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Real-Time PCR Quantification of the Plant Growth Promoting Bacteria Herbaspirillum seropedicae Strain SmR1 in Maize Roots

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Abstract

The plant growth promoting bacteria Herbaspirillum seropedicae SmR1 is an endophytic diazotroph found in several economically important crops. Considering that methods to monitor the plant–bacteria interaction are required, our objective was to develop a real-time PCR method for quantification of PGPB H. seropedicae in the rhizosphere of maize seedlings. Primer pairs were designed, and their specificity was verified using DNA from 12 different bacterial species. Ten standard curves of qPCR assay using HERBAS1 primers and tenfold serial dilutions of H. seropedicae SmR1 DNA were performed, and PCR efficiency of 91 % and correlation coefficient of 0.99 were obtained. H. seropedicae SmR1 limit of detection was 101 copies (corresponding to 60.3 fg of bacterial DNA). qPCR assay using HERBAS1 was used to detect and quantify H. seropedicae strain SmR1 in inoculated maize roots, cultivated in vitro and in pots, harvested 1, 4, 7, and 10 days after inoculation. The estimated bacterial DNA copy number per gram of root was in the range 107–109 for plants grown in vitro and it was around 106 for plants grown in pots. Primer pair HERBAS1 was able to quantify H. seropedicae SmR1, and this assay can be useful for monitoring plant–bacteria interaction.

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Acknowledgments

We would like to express our gratitude to Fábio de Oliveira Pedrosa, Leda Chubatsu, and Michelle Tadra-Sfeir, Universidade Federal do Paraná, for providing A. brasilense strain FP2, H. seropedicae strain SmR1, and other Herbaspirillum, to Luciane Passaglia for providing Rhizobium, Microbacterium, and Pseudomonas. This work was financially supported by the National Institute of Science and Technology—Biological Nitrogen Fixation, INCT-FBN, CNPq, Ministry of Science and Technology, Brazil. TPP, FPA, and PD were recipients of Master and PhD fellowships from CAPES, Ministry of Education, Brazil. FCAB is recipient of post-doctoral fellowship from CAPES PNPD, and ACMA is recipient of research fellowship from CNPq (PQ-2).

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  1. Departamento de Ciência e Tecnologia de Alimentos, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Rod. Admar Gonzaga, 1346, Florianópolis, SC, 88034-001, Brazil

    Tomás Pellizzaro Pereira, Fernanda Plucani do Amaral, Pamela Dall’Asta, Fábio Cristiano Angonesi Brod & Ana Carolina Maisonnave Arisi

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  1. Tomás Pellizzaro Pereira
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  2. Fernanda Plucani do Amaral
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Correspondence to Ana Carolina Maisonnave Arisi.

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Pereira, T.P., do Amaral, F.P., Dall’Asta, P. et al. Real-Time PCR Quantification of the Plant Growth Promoting Bacteria Herbaspirillum seropedicae Strain SmR1 in Maize Roots. Mol Biotechnol 56, 660–670 (2014). https://doi.org/10.1007/s12033-014-9742-4

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