Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings - Université Claude Bernard Lyon 1 Accéder directement au contenu
Article Dans Une Revue Soil Biology and Biochemistry Année : 2010

Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings

Résumé

Azospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 104 CFU equivalents g−1 lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 104 CFU equivalents g−1. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants.

Dates et versions

hal-00874738 , version 1 (18-10-2013)

Identifiants

Citer

Olivier Couillerot, Marie-Lara Bouffaud, Baudoin Ezékiel, Daniel Muller, Jesus Caballero-Mellado, et al.. Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings. Soil Biology and Biochemistry, 2010, 42 (12), pp.2298-2305. ⟨10.1016/j.soilbio.2010.09.003⟩. ⟨hal-00874738⟩
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