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

Fumito Maruyama,Joaquin I Rilling,Jacquelinne J Acuña,Milko A Jorquera,Michael J Sadowsky

doi:10.3390/microorganisms9071351

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.

Highlights

Biofertilizer products based on microbial inoculants have been marketed since the late 1800’s and are currently commercialized in many countries
We report the development of a CRISPRloci -PCR approach based on the combined use of a designed primer set for clustered regularly interspaced short palindromic repeats (CRISPR) loci and a standardized endpoint PCR reaction for the strain-specific detection of Azospirillum sp. strain B510 (Figure S1)
Results this analysis only for Azospirillum s as of determined by flowrevealed cytometry, amplification and this was equivalent to ~15.1 ngfrom μL DNA

Summary

Introduction

Biofertilizer products based on microbial inoculants have been marketed since the late 1800’s and are currently commercialized in many countries. Several types of microbial inoculants containing nitrogen-fixing microorganisms are marketed and include symbiotic legume root and stem nodule bacteria (rhizobia) as well as free-living diazotrophic plant growthpromoting rhizobacteria (PGPR), such as Azospirillum. In this context, members of the genus Azospirillum are among the most studied and used soil microbial inoculants for non-legume crops (e.g., cereals). Members of the genus Azospirillum are among the most studied and used soil microbial inoculants for non-legume crops (e.g., cereals) These inoculants are extensively used because of their growth-promoting functions, mainly due to their ability to fix atmospheric nitrogen (N).

Methods

Results

Conclusion