& C. Tul.) Trappe d.A CMI-Unibo 4231 Marmora AZD3965 mw forest, Morocco Tuber rufum Pico d.A CMI-Unibo 1798 Emilia Romagna, Italy Terfezia claveryi Chatin d.A CMI-Unibo 4231 Cappadocia, Turkey Choiromyces meandriformis Vittad. d.A CMI-Unibo 1432 Emilia Romagna, Italy Balsamia vulgaris Vittad. d.A CMI-Unibo 3460 Emilia Romagna, Italy Genea klotzschii Berk. & Broome d.A CMI-Unibo 1944 Emilia Romagna, Italy Ganoderma lucidum (Curtis) P. Karst. M Glu5039 Armenia Hymenogaster luteus Vittad. d.B CMI-Unibo 1947 Emilia Romagna, Italy Valsa ceratosperma
(Tode) Maire M Vce155 Emilia Romagna, Italy Cryphonectria parasitica (Murrill) M.E. Barr. M Cpa5 Emilia Romagna, Italy Monilia laxa (Ehrenb.) Sacc. & Voglino M Mla95 Emilia Romagna, Italy Aspergillus flavus Link M Afl7 Emilia Romagna, Italy Penicillium expansum Link M Pex25 Emilia Romagna, Italy 1 d.A = dried ascoma; d.B = dried basidioma; M = mycelium in pure culture. 2 CMI-Unibo = Center of mycology of Bologna University. Estrogen/progestogen Receptor modulator 3 Bonuso et al. . Figure 1 PCR sensitivity of the primer pairs selected from ITS1
and ITS2 regions. Reactions carried out using serial dilutions of T. magnatum DNA (TM-DNA) in pooled non-target fungal DNAs (F-DNA): lane M, Mass ruler marker (GSK2118436 cell line Fermenats); lanes 1, 3, 5 and 7, ITS1for-ITS1rev primer pair; lanes 2, 4, 6 and 8, ITS2for-ITS2rev primer pair. Lanes 1–2, 10 ng TM-DNA/90 ng F-DNA; lanes 3–4, 1 ng TM-DNA/99 ng F-DNA; lanes 5–6, 0.1 ng TM-DNA/99.9 ng F-DNA; lanes 7–8, 0.01 ng TM-DNA/99.99 ng F-DNA. Real time quantification of T. magnatum DNA The real-time assay showed reliable amplification over the 6 orders of magnitude generating Florfenicol almost identical standard curves from each run quantifying T. magnatum DNA in soil samples. The correlation coefficients (R2 values) were always higher than 0.99 and amplification efficiency
was about 85%. The mean standard curve resulting from 18 independent plates is shown in Figure 2. The detection limit for real-time PCR with the ITS1 primer/probe set was approximately 10 fg. However, since standard replicates containing less than 100 fg of T. magnatum DNA gave inconsistent amplifications, to avoid the inclusion of false positive test results, values lower than this threshold were considered as 0. Figure 2 Real-time PCR standard curve for T. magnatum DNA quantification. The curve was generated by plotting the means of the Ct values obtained against the logarithm of a known quantity of genomic DNA. Variability is shown as the mean Ct value ± SD. Detection of T. magnatum ascomata and DNA Truffle production was scattered and localized in only 17 of the 39 plots examined. A total of 74 T. magnatum ascomata, for a total weight of 1184.3 g, were collected over the 3-year period of investigation in the 4 experimental truffières (Additional file 1). There was a high variation in the concentration of T.