Nematoda environmental sample from conventional vineyard plot in the vine rows  depth 8 Rep 1
Free-living soil nematodes determination: a) One hundred grams from each soil aggregate size was used for nematode extraction, following Cairns (1960).  b) The recovered nematodes were transferred to an Eppendorf tube after being centrifuged for 10 min at 15,000 rpm to reduce the remaining water (personal information). The pellets were used for DNA extraction by using Kit PureLink Genomic DNA Mini. The eluted DNA was stored at -20C until use as a polymerase-chain-reaction (PCR) template. The 3' part of SSU rDNA was amplified by PCR using Integrated DNA Technologies forward primer sequence NF1: 5'-GGT GGT GCA TGG CCG TTC TTA GTT-3', and reverse primer sequence 18Sr2b: 5'-TAC AAA GGG CAG GGA CGT AAT-3'. Reactions were carried out in a 25 L final volume including 9.5 L ultra-pure water, 12.5 L PCRBIO HS Taq Mix Red, 1 L F primer, 1 L R primer and 1 L of DNA from each sample.  The PCR program involved a five stage procedure: 1) denaturation step of 30 s at 98C; 2) a denaturation step of 10 sec at 98C; 3) an annealing step of 30 s at 58C; 4) an extension step of 1.5 min at 72C and 5) a final extension step of 10 min at 72C. The step 2-4 x 20 cycles.