J Appl Genetics (2012) 53:237–247 was carried out in a Biometra Tpersonal 48 thermocycler (Whatman Biometra, Goettingen, Germany). Additionally, for 12 representative isolates (forming the highest and the lowest amount of FB1 and MON), a part of the translation elongation factor 1-α (TEF) was amplified with the following primer pair: EF1-forward: 3′-ATGGGTAAG GARGACAAGAC-5′ (O’Donnell et al. 1998) and TEF_WI- reverse: 3′- GCTCACGRGTCTGGCCATCCTTG′ (designed in this study). Primer TEF_WI is located about 90 nt down- stream of the sequence of the reverse primer designed by O’Donnell et al. (1998). The reaction mixture contained: 20 ng of fungal DNA, 0.2 mM of each dNTP, 1 μM of each primer and 0.5 U of DreamTaq™ polymerase (Fermentas GMBH, St. Leon-Rot, Germany) in 1× reaction buffer with 2 mM MgCl2. Amplification was carried out using the fol- lowing programme: an initial denaturation for 2 min at 94°C, followed by 45 cycles of denaturation at 94°C for 30 s, primer annealing at 58°C for 30 s and extension at 72°C for 1 min. The amplification was ended with an additional extension at 72°C for 5 min. Amplification products were cleaned and then sequenced on ABI PRISM® 310 Analyzer (Applied Biosystems), according to the procedure described previously (Irzykowska et al. 2005). The resulting sequences were checked manually and were aligned using the computer software package CLUSTAL_X (Thompson et al. 1997). The obtained TEF sequences were compared with all the sequences deposited at the Fusarium ID Database (http:// www.fusariumdb.org/index.php). Diagnostic PCR for mating type detection To determine the mating type of the F. oxysporum isolates, conserved portions of the ALPHA or HMG boxes of the MAT1-1 and MAT1-2 idiomorphs were amplified with degen- erate oligonucleotide primer pairs: forward (ALPHA) 5′- CGCCCTCTKAAYGSCTTCATG-3′ and reverse (ALPHA) 5′-GGARTARACYTTAGCAATYAGGGC-3′ for MAT1-1 and forward (HMG) 5′-CGACCTCCCAAYGCYTACAT-3′ and reverse (HMG) 5′-TGGGCGGTACTGGTARTCRGG-3′ for MAT1-2 (Kerényi et al. 2004). The PCRs were carried out using a Taq PCR Core Kit (QIAGEN, Inc., Hilden, Germany) with a reaction mixture containing: 5 ng of fungal DNA, 0.2 mM of each dNTP, 0.5 μM of each primer and 0.5 U of Taq DNA polymerase in 1× reaction buffer with 2.5 mM magnesium chloride. The PCR profile was as described previ- ously (Irzykowska and Kosiada 2011). Genetic fingerprinting of F. oxysporum Random amplified polymorphic DNA PCRs (RAPD-PCRs) were carried out using a Taq PCR Core Kit (QIAGEN, Inc., Hilden, Germany). The reaction mixture was as described previously (Irzykowska and Bocianowski 2008). Eight random 239 10-mer and one 11-mer primers, OPB-07, OPC-02, OPC-04, OPC-08, OPC-15, OPJ-10+A, OPL-11, OPL-12 and OPL-19 (Qiagen Operon, Cologne, Germany), were used to screen the isolates for DNA polymorphism. Amplification was carried out using the following programme: an initial denaturation for 2 min at 94°C, followed by 40 cycles of denaturation at 94°C for 30 s, primer annealing at 37°C for 1 min and exten- sion at 72°C for 2 min. The amplification was ended with an additional extension at 72°C for 5 min. PCR was repeated twice in order to check reproducibility. The comparison of each band profile for each primer was performed on the basis of the presence (1) versus the absence (0) of RAPD products of the same length. Electrophoresis conditions The PCR products were separated by electrophoresis (4 V cm−1) in 1.5% agarose gels with 1× TBE buffer (89 mM Tris-borate and 2 mM EDTA, pH 8.0) and visualised under UV light following ethidium bromide staining. A Gene Ruler™ 100 bp DNA Ladder Plus (Fermentas GMBH, St. Leon-Rot, Germany) was used as a molecular size standard for PCR products. Fungal cultures for mycotoxin estimation For toxin quantification, rice cultures were prepared for indi- vidual F. oxysporum isolates. Long-grain white rice (RANI) tested as being mycotoxin-free was used as a medium for the formation of mycotoxins by F. oxysporum isolates. Rice sam- ples of 65 g were soaked for 16 h with 81 ml of distilled water in 500-ml Erlenmeyer flasks and then sterilised for 15 min at 120°C. Such samples were inoculated with 5-mm disks of PDA medium overgrown by F. oxysporum mycelium. On rice substrate, fungal cultures were incubated for 3 weeks at 20°C. All samples for analyses were prepared and cultured in tripli- cate (three Erlenmeyer flasks with the prepared medium) and the mean values of the results were calculated. Mycotoxin analyses Chemicals and reagents Standards of pure FB1 and MON were purchased from Sigma (St. Louis, MO, USA). Acetonitrile, methanol (high- performance liquid chromatography [HPLC] grade), disodium tetraborate, 2-mercaptoethanol and t-butyl-ammonium hydrox- ide were purchased from Sigma-Aldrich. Sodium dihydrogen phosphate, acetic acid, n-hexane, o-phosphoric acid and dichloromethane were purchased from POCh (Gliwice, Poland). Water for the HPLC mobile phase was purified using a Milli-Q system (Milipore, Bedford, MA, USA).