Quantification of Gram-negative sulphate-reducing bacteria in rice field soil by 16S rRNA gene-targeted real-time PCR

Abstract

For the quantification of Gram-negative sulphate reducers in rice fields, 11 real-time PCR assays were established targeting 16S rRNA genes combined with SybrGreen™ detection. Three of these assays were specific for the “main” groups, i.e. the Desulfovibrionaceae, the Desulfobacteraceae and Desulfobulbus sp., whereas eight assays were developed for subgroups within the first two main groups. The detection limits of the assays were between 2×10 5 and 4×10 3 targets g −1 (wet weight) or less than 0.02% of the eubacterial 16S rDNA targets in bulk soil, rhizosphere soil and rice root DNA extracts. Analysis of soil spiked with defined cell numbers of sulphate-reducing bacteria showed good correlation of measured target numbers to amended cells. In rice field bulk and rhizosphere soil, the Desulfobacteraceae were the predominant main group with target numbers of 6.4×10 7 (±1.0×10 7) and 7.5×10 7 (±1.7×10 7), respectively. Within this group the Desulforhabdus/ Synthrophobacter assemblage and Desulfobacterium sp. were predominant. At the rice roots, the three main groups were abundant in similar numbers (approx. 1.0×10 8) indicating that the relative abundance of the Desulfovibrionaceae and also of Desulfobulbus sp. was increased, relatively to the Desulfobacteraceae. Within the Desulfovibrionaceae the subgroup was predominant that was detected by assay DSV-II. This assay detects many from rice field soil isolated Desulfovibrio-strains and molecular retrieved sequences. Therefore these organisms that were already detected in the rice field environment by isolation and by molecular techiques are indeed best adapted to the conditions provided by the rice roots.