Long term effect of methylparathion contamination on soil microbial community diversity estimated by 16S rRNA gene cloning

The long-term effects of methylparathion contamination on the diversity of soil microbial community was investigated by a culture-independent approach using small subunit ribosomal RNA (SSU rRNA) gene-based cloning. Microbial DNA extracted from both the control soil sample and methylparathion contaminated soil sample was subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences. From the PCR amplification product, clone libraries were constructed for both samples. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and HhaI. A total of 603 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, the phylotype richness, frequency distribution (evenness) of the two clone libraries were compared by using a variety of diversity indices. Phylogenetic analysis of the sequences of the dominant phylotypes revealed that the bacterial communities changed noticeably. In the control soil, the dominant bacterial groups included a member of a novel bacterial division, the bacillus genus, and a member of alpha-proteobacteria, while in methylparathion contaminated soil, the dominant phylotypes were replaced by a member of the flexibactera-cytophaga-bacteroides division and two members of the gamma-proteobacteria subdivision. This is the first report of the long-term effects of methylparathion (one of the major pesticides widely used in developing countries) on soil microbial community diversity and structure by a culture-independent method, and provides the evidences to assess the long-term environmental toxicological effects of methylparathion from the microbial community viewpoint.