The specificity and the efficiency of the primer pairs was verified by melting curves and the construction of standard curves based on a Olaparib ic50 serial two-fold dilution (20 – 2-5) using soil DNA as the template. Template plasmids were used to generate a standard curve that was used as an external

standard. The target DNA sequence was cloned into the pGEM-T vector selleck products (Promega) and the resulting plasmids were purified. All plasmids were quantified by spectrometry using a Nanodrop ND-1000 instrument (Thermo Scientific) and copy numbers were estimated based on the molecular weight of the template. The number of copies of the cloned target DNA in the dilution series ranged from 106 to 101. Real-Time

PCR assays Real-time PCR was performed using the iQ SYBR Green Supermix (Bio-Rad). The reaction mixtures PD-0332991 purchase contained 7.5 μl of iQ SYBR Green Supermix, 1 μl of DNA solution (corresponding to 1 ng of DNA), and 350 nmol of each gene-specific primer. The experiments were conducted in 96-well plates with an iQ 5 Multicolour Real-Time PCR Detection System (Bio-Rad). PCR was always performed with three biological and three technical replicates. The cycling conditions were 10 s at 95°C, 30 s at 55°C or 62°C. Template abundances were determined based on the Ct values (which measure the number of cycles at which the fluorescent signal exceeds the background level and surpasses HA-1077 supplier the threshold established based on the exponential phase of the amplification plot). The significance of differences between the Ct values of different treatments were determined by one way analyses of variance ( p < 0.05) and grouped according to the Tukey HSD test in R (R Core team, 2012). Acknowledgments We thank D. Krüger for advice on fungal PCR primer construction. We thank K. Hommel, I. Krieg and B. Krause for oak micropropagation

and S. Recht for her role in setting up the soil microcosms. Financial support was supplied by the German Science Foundation (DFG) (TA 290/4-1) and by the Helmholtz Gemeinschaft. This work was kindly supported by Helmholtz Impulse and Networking Fund through Helmholtz Interdisciplinary Graduate School for Environmental Research (HIGRADE). The authors thank the Laboratory of Electron Microscopy BC AS CR, v.v.i. – Parasitology Institute České Budějovice for a productive collaboration on scanning electron microscopy. Electronic supplementary material Additional file 1: Experimental setup for quantification of AcH 505 and P. croceum under different culture conditions. (PDF 310 KB) Additional file 2: qRT-PCR melting and standard curves obtained using the AcH107 primer pair. (PDF 362 KB) Additional file 3: qRT-PCR melting and standard curves obtained with the ITS-P primer pair.