240 Fumonisin B1 analysis Samples (10 g) of dry 3-week-old cultures were homoge- nised for 3 min in 20 ml of methanol–water (3:1, v/v) and filtered through Whatman no. 4 filter paper, in accordance with the method described by Sydenham et al. (1990). The detailed procedure of the extraction and purification of FB1 has been reported previously (Waśkiewicz et al. 2010a). Purified FB1 was quantitatively determined by the HPLC method. The o-phosphoric acid (OPA) reagent (20 mg per 0.5 ml methanol) was prepared and diluted with 2.5 ml of 0.1 M disodium tetraborate (Na2B4O7 × 10H2O) and then combined with 25 ml of 2-mercaptoethanol added to the solution. The FB1 standard (5 μl) or extracts (20 μl) were derivatised with 20 or 80 μl of the OPA reagent. After 3 min, the reaction mixture (10 ml) was injected onto an HPLC column. Methanol–sodium dihydrogen phosphate (0.1 M in water) solution (77:23, v/v), adjusted to pH 3.35 with o-phosphoric acid, after filtration through a 0.45-mm Waters HV membrane, was used as the mobile phase with a flow rate of 0.6 ml min−1. A Waters 2695 apparatus (Waters Division of Millipore, Milford, MA, USA), with a C-18 Nova Pak column (3.9 × 150 mm) and a Waters 2475 fluorescence detector (λex0 335 nm, λem0 440 nm), was used in the metab- olite quantitative determination. The identification and quanti- fication of FB1 was observed by comparison of the retention times and peak areas in the samples with those observed for FB1 standard and using the relevant calibration curve (correlation coefficients for FB1 was 0.9967). The limit of detection (LOD) was 10 ng g−1 for FB1 and was defined as the concentration that was three times higher than the standard deviation of the blank signal. Recovery for FB1 measured by the mycotoxins extrac- tion from blank samples spiked with 10–100 ng g−1 of the compound was equal to 93%. The relative standard deviations (RSDs) were lower than 8%. Moniliformin analysis Culture samples (15 g) of each strain were homogenised with 75 ml of acetonitrile–methanol–water (16:3:1, v/v/v) and fil- tered (Whatman no. 4 filter paper). Moniliformin was extracted and purified according to the methods described by Waśkiewicz et al. (2010b). MON was quantified using a Waters 501 apparatus (Waters Division of Millipore) with a C-18 Nova Pak column (3.9 × 300 mm) and a Waters 486 UV detector (λmax0 229 nm). Acetonitrile–water (15:85, v/v) buffered with 10 ml of 0.1 M K2HPO4 in 40% t-butyl- ammonium hydroxide in 1 L of solvent (Sharman et al. 1991) was used as the mobile phase (flow rate0 0.6 ml min−1). The MON detection limit was 25 ng g−1. Positive results (on the basis of retention time) were confirmed by the HPLC analysis and by comparison with the relevant calibration curve (the J Appl Genetics (2012) 53:237–247 correlation coefficient for MON was 0.9990). The recovery for MON was 90%. The RSD was below 7%. Statistical analysis The one-way analysis of variance (ANOVA) of F. oxysporum occurrence was carried out in order to determine the differen- tiation of isolates regarding the concentrations of FB1 and MON. Tukey’s least significant differences (LSDs) for each trait were calculated. Homogeneous groups (not significantly different from each other) for the analysed traits were deter- mined on the basis of least significant differences. The rela- tionship between FB1 and MON concentrations was estimated using correlation coefficients (Kozak et al. 2010). The coefficients of genetic similarity (S) of the investigated isolates were calculated using the following formula (Nei and Li 1979): Sij ¼ 2Nij Ni þ Nj where Nij is the number of alleles present at the i-th and j-th isolates, Ni is the number of alleles present at the i-th isolate, Nj is the number of alleles present at the j-th isolate, and i, j0 1, 2, …, 31. On the basis of the calculated coefficients, isolates were grouped hierarchically using the unweighted pair group method of arithmetic means (UPGMA). The relationship among isolates was presented in the form of a dendrogram (Kumar et al. 2004). The association between RAPD markers and the FB1 and MON concentrations of F. oxysporum isolates was estimated by regression analysis (Bocianowski and Seidler-Łożykowska 2012). The molecular marker observations were treated as independent variables and considered in individual models. The relationship between MAT idiomorphs and the biosynthe- sis of FB1 and MON were performed by the t-test (Snedecor and Cochran 1989). All statistical analyses were performed using the package GenStat v.7.1. (Payne et al. 2003). Results Identification of F. oxysporum isolates All of the tested isolates displayed morphology typical for F. oxysporum, quite similar to standard F. oxysporum f. sp. asparagi isolates DSM-62287 (here, O-31). Species-specific PCR assay targeting the calmodulin gene confirmed morpho- logical species identification. Specific PCR products obtained for each isolate, including the reference one, was 534 bp in length. Additionally, for representative isolates producing and non-producing mycotoxins, a 810-bp fragment of the TEF