0) was between F. oxysporum f. sp. melonis and F. oxysporum f. sp. radicis lycopersici. The sequence of ITS2 for the F. oxysporum f. sp. studied has been

obtained from NCBI and aligned. The alignment between F. oxysporum formae speciales is depicted (Supporting information, Fig. S1). The sequences of the F. oxysporum formae speciales check details used in this study possess variations capable of producing differences in the HRM curves. Specifically, we observed point mutations for most of the species while F. oxysporum f. sp. phaseoli was the most diverse, having on top of SNPs insertions and deletions responsible for the differences in the observed melting curves between the different formae speciales amplicons (Fig. S1). The ITS region of rRNA genes is a useful marker for discriminating species because it contains stretches of high sequence conservation, while at the same time, the size of the sequence varies in different Fusarium formae speciales (Suga et al., 2000; Visentin et al., 2010). It has been successfully used before for the identification of Aspergillus (Henry et al., 2000) and Fusarium (Gurjar et al., 2009) species. It has been previously reported that the ITS region is suitable for the identification of F. oxysporum formae speciales complex with high sensitivity and specificity (Alves-Santos et al., 2002; Visentin et al., 2010). Thus, several Fusarium species have been discriminated using

the ITS of the ribosomal DNA based on amplicon length (Visentin et al., 2010). Sequence variation at the ITS region of rRNA genes allowed a very clear and reproducible HRM curve

analysis differentiation 5-Fluoracil price among all seven Fusarium formae speciales that were analyzed in the present study as depicted in Fig. 1b. Another conserved region, the TEF1, Metalloexopeptidase was also tested but failed to generate high-quality discriminatory results (data not shown). A significant aspect of our results is that we were able to transfer and adapt the universal primers and standard PCR conditions previously designed to discriminate species by DNA sequencing to more rapid, closed-tube, melting curve assays like the HRM analysis; this was exemplified by successful genotyping of the seven F. oxysporum species tested. In conclusion, this is the first study describing the application of HRM curve analysis for differentiation of Fusarium formae speciales. Universal marker ITS was able to differentiate between the seven F. oxysporum formae speciales, following HRM curve analysis. The current study demonstrated that the real-time PCR-HRM method is a cheap, accurate, rapid close-tubed assay for the differentiation and genotyping of Fusarium oxysporum formae speciales complexes in pure cultures. Although our genotyping method shows the potential for being used to assign new unclassified strains to a f. sp., as yet that potential has not yet been fulfilled because we do not know enough about genetic variation within versus between formae speciales.