Development of DNA markers for detection of inoculated bacteria in the rhizosphere of wheat (Triticum aestivum L.)

Abstract

Plant growth-promoting rhizobacteria (PGPR) are a group of soil microorganisms that improve plant growth and yield through a number of diverse mechanisms such as phosphate solubilization, nitrogen fixation, production of phytohormones, and repression of soil borne pathogens. Due to high cost of chemical fertilizers and negative environmental effects, the use of PGPR as biofertilizer is advantageous for development of sustainable agriculture. The objective of this study was to develop DNA-based markers for five PGPR strains to detect these bacteria in the rhizosphere of inoculated wheat. The rhizobacterial strains included four phosphate solubilizer strains (Arthrobacter strain WP-2, Bacillus strain MP5, Rhodococcus strain M28 and Serratia strain 5D) and one phytohormone producer Azospirillum strain WS1. DNA-based markers were developed using 16S rRNA gene restriction patterns, Random Amplified Polymorphic DNA-PCR (RAPD-PCR), Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) and BOX-PCR. In this study, the most differentiating DNA patterns for all strains were obtained by using the BOX primer in PCR. All the strains tested as single-strain inocula resulted in improved growth of wheat plants. The inoculated strains were successfully re-isolated from wheat rhizosphere and confirmed by BOX-based DNA markers. These results indicated that BOX-PCR is a useful and highly discriminatory fingerprinting technique for rapid detection of bacterial strains.