Detection of Desulfotomaculum in an Italian rice paddy soil by 16S ribosomal nucleic acid analyses

Two specific primers were developed for the amplification of 16S rRNA genes of Desulfotomaculum lineage 1 to detect members of the genus Desulfotomaculum in rice field soil. The combination of both primers in PCR allowed the specific amplification and cloning of ten 16S rDNA sequences of this group from rice paddy soil DNA extracts. The phylogenetic analysis showed that these sequences formed a deeply branching cluster within Desulfotomaculum lineage 1, together with two sequences from the database and two sequences from a hydrocarbon-contaminated aquifer. Dissimilarity values to validly described species, including recently isolated strains of Desulfotomaculum from rice paddy microcosms, were higher than 12%. Within the new cluster the cloned sequences formed three separate groups which were each represented by at least two sequences with identities of greater than or equal to 99% while one sequence represented an additional group. The sequences should represent sulfate-reducing organisms because they clearly fell into the physiologically coherent group of Gram-positive sulfate reducers. The relative abundance of bacteria of the Desulfotomaculum lineage 1 in rice paddy soil and root samples was estimated with rRNA dot blot hybridizations of extracted RNA. The relative RNA content of Desulfotomaculum lineage 1 was 0.55% in the bulk soil and 1% in the rice root samples, respectively, of the total 16S rRNA content (probe Eub338). Hybridization of rRNA with a probe targeting the new cluster represented by the cloned sequences confirmed the high abundance of 16S rRNA sequences from this cluster in the rice paddy field samples. Another hybridization probe detecting Desulfotomaculum acetoxidans and two closely related Desulfotomaculum isolates from rice paddy soil indicated that these bacteria were less abundant.