Couillerot O., Bouffaud M.L., Baudoin Ezékiel, Muller D., Caballero-Mellado J., Moënne-Loccoz Y. (2010). Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings. Soil Biology and Biochemistry, 42 (12), 2298-2305. ISSN 0038-0717.
Titre du document
Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings
Couillerot O., Bouffaud M.L., Baudoin Ezékiel, Muller D., Caballero-Mellado J., Moënne-Loccoz Y.
Source
Soil Biology and Biochemistry, 2010,
42 (12), 2298-2305 ISSN 0038-0717
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 x 10(4) CFU equivalents g(-1) lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 10(4) 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.
Plan de classement
Sciences du monde végétal [076]
;
Biotechnologies [084]
