| Summary: | <i>Azospirillum</i>-based plant and soil inoculants are widely used in agriculture. The inoculated <i>Azospirillum</i> 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 <i>Azospirillum</i> species. Here, we investigated the use of CRISPR loci to track specific <i>Azospirillum</i> strains in soils systems by PCR. Primer sets for <i>Azospirillum</i> sp. strain B510 were designed and evaluated by colony and endpoint PCR. The CRISPR<sub>loci</sub>-PCR approach was standardized for <i>Azospirillum</i> sp. strain B510, and its specificity was observed by testing against 9 different <i>Azospirillum</i> strains, and 38 strains of diverse bacterial genera isolated from wheat plants. The CRISPR<sub>loci</sub>-PCR approach was validated in assays with substrate and wheat seedlings. <i>Azospirillum</i> 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 CRISPR<sub>loci</sub>-PCR approach was found to be a useful molecular tool for specific tracking of <i>Azospirillum</i> 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.
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