Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

作者：Couillerot O; Poirier M A; Prigent Combaret C; Mavingui P; Caballero Mellado J; Moenne Loccoz Y^*

来源：Journal of Applied Microbiology, 2010, 109(2): 528-538.

DOI：10.1111/j.1365-2672.2010.04673.x

• Azospirillum
• inoculant quantification
• plant growth-promoting rhizobacteria
• real-time PCR
• root colonization
• sequence characterized amplified region markers

Aims:
To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real-time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP-154 and CFN-535 in the rhizosphere.
Methods and Results:
Primers were designed based on strain-specific SCAR markers and were screened for successful amplification of target strain and absence of cross-reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real-time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 x 103 (for UAP-154) and 4 x 104 CFU g-1 (for CFN-535) in the maize rhizosphere. Inoculant quantification was effective from 104 to 108 CFU g-1 soil.
Conclusion:
BOX-based SCAR markers were useful to find primers for strain-specific real-time PCR quantification of each A. brasilense inoculant in the maize rhizosphere.
Significance and Impact of the Study:
Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation-independent monitoring methods were lacking. The real-time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP-154 and CFN-535.

• 出版日期2010-8