Soil samples at pre-vegetation and post-harvest stage, were collected from 0–10 cm depth using a 5 cm diameter soil corer . To ensure the spatial homogeneity, soil samples were pooled and homogenously mixed prior to subsequent analyses. After removal of plant debris, samples were sieved through a 2-mm sieve and divided into two sub-samples. One sample LY2874455 purchase was this website stored for 7 days (4°C) to prevent from sunlight and to reduce the microbial activity for molecular biological analyses (microbial density and diversity), and the other air dried for soil analyses. Soil pH was determined using pH meter (Systronics-model 361). Organic carbon content was determined by wet digestion method of Walkey
and Black . The available Zn, Fe, and Mn in the GF120918 soil samples were extracted with a diethylene triamine penta-acetic acid (DTPA) solution (0.005 M DTPA + 0.01 M CaCl2 + 0.1 M triethanolamine, pH 7.3 . The respective micro-nutrients studied were Zn2+, Fe2+ and Mn2+. The available sulphur was determined using the method of Comb et al. , and available K2O by the method of Licina and Markovic . Soil DNA extraction Total genomic DNA (in triplicate at each sampling stage) was extracted from 0.5 g rhizosphere soil using Fast DNA® spin kit (MP Biol, USA) combined with Fast DNA prep bead beater according to manufacturer’s protocol. The genomic DNA was eluted in 50 μl DNA eluting solution (DES) and stored (-20°C) for subsequent
analysis. The concentration and purity of extracted DNA was determined using Nanodrop spectrophotometer (ND 1000, Nano Drop Technologies, Inc., Wilmington, DE, USA). Real time PCR for total actinomycetes 16S rRNA gene copy number Real Time Quantitative
PCR (qPCR) amplification was performed using Applied Biosystems 7500 Fast Real –Time PCR system containing 96-well plate (ABI 7500) to quantify the abundance of total actinomycetes specific 16S rRNA gene copy number using universal primer sets, 517 F (5’-CCA GCA GCC GCG GTA AT-3’) and Act704R (5’-TCT GCG CAT TTC ACC GCT AC-3’) . The amplifications were carried out in triplicate in a final 25 μl volume containing 10X SYBR Green PCR master mix (Fermentas, USA). The reaction mixture (25 μl) comprised of 7.5 μl master mix (2X), 10 pmol each of primer (517 F and Act704R) and 45 ng genomic DNA template. The two-step many Amp + Melt protocol was as follows: (i) amplification step: denaturing at 95°C for 4 min, 40 cycles of 30 s at 94°C and 30 s at 55°C, 1 min at 95°C, 1 min at 55°C, and (ii) melting curve analysis step: 81 cycles of 30s at 55°C. Plasmid DNA containing target gene (actinomycetes- specific 16S rRNA) was used as the standard DNA in real time PCR assay, was obtained by PCR-cloning using the universal actinomycetes-specfic primers . Standard curves were generated by plotting the threshold cycle for each standard, calculated with ABI Prism 7900 SDS 2.2.2 software (Applied Biosystem, USA), against the gene copy number.