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


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 10(3) (for UAP-154) and 4 x 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.


CONCLUSIONS
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.


CONCLUSIONS
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.
Couillerot
O
O
 Université de Lyon, F-69622, Lyon, France.| Université Lyon 1, Villeurbanne, France.| CNRS, UMR5557, Ecologie Microbienne, Villeurbanne, France.
Poirier
M-A
MA
 Université de Lyon, F-69622, Lyon, France.| Université Lyon 1, Villeurbanne, France.| CNRS, UMR5557, Ecologie Microbienne, Villeurbanne, France.
Prigent-Combaret
C
C
 Université de Lyon, F-69622, Lyon, France.| Université Lyon 1, Villeurbanne, France.| CNRS, UMR5557, Ecologie Microbienne, Villeurbanne, France.
Mavingui
P
P
 Université de Lyon, F-69622, Lyon, France.| Université Lyon 1, Villeurbanne, France.| CNRS, UMR5557, Ecologie Microbienne, Villeurbanne, France.
Caballero-Mellado
J
J
 Centro de Ciencias Genomicas, UNAM, Apdo. Postal No. 565-A, Cuernavaca, Mor., México.
Moënne-Loccoz
Y
Y
 Université de Lyon, F-69622, Lyon, France.| Université Lyon 1, Villeurbanne, France.| CNRS, UMR5557, Ecologie Microbienne, Villeurbanne, France.
D015806:Azospirillum brasilense / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry; D005819:Genetic Markers; D018517:Plant Roots / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D019105:Random Amplified Polymorphic DNA Technique; D058441:Rhizosphere; D012988:Soil Microbiology; D003313:Zea mays / Q000382:microbiology*
D017931:DNA Primers; D004269:DNA, Bacterial; D005819:Genetic Markers