Nested PCR products generated by Gol1 and NS31-GC primer
pairs were subsequently analyzed by DGGE using a Dcode
Universal Mutation Detection System (Bio-Rad, USA), where
amplicons (200 ng) were loaded onto the 1 mm-thick gel. Gels
were prepared and run under the following conditions: 8% (w/v)
polyacrylamide (37.5:1 acrylamide/bis-acrylamide), 1 TAE buffer,
linear gradient from 35% to 55% denaturant (where 100%
denaturant is defined as 7 M urea and 40% v/v deionized
formamide). Electrophoresis were performed at 60 C and 80 V
for 10 h. DGGE gels were stained using 20 ml of 1 TAE containing
2ml Sybr Green I, scanned and finally analyzed using QuantityOne
software (version 4.5, Bio-Rad, USA). The Shannon index H was
used to calculate the band pattern diversity of AMF based on the
following equation: