Azospirillum brasilenseviable cells enumeration using propidium monoazide-quantitative PCR

Azospirillum brasilenseis a plant growth promoting bacteria used as an inoculant in diverse crops. Accurate analytical methods are required to enumerate viable cells in inoculant formulations orin planta. We developed a quantitative polymerase chain reaction (qPCR) assay associated to propidium monoazide (PMA) to evaluate the cell viability ofA. brasilensein inoculant and in maize roots.A. brasilensewas grown in culture medium and was exposed to 50 celcius. Maize roots were grown in vitro and harvested 7 days after inoculation. Quantification was performed by qPCR, PMA-qPCR, and plate counting. Standard curves efficiency values ranged from 85 to 99%. The limit of detection was 10(4) CFU per gram of fresh root. Enumeration obtained in maize roots by qPCR where higher than enumeration by PMA-qPCR and by plate counting. PMA-qPCR assay was efficient in quantifying inoculant viable cells and provides reliable results in a quickly and accurately way compared to culture-dependent methods.