Aliquots of PCR products were checked by electrophoresis on a 2% agarose gel. The PCR products obtained for the six strains were sequenced and aligned using clustalx (Thompson et al., 1997). Variable nucleotides between the sequence of Fo47 and those of other strains were used to design two primers potentially specific

for Fo47. The specificity of these primers was tested in conventional PCR reactions as described above. Real-time PCR reactions were performed on an ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems®, Foster City, CA). The PCR mixtures were set up as follows: 5 μL of DNA (5 ng), 0.3 μM of each primer P47C and P47D, 12.5 μL of the SYBR green master mix (Quanti Tech SYBR Green kit, Qiagen Gmbh, Hilden, Germany), 0.5 μg of T4 gene

32 protein (Quantum-Appligene, France) and Mol Bio grade water (5 Prime Gmbh, Hamburg, Germany) in a final volume of 25 μL. In the negative click here and positive controls, DNA was replaced by Mol Bio grade water and Fo47 DNA, respectively. The program used for PCR was: 1 min at 95 °C, followed by www.selleckchem.com/products/FK-506-(Tacrolimus).html 35 cycles of denaturation for 15 s at 95 °C, annealing for 30 s at 60 °C, extension for 30 s at 72 °C and data collection after 30 s at 78 °C to eliminate parasitic peaks. A dissociation curve was included at the end of the PCR program to evaluate potential primer–dimers and nonspecific amplification products. A standard curve based on Ct values vs. known quantities of target DNA was constructed using the plasmid that contained the cloned genomic fragment of the strain Fo47. Plasmid DNA was extracted using the QIAfilter

plasmid purification kit (Qiagen, Courtaboeuf, France) and linearized using the restriction enzyme SalI (Q-BIOgene). A standard curve in each microplate was obtained by amplification of 10-fold dilution series (102–108) of the linearized plasmid containing the SCAR from Fo47. Similarly standard curves were constructed by mixing dilution series of plasmid (102–108) or fungal DNA (10–104 pg) with 5 ng of plant DNA. The specificity of primers for the real-time PCR assay was tested with DNA extracted from the five strains used to design the primers, 12 additional strains of F. oxysporum, and three strains belonging to other Fusarium spp.: Fusarium redolens, Fusarium moniliforme, Fusarium solani (Table S1). Both Fo47 and F. oxysporum f. sp. lycopersici 8 (Fol8, ATCC number MYA-1199) strains were used. Inoculum CYTH4 was prepared according to L’Haridon et al. (2007). The concentration of the conidial suspension was adjusted to the desired concentration using sterile-distilled water. Tomato (Solanum esculentum) cv. Montfavet 63-5, which is susceptible to Fusarium wilt, was used to analyze the root colonization by Fo47. Tomato plants were cultivated in soil originating from a field in Epoisses (Bretennières, France) passed through a 4-mm sieve. It is a silt loamy soil with 50% silt, 41% clay, 9% sand and 2.6% organic matter, pH 8.2. The soil was used either nontreated or heat-treated at 100 °C for 1 h.