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Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize
Author(s) -
Couillerot O.,
Poirier M.A.,
PrigentCombaret C.,
Mavingui P.,
CaballeroMellado J.,
MoënneLoccoz Y.
Publication year - 2010
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2010.04673.x
Subject(s) - microbial inoculant , azospirillum brasilense , biology , horticulture , geography , humanities , art , inoculation
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 × 10 3 (for UAP‐154) and 4 × 10 4 CFU g −1 (for CFN‐535) in the maize rhizosphere. Inoculant quantification was effective from 10 4 to 10 8 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.