Enumeration of plant-growth promoting bacteria Herbaspirillum seropedicae viable cells by a new propidium monoazide combined with quantitative PCR (PMA-qPCR) assay

Abstract

Abstract The plant growth-promoting bacteria Herbaspirillum seropedicae is an endophytic diazotroph found in several economically important crops. Reliable, high-yield, and cost-effective methods are needed to determine bacterial viability in inoculant formulations or in plant. The present study aims to develop a PMA-qPCR assay to evaluate viable cells of H. seropedicae in pure culture and maize roots grown in vitro. H. seropedicae grown in a culture medium was submitted to heat treatment at 48°C for different periods of time. Maize roots were inoculated, grown in vitro and collected seven days after inoculation. The bacteria viable cells were quantified using qPCR, PMA-qPCR assays, and plate counting. Standard curves were prepared, and the efficiency obtained ranged from 85 to 99%. The limit of detection (LOD) was 101 genome copies, corresponding to 60.3 pg of DNA. Enumeration obtained in pure cultures by qPCR, PMA-qPCR and plate count were 8.85 ± 0.16, 6.51 ± 0.12 and 2.25 ± 0.30 log CFU.mL− 1 after heat treatment, respectively. These results showed that PMA-qPCR is a powerful approach for quantifying viable and viable but non-culturable cells in inoculants and plants. PMA-qPCR allowed reliable obtained results much faster than culture-dependent methods.