J Appl Genetics (2012) 53:227–236 Table 1 Fusarium equiseti isolates used in this study, along with their country of origin, presence of species-specific and TRI5 gene-specific markers, as well as the amplicon sizes (in base pairs) of partial coding sequences of PKS4 and PKS13 genes from the zearalenone (ZEA) biosynthetic cluster Isolate Host Origin FeF/R TRI5 TRI4 PKS4* PKS13* ITEM 4323 Equisetum arvense Argentina + ITEM 2753 F. avenaceum Allium cepa Italy − ITEM 3190 Chenopodium Italy + ITEM 3673 Oryza sativa Italy + ITEM 3699 O. sativa Italy − ITEM 4586 Pinus root Italy + ITEM 4743 Solanum lycopersicum Italy + ITEM 5104 Orobanche ramosa Italy + ITEM 6164 O. ramosa Italy − ITEM 6461 Triticum sp. Italy + ITEM 6463 Triticum sp. Italy + ITEM 7502 Triticum sp. Italy + KF 2650 Soil Italy − KF 2651 Soil Italy − KF 8 Triticale Poland − KF 13 T. aestivum Poland + KF 24 Zea mays Poland + KF 72 Triticale Poland − KF 89 T. aestivum Poland − KF 1011 Lycopersicon esculentum Poland + KF 1017 L. esculentum Poland − KF 2652 Soil Poland + KF 2653 Soil Poland + KF 2654 Soil Poland + KF 2655 Soil Poland + KF 2656 Soil Poland − KF 2640 F. langsethiae T. aestivum Poland − − − − − + − − − + − − − + + + − + + + + − + − + + + + − − − − − − − − − − − − − + − + − + − − − − − − − − + 229 − − 500 − − − 300 − − − − − − − 750 − − 700 − − 400 532 − − 200/355 532 355 532 355 532 750 − 750 532 355 532 355 532 750 532 750 532 200/700 532 355/750 200/532 150/750 532 200/750 532 355 532 − 700 *In base pairs City, CA, USA) according to Błaszczyk et al. (2005) and precipitated with ethanol. Sequence reading was performed using Applied Biosystems equipment. Sequence analysis and phylogeny reconstruction The sequences of the PCR products were compared to the National Center for Biotechnology Information (NCBI) nucleotide collection using MEGABLAST and initially aligned with the ClustalW algorithm. Phyloge- netic relationships were reconstructed with the MEGA4 software package (Tamura et al. 2007) using the maxi- mum parsimony approach (closest neighbour interchange heuristics). No gap-containing positions were considered in the phylogeny analysis. All reconstructions were test- ed by bootstrapping with 10,000 replicates. Zearalenone analysis Rice cultures were prepared from individual Fusarium iso- lates (Kostecki et al. 1999; Moretti et al. 2008). Long-grain white rice was used (50 g per flask with the addition of 12.5 ml of sterile water), left overnight and sterilised by autoclaving the next day. Samples were inoculated with 4 cm2 of 7-day-old mycelium on PDA medium. The average culture humidity was kept at around 30% and maintained for 14 days. Then, the cultures were dried at room temperature, ground and homogenised for 3 min with methanol–water (3:1, v/v) mixture (10 ml for 5 g of rice culture) and, finally, filtered through Whatman no. 4 filter paper (Sydenham et al. 1990). ZEA content was determined by the high-performance liquid chromatography (HPLC) method according to the method of Visconti and Pascale (1998). The chromatograph- ic system consisted of Waters HPLC 2695 apparatus with a Waters 2475 Multi λ Fluorescence Detector and a Waters 2996 Array Detector. The excitation and emission wave- lengths were 274 and 440 nm, respectively. The reserved- phase C-18 Nova Pak column (3.9× 150 mm) and acetoni- trile–water–methanol (46:46:8, v/v/v) as the mobile phase at a flow rate of 0.5 ml/min were used. The detection limit of ZEA was 3 ng/g.