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

• Published in: Journal of applied microbiology Vol. 109; no. 2; pp. 528 - 538
• Main Authors: Couillerot, O, Poirier, M.-A, Prigent-Combaret, C, Mavingui, P, Caballero-Mellado, J, Moënne-Loccoz, Y
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-08-2010; Blackwell Publishing Ltd; Blackwell; Oxford University Press; Wiley
• Subjects: Amplification; Azospirillum; Azospirillum brasilense; Azospirillum brasilense - classification; Azospirillum brasilense - genetics; Azospirillum brasilense - growth & development; Biochemistry, Molecular Biology; Biodiversity and Ecology; Biological and medical sciences; Colonization; Corn; Deoxyribonucleic acid; DNA; DNA Fingerprinting; DNA Primers - chemistry; DNA, Bacterial - chemistry; Environmental conditions; Environmental effects; Environmental Sciences; Fundamental and applied biological sciences. Psychology; Genetic Markers; inoculant quantification; Life Sciences; Markers; Microbiology; Molecular biology; Monitoring methods; plant growth-promoting rhizobacteria; Plant Roots - microbiology; Polymerase chain reaction; Polymerase Chain Reaction - methods; Primers; Random amplified polymorphic DNA; Random Amplified Polymorphic DNA Technique; real-time PCR; Rhizosphere; root colonization; sequence characterized amplified region markers; Soil Microbiology; Zea mays; Zea mays - microbiology; Monocotyledones; Applied microbiology; Scar; Spirillaceae; Primer; Azospirillum brasilense; real-time PCR; plant growth-promoting rhizobacteria; Design; Gramineae; Angiospermae; Bacteria; Plant growth promoting rhizobacteria; Inoculant; Quantitative analysis; Zea mays; Spirillales; Root; Azospirillum; Corn; sequence characterized amplified region markers; Real time; Polymerase chain reaction; Rhizosphere; inoculant quantification; root colonization; Spermatophyta; Colonization
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2010.04673.x

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. 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. 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. 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.