Assessment of SCAR markers to design real‐time PCR primers for rhizosphere quantification of
            <i>Azospirillum brasilense</i>
            phytostimulatory inoculants of maize

Couillerot, Olivier; Poirier, Marie-Andrée; Prigent‐Combaret, Claire; Mavingui, Patrick; Caballero‐Mellado, Jesús; Moënne‐Loccoz, Yvan

doi:10.1111/j.1365-2672.2010.04673.x

Cited by 40 publications

(24 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The detection limit of the primers (10 3 CFU ml −1 ) was the same between pure and mixed cultures and was similar to or better than those reported previously for other Azospirillum ‐targeted primers (Baudoin et al. 2010; Couillerot et al. 2010).…”

Section: Discussionsupporting

confidence: 86%

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

Shime-Hattori

Kobayashi

Ikeda³

et al. 2011

Journal of Applied Microbiology

View full text Add to dashboard Cite

Aims: To develop a rapid and simple genus‐specific polymerase chain reaction (PCR) method for detecting and identifying isolates of the genus Azospirillum which is well‐recognized as plant growth‐promoting rhizobacterium. Methods and Results: Nine pairs of PCR primers were designed based on the Azospirillum 16S rRNA, ipdC, nifA and nifH genes to assess their genus specificity by testing against 12 Azospirillum (from seven species) and 15 non‐Azospirillum reference strains, as compared with the fAZO/rAZO pair reported by Baudoin et al. (J Appl Microbiol, 108, 2010, 25). Among the primer pairs assessed, the Az16S–A pair designed on the 16S rRNA gene sequence showed the highest genus specificity: it successfully yielded a single amplicon of the expected size in all the 12 Azospirillum strains and for a close relative, Rhodocista centenaria. The PCR with the Az16S–A primers generated a detectable amount of the amplicon from ≥103 CFU ml−1 of Azospirillum cell suspensions even in the presence of contaminants and accurately discriminated Azospirillum and non‐Azospirillum species in both 35 Azospirillum‐like and 70 unknown isolates from plant roots and rhizosphere soils. Conclusions: We developed a rapid and simple PCR method for detecting and identifying Azospirillum isolates within populations of rhizosphere bacteria. Significance and Impact of the Study: The method developed would serve as a useful tool for isolating a variety of indigenous Azospirillum bacteria from agricultural samples.

show abstract

Section: Discussionsupporting

confidence: 86%

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

Shime-Hattori

Kobayashi

Ikeda³

et al. 2011

Journal of Applied Microbiology

View full text Add to dashboard Cite

show abstract

“…The standard curves used for the quantification of A. brasilense FP2 in wheat were constructed as described previously (22), with the following modifications. Wheat plants were grown under axenic condition as described below for 7 days, and roots were collected and crushed in liquid nitrogen using a mortar and pestle.…”

Section: Methodsmentioning

confidence: 99%

“…Quantitative PCR (qPCR) has been the method of choice to quantify rhizosphere populations because it allows high specificity, sensitivity, and speed (17,22,23). This technique has successfully been used to quantify several bacteria associated with plants.…”

mentioning

confidence: 99%

“…The application of strainspecific primers is difficult in field experiments, where closely related indigenous bacteria may interfere with the amplification and quantification. For strain-specific molecular monitoring, sequence-characterized amplified region (SCAR) markers obtained from BOX-PCR, enterobacterial repetitive intergenic consensus sequence-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR fragments were recently applied to design primers for the qPCR quantification of A. brasilense and Azospirillum lipoferum at the strain-specific level (17,22).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR

Stets

Alquéres

Souza

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

bAzospirillum 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 N 2 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 (ϳ10 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.A zospirillum is one of the most important genera of plant growth-promoting rhizobacteria found worldwide under a variety of environmental and soil conditions (1). The diazotroph Azospirillum brasilense is the best-studied species of the genus, is found in close association with many agriculturally important crops, and exerts beneficial effects on plant growth and productivity (2-4). Nitrogen fixation (5, 6) and the production of the auxin 3-indoleacetic acid (IAA) by many representatives of the genus Azospirillum are related to the growth promotion effects observed in inoculated plants, such as increases in root length and the numbers of root hairs and lateral roots (3).The biotechnological use of A. brasilense inoculants in Latin American and in Brazil, in particular, has increased in recent years (7). Strain FP2 is a spontaneous mutant of A. brasilense Sp7 (8). Strain Sp7 has been shown to be capable of stimulating the growth of several members of the family Poaceae and increasing the productivities of wheat and maize crops (2). Strain FP2 can also promote the growth of wheat (9) and enhance maize and wheat productivity under field conditions (unpublished data). Most of the A. brasilense inoculants in B...

show abstract

“…Starting with the assumption that information on the monitoring and quantification of bacteria after inoculation is scarce, and that available techniques typically require sophisticated equipment (COUILLEROT et al, 2010), the concentration of Azospirillum can be determined indirectly by the analysis of plant development and production, varying the doses of inoculant.…”

Section: Introductionmentioning

confidence: 99%

Inoculation of maize withAzospirillum brasilensein the seed furrow

Morais

Brito

Brandão

et al. 2016

Revista Ciência Agronômica

View full text Add to dashboard Cite

-Several studies addressing the inoculation of cereals with diazotrophic microorganisms can be found in the literature. However, in many experiments, investigators have overlooked the feasibility of applying these microorganisms to the furrow together with the seed, and the effect of bacterial concentration on phytostimulation. The aim of this work was to evaluate the effect of doses of an inoculant based on Azospirillum brasilense, applied to the seed furrow when planting maize, combined with different doses of nitrogen fertiliser. The experiment was carried out in the field, in soil of the cerrado region of Brazil. An experimental design of randomised blocks in bands was adopted, comprising nitrogen (40, 100, 200 and 300 kg ha -1 ) and doses of an A. brasilense-based liquid inoculant applied to the seed furrow (0, 100, 200, 300 and 400 mL ha -1 ). The dose of 200 mL ha -1 Azospirillum was noteworthy for grain production. This is the first report of the effective application of Azospirillum in the seed furrow when planting maize in the cerrado region of Brazil.

show abstract

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

Cited by 40 publications

References 52 publications

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

A rapid and simple PCR method for identifying isolates of the genus Azospirillum within populations of rhizosphere bacteria

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR

Inoculation of maize withAzospirillum brasilensein the seed furrow

Contact Info

Product

Resources

About