16s rDNA Based Identification of Bacteria in the Organophosphates Treated Agricultural Soil

Abstract

This work was undertaken with the aim to isolate and identify the bacteria from agricultural soil, untreated and treated with four organophosphates (Malathion, Chlorpyrifos, Phorate and Dimethoate) and find the influence of these organophosphates on them. Conventional methods of bacterial identification have major drawbacks. Therefore species level identification was carried out based on 16S rDNA sequence and GenBank database. The 16S rDNA gene is highly conserved within a species and among species of the same genus, and hence, used as the new technique for identification of bacteria to the species level. Fragment of 16S rDNA gene of the DNA of isolated bacteria was amplified by PCR . Forward and reverse DNA sequencing reaction of purified PCR amplicon was carried out with 16sF and 16sR primers. BLAST was performed and phylogenetic tree constructed using the sequences. The evolutionary distances were computed using the Kimura 2-parameter method and expressed in the units of the number of base substitutions per site. Phylogenetic analyses were conducted in MEGA4. 1359nt, 1458nt and 1309nt contig region of sample A, B and E was homologous with sequence with Genbank accession no. FJ158842.1, DQ178226.1 and HQ190844.1 which were Sphingomonas sp.SZL-1, Pseudomonas mendocina strain PC19 and Brevundimonas sp.XJ-412 respectively. Sphingomonas was affected the most followed by Pseudomonas by all the four organophosphates considered for study and was absent in most of the treated soil plates upto 21 st day whereas Brevundimonas was least affected, present in almost all the treated plates as compared with controls. The three of the above characterized bacteria is found to play a significant role in bioremediation and soil fertility maintenance. Our results emphasizes on less use of organophosphates in agroecosystem to reduce biotic stress on normal soil microfauna, that would ultimately be helpful in better crop yield, thereby maintaining natural ecological balance.