004; Proctor et al. 2009; Stępień et al. 2011a). Based on the obtained tef-1α sequences, an alignment was prepared and the dendrogram of similarities was calculated (Fig. 1). Concerning only the country of origin, groups of isolates are dispersed, although a group of seven Polish strains from soil, L. esculentum and Z. mays is distinguishable: KF 2652, KF 2653, KF 2654, KF 2655, KF 24, KF 1011 and KF 1017. What is even more interesting, a group of four isolates (KF 72, KF 89, KF 2650 and KF 2651) is clearly sharing a rather uncommon variant of the gene, regardless of the origin (two are IT and the other two PL, see Fig. 1). A high level of diversity among F. equiseti strains is not surprising, as it was already shown that the tef-1α gene displays a considerable level of polymorphism among the genotypes of this species (Punja et al. 2008). Also, using different techniques, like restriction fragment length polymorphism (RFLP) and thin layer chromatography (TLC) of the synthesised metabolites, it was possible to differentiate the isolates of common geo- graphical origin (Kosiak et al. 2005). Another remarkable observation is a similarity between the strain ITEM 2753, which was confirmed as F. avenaceum, and ITEM 5104. The most likely explanation for this is the misidentification of the strain. However, concerning morphological traits typi- cal for F. equiseti, as well as the presence of the FeF/FeR and PKS4 gene markers (Table 1) and the high level of the in vitro ZEA biosynthesis (Fig. 3), it is also possible that ITEM 5104 strain represents the “off-type” F. equiseti genotype. As far as growth speed analysis is concerned, the majority of Polish F. equiseti strains grew slightly faster than the Italian ones (Fig. 2). There were two exceptions, however: the strains ITEM 3699 and ITEM 6164 were the fastest growing from the IT population. It seems likely that populations originating