CRISPR loci-PCR as Tool for Tracking <i>Azospirillum</i> sp. Strain B510

• Main Authors: Joaquin I. Rilling, Fumito Maruyama, Michael J. Sadowsky, Jacquelinne J. Acuña, Milko A. Jorquera
• Format: Article
• Language: English
• Published: MDPI AG 2021-06-01
• Series: Microorganisms
• Subjects: <i>Azospirillum</i>; microbial inoculants; strain tracking methods; CRISPR_loci-PCR
• Online Access: https://www.mdpi.com/2076-2607/9/7/1351

<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_loci-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_loci-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_loci-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.