title: Development of sequence-characterized amplified region (SCAR) markers as a quality standard of inoculants based on Azospirillum.
abstract: An attempt was made in this work to develop a strain-level molecular marker for unambiguous authentication of two Azospirillum inoculants, viz. A. lipoferum (strain Az204) and A. brasilense (strain Sp7). The sequence-characterized amplified region (SCAR) markers obtained from DNA fingerprints were designed for discrete detection of the strains. The SCAR primers could successfully amplify the target strain without cross-reaction with other Azospirillum strains, native isolates and other inoculants. The detection limit of SCAR primer for Az204 was 8.00 pg of DNA (approximately 10(5) cells per mL), and for Sp7, it was 0.49 pg of DNA (equal to 10(4) cells per mL). A simplified Sephadex G100-based crude DNA extraction protocol developed in this study was found suitable for SCAR marker-based strain authentication. Further, SCAR primers were assessed for simultaneous authentication as well as quantification of commercially prepared Azospirillum inoculants by quantitative real-time PCR (RT-PCR) and most-probable-number PCR (MPN-PCR). The RT-PCR assay can be able to quantify the commercial formulations as equal to culturable MPN method, while MPN-PCR failed for Az204. The SCAR marker-based strain authentication and presumptive quantification developed in the present work can contribute to improving the quality standard of commercial inoculants.
authors.affiliation: Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: Pasupuleti
authors.initials: P
authors.lastname: Reddy Priya
authors.affiliation: Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: Raju
authors.initials: R
authors.lastname: Selastin Antony
authors.affiliation: Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, 641003, India.
authors.forename: Ganesan
authors.initials: G
authors.lastname: Gopalaswamy
authors.affiliation: Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, 641003, India. dbalu@tnau.ac.in.
authors.forename: Dananjeyan
authors.initials: D
authors.lastname: Balachandar
mesh_terms: D016949:Azospirillum / Q000145:classification* / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000379:methods* / Q000592:standards; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000235:genetics; D005819:Genetic Markers; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D005819:Genetic Markers
keywords: Azospirillum; Inoculants; Quality standards; Sequence-characterized amplified region markers; Strain authentication