CRISPR loci-PCR as Tool for Tracking Azospirillum sp. Strain B510

Abstract

Azospirillum-based plant and soil inoculants are widely used in agriculture. The inoculated Azospirillum strains are commonly tracked by both culture-dependent and culture-independent methods, which are time-consuming or expensive. In this context, clustered regularly interspaced short palindromic repeats (CRISPR) loci structure is unique in the bacterial genome, including some Azospirillum species. Here, we investigated the use of CRISPR loci to track specific Azospirillum strains in soils systems by PCR. Primer sets for Azospirillum sp. strain B510 were designed and evaluated by colony and endpoint PCR. The CRISPRloci-PCR approach was standardized for Azospirillum sp. strain B510, and its specificity was observed by testing against 9 different Azospirillum strains, and 38 strains of diverse bacterial genera isolated from wheat plants. The CRISPRloci-PCR approach was validated in assays with substrate and wheat seedlings. Azospirillum sp. strain B510 was detected after of two weeks of inoculation in both sterile and nonsterile substrates as well as rhizosphere grown in sterile substrate. The CRISPRloci-PCR approach was found to be a useful molecular tool for specific tracking of Azospirillum at the strain level. This technique can be easily adapted to other microbial inoculants carrying CRISPR loci and can be used to complement other microbiological techniques.

Current Techniques to Study Beneficial Plant-Microbe Interactions

“…While these techniques work quite well under laboratory conditions, they are often much less effective under field conditions. To get around any constraints or limitations that may exist when using the above-mentioned laboratory approaches, one group of scientists first identified CRISPR loci that existed in a strain of Azospirillum that they were studying [ 206 ]. CRISPR loci are short palindromic repeats interspersed with spacer sequences found within the chromosomal DNA of an organism.…”