238 (MON) producer (Logrieco et al. 2003). Recent studies suggest that some F. oxysporum strains can also biosynthesise fumoni- sin B1-FB1 (Waśkiewicz et al. 2010a; Stępień et al. 2011a, b). MON exhibits cytotoxic and cardiotoxic activity, causes de- velopmental disorders and may also induce the development of Keshan disease, human myocardial impairment reported in rural areas of China and South Africa (Pineda-Valdes and Bullerman 2000). Fumonisins are polyketide-derived myco- toxins inhibiting a ceramide synthase, the key enzyme in the sphingolipid biosynthetic pathway, resulting in serious myco- toxicoses (Soriano and Dragacci 2004; Voss et al. 2007). Contamination of food with FB1 has been associated with an oesophageal cancer of humans in Africa, China and the United States (Nicholson 2004). Considering an available toxicologi- cal evidence, the International Agency for Research on Cancer (IARC) describe FB1 as a probable carcinogenic to humans (IARC 2002). Food processing does not solve the problem because the hydro-thermal stability of fumonisin is high and decomposition products are more toxic than fumonisin itself (Bullerman et al. 2002). F. oxysporum is a large taxonomic unit, described as a species complex. So far, no known sexual stage of the F. oxysporum is available and the concept of a biological species and the sources of genetic variation are still discussed (Kistler 1997; O’Donnell et al. 2009; Kück and Pöggeler 2009). In most fungi, the mating type locus conferring mating behaviour consists of dissimilar DNA sequences (idiomorphs) in the mating partners (Pöggeler 2001). In heterothallic species, the mating type is controlled by a single locus with two idiomor- phic alleles, designated MAT1-1 and MAT1-2 (Kerényi et al. 1999; Turgeon and Yoder 2000). Each MAT idiomorph carries one gene encoding a single MAT-specific DNA binding pro- tein. These proteins probably play a crucial role in the path- ways of cell speciation and sexual morphogenesis as regulatory transcription factors. The two MAT alleles contain a conserved alpha (ALPHA) box domain or a high mobility group (HMG) box domain, respectively (Yun et al. 2000). Evolutionary theory indicates that sexual reproduction plays an important role in a pathogen’s evolution, i.e. the develop- ment of a new pathogenic race compatible with a disease- resistant cultivar and of a new strain resistant to a fungicide. Recently, it was shown that the presence of the MAT1-2 idiomorph in the F. culmorum genome significantly affects mycelium growth (unpublished data). Assessing the pos- sibility of mating by toxigenic strains is important for the design of successful control strategies, since these strategies are different for clonally and sexually reproducing organisms (McDonald and McDermott 1993). Moreover, there is poor information on the toxicity of pathogenic F. oxysporum iso- lates with different mating type alleles. The present study focused on the genetic and toxigenic differentiation of F. oxysporum isolates. The principal aims of the study were to estimate mycotoxins biosynthesis yield J Appl Genetics (2012) 53:237–247 and to detect mating type idiomorphs in the F. oxysporum genome. Additionally, the relationship between these features was assessed. Moreover, the level of intra-species genetic diversity were estimated and associations between molecular markers and mycotoxins (FB1 and MON) formation were examined. Materials and methods Fungal isolates Fungi were collected from green (O-02, O-11, O-30) and white (remaining isolates) spears of asparagus ‘Gijnlim’ and ‘Eposs’ cultivated in Western Poland. Fungi were isolated from the basal part of spears (one spear per plant) on the borderline between discoloured and healthy tissues. After disinfection with 1% sodium hypochlorite, five sections (about 1 mm in diameter) of tissue were cut from spears and such a set sections was transferred onto a Petri dish with potato dextrose agar (PDA) medium. One of these fungal cultures from spears was randomly chosen for further work. Thirty single-spore cultures of F. oxysporum were used in the chemical and molecular analyses. All fungal isolates (O-01– O-30) were identified on the basis of their morphology (Booth 1971; Gerlach and Nirenberg 1982; Kwasna et al. 1991). The morphological features of the identified isolates were compared with standard isolates DSM-62287 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Germany) of F. oxysporum f. sp. asparagi. Isolates of DSM-62287 in this study were designated as O-31. DNA preparations and molecular identification of F. oxysporum isolates by PCR and sequencing procedures Mycelia from 9-day-old single-spore cultures of F. oxysporum grown on liquid medium (5 g L−1 of glucose, 1 g L−1 of yeast extract) were separated from solution by vacuum filtration with a Büchner funnel. DNA was extracted and purified using a DNeasy Mini Kit (QIAGEN Inc., Hilden, Germany), according to the manufacturer’s recommendations. Polymer- ase chain reaction (PCR) with species-specific primers designed based on a partial sequence of calmodulin gene (Mulè et al. 2004a, b) was used to confirm the morphological identification of each fungal isolate. For the PCR analysis, a forward primer CLOX1: 5′-CAGCAAAGCATCAGACCAC TATAACTC-3′ and a reverse primer CLOX2: 5′-CTTGTC AGTAACTGGACGTTGGTACT-3′ (Sigma, Pampisford, UK) were used. The amplification reactions were performed with a Taq PCR Core Kit (QIAGEN, Inc., Hilden, Germany). The reaction mixture and amplification conditions were as described previously (Waśkiewicz et al. 2010b). Amplification