Abstract
Purpose
Bacterial quantification by qPCR is considered the gold standard for microbial molecular diagnosis. However, a fundamental pre-requisite in this methodology is the designing of specific primers for the bacterium of interest. With the increase in bacterial genome sequencing data in the recent years, it has become possible to design specific primers that can be used to quantify different strains of the same bacterial species.
Methods
To develop a real-time PCR (qPCR) protocol for the specific quantification of Azospirillum baldaniorum Sp245 strain (old Azospirillum brasilense), the Sp245 genome sequence was fragmented into small contigs with 500 base pairs each, and analyzed for similarity against the NCBI non-redundant database. A. baldaniorum-specific contigs were used to design the primers. The best pair of primers was used to quantify these bacteria after inoculation in different cultivars of Brachiaria, grown under field conditions.
Results
Our results showed that the primer pair Sp245p10 was highly specific for the Sp245 strain in the Brachiaria root and shoot field under different conditions. The qPCR assay using these primers showed differences among cultivars in the number of bacteria detected in plants after inoculation. Additionally, the number of bacteria observed in the roots was higher than that in the shoots.
Conclusion
The qPCR methodology using a Sp245 strain-specific primer may be used to monitor A. baldaniorum inoculated into other plants and may find potential application in field experiments.
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Acknowledgements
We would like to express our gratitude to Johanna Döbereiner Biological Resource Center (https://www.embrapa.br/agrobiologia/crb-jd) at Embrapa Agrobiology for providing bacterial strains for this study.
Funding
This research was supported by a research grant from Embrapa (n° 12.13.08.004.00.02.006). Isis C. Soares and Rafael Sanches Pacheco were supported by fellowship from the Coordination of Personnel Improvement of Higher Education (CAPES—finance Code 001), Ministry of Education, Brazil, while Jean Luiz Simões-Araujo is recipient Researcher fellowship from the National Council of Scientific and Technological Development (CNPq, process n° 310642/2019–1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Soares, I.C., Pacheco, R.S., da Silva, C.G.N. et al. Real-time PCR method to quantify Sp245 strain of Azospirillum baldaniorum on Brachiaria grasses under field conditions. Plant Soil 468, 525–538 (2021). https://doi.org/10.1007/s11104-021-05137-y
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DOI: https://doi.org/10.1007/s11104-021-05137-y