title: Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR.
abstract: Azospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N2 fixation. However, one of the most important factors for achieving an increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for the strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on a comparison of the genomic sequences within the same species. The draft genome sequences of A. brasilense FP2 and Sp245 were aligned, and FP2-specific regions were filtered and checked for other possible matches in public databases. Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were specific for the A. brasilense FP2 strain. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and nonsterile growth conditions. In addition, coinoculations with other plant growth-promoting bacteria in wheat were performed under nonsterile conditions. The results showed that A. brasilense FP2 inoculated into wheat roots is highly competitive and achieves high cell numbers (∼10(7) CFU/g [fresh weight] of root) in the rhizosphere even under nonsterile conditions and when coinoculated with other rhizobacteria, maintaining the population at rather stable levels for at least up to 13 days after inoculation. The strategy used here can be applied to other organisms whose genome sequences are available.
authors.affiliation: Department of Biochemistry and Molecular Biology, Federal University of Parana (UFPR), Curitiba, PR, Brazil Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Department for Environmental Sciences, Research Unit Microbe-Plant Interactions, Neuherberg, Germany.
authors.forename: Maria Isabel
authors.initials: MI
authors.lastname: Stets
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Department for Environmental Sciences, Research Unit Microbe-Plant Interactions, Neuherberg, Germany.
authors.forename: Sylvia Maria Campbell
authors.initials: SM
authors.lastname: Alqueres
authors.affiliation: Department of Biochemistry and Molecular Biology, Federal University of Parana (UFPR), Curitiba, PR, Brazil.
authors.forename: Emanuel Maltempi
authors.initials: EM
authors.lastname: Souza
authors.affiliation: Department of Biochemistry and Molecular Biology, Federal University of Parana (UFPR), Curitiba, PR, Brazil.
authors.forename: Fábio de Oliveira
authors.initials: Fde O
authors.lastname: Pedrosa
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Department for Environmental Sciences, Research Unit Microbe-Plant Interactions, Neuherberg, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schmid
authors.affiliation: Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Department for Environmental Sciences, Research Unit Microbe-Plant Interactions, Neuherberg, Germany.
authors.forename: Anton
authors.identifier: http://orcid.org/0000-0002-8152-8098
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: Department of Biochemistry and Molecular Biology, Federal University of Parana (UFPR), Curitiba, PR, Brazil leonardo@ufpr.br.
authors.forename: Leonardo Magalhães
authors.identifier: http://orcid.org/0000-0002-1380-576X
authors.initials: LM
authors.lastname: Cruz
mesh_terms: D015806:Azospirillum brasilense / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D018517:Plant Roots / Q000254:growth & development / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity; D014908:Triticum / Q000254:growth & development / Q000382:microbiology*
chemical_list: D017931:DNA Primers