230 Table 2 Primers used in this study, expected amplicon sizes, references and GenBank acces- sion numbers of the sequences amplified Marker 5′>3′ sequence FeF1 FeR1 FaF FaR Flang Lanspo Ef728M Tef1R PKS4_F PKS4_R PKS13_1 PKS13_2 TRI4F1506 T4EndR2 TRI5_1 TRI5_2 TRI13DON-F TRI13DON-R TRI13NIV-F TRI13NIV-R Esyn_1 Esyn_2 CATACCTATACGTTGCCTCG TTACCAGTAACGAGGTGTATG AGCATTGTCGCCACTCTC GTTTGGCTCTACCGGGACTG CAAAGTTCAGGGCGAAAACT TACAAGAAGAGCGTGGCGATAT CATCGAGAAGTTCGAGAAGG GCCATCCTTGGAGATACCAGC AGACGGCGCAACAAGGGCTG GCAGTTGCCCGTGTCGGACA CCCAGCCAAGCCCAGTACGC ACAGCGGCTGACCTGGGTCA CCCCTGGCTACTCTCGAGA AAGCTTTGAGAACCTTCAC AGCGACTACAGGCTTCCCTC AAACCATCCAGTTCTCCATCT CATCATGAGACTTGTKRAGTTTGGG GCTAGATCGATTGTTGCATTGAG CCAAATCCGAAAACCGCAG TTGAAAGCTCCAATGTCGTG GCCGTTGGCGAGCTGGTCAT GCAAAGCACGCGTCAACGCA J Appl Genetics (2012) 53:227–236 Amplicon size (bp) 389 920 310 600 355 532 550 545 216 308 997 Accession number/reference Mishra et al. 2003 Doohan et al. 1998 Wilson et al. 2004 Multiple AY495638.1 DQ019316.1 Nicholson et al. 2004 EF661664.1 Nicholson et al. 2004 Nicholson et al. 2004 Z18755.3 Results Species-specific DNA marker detection, tef-1α sequence analysis and phylogeny reconstruction Three species-specific DNA markers FeF1/R1, FaF/R and Flang/Lanspo (Table 2) were used to identify the strains included in the study. Of the 25 F. equiseti strains studied, 16 displayed the amplicon of expected size, while nine genotypes did not amplify the fragment (Table 1). In the cases where, based on the molecular data from species- specific marker detection, we were not able to confirm the species identification, we still could prove it thanks to the sequence similarities to the tef-1α sequences of F. equiseti available in the GenBank sequence database using BLASTn. The primers Ef728M/Tef-1R were used success- fully in many fungal species and they worked fine in our case, amplifying the fragment of tef-1α gene using template from all isolates except one (ITEM 6164). Among 27 iso- lates studied, 22 presented the highest similarity to GenBank accessions of F. equiseti (90–99%). The isolate ITEM 2753 appeared to be F. avenaceum and KF 2640 was confirmed as F. langsethiae. The ITEM 3699 strain was the most similar to F. langsethiae and the ITEM 5104 strain to F. avenaceum (Fig. 1). To investigate the strain ITEM 6164 in more detail, a tef-1α gene fragment adjacent to the standard one was amplified and sequenced using an alternative pair of pri- mers. It was dedicated to identifying other fungal genera, but also with some Fusarium genotypes (results not shown). It displayed the highest level of similarity to F. sporotrichioides (97%), F. graminearum (97%) and F. fujikuroi (97%). All the obtained tef-1α sequences (except ITEM 6164) were submit- ted to the NCBI GenBank and are publicly available under accession numbers JF966231–JF966256. Growth rate Petri plates with PDA medium were used for the analysis of the growth speed of 25 F. equiseti strains used in the study. In the group of Polish strains, only two genotypes measured 70 mm (KF 2652 and KF 2655), while all the remaining isolates ranged from 40 to 55 mm (Fig. 2). The majority of Italian strains grew more slowly than the Polish group, form- ing a colony of about 25–30 mm in diameter after four days. However, some exceptions from this rule were recorded (ITEM 3699 and ITEM 6164 reached 64 and 75 mm, respec- tively). Also, two isolates originating from Italy (KF 2650 and KF 2651) presented growth speed on the level of Polish strains rather than of the Italian group (Fig. 2). Quantification of zearalenone ZEA biosynthesis levels varied among all the F. equiseti strains analysed (Fig. 3). ZEA was detected in 20 of the 27 tested isolates. ZEA was produced by F. equiseti in concen- trations ranging between 1.34 and 34,237 ng/g (>34 μg/g).