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

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 useful to determine bacterial viability in inoculant formulations or in plant. We developed a PMA-qPCR assay to enumerate viable cells of Herbaspirillum seropedica in pure culture and inoculated maize roots. The results obtained using qPCR, PMA-qPCR and plate counting were compared. The limit of quantification (LOQ) using qPCR was 2 log genome copies. Enumeration obtained in pure cultures by qPCR, PMA-qPCR and plate count were 8.85, 6.51 and 2.25 log CFU.mL−1, respectively, after 180 min of heat treatment at 48 °C. Enumeration obtained in inoculated roots by qPCR, PMA-qPCR and plate count were 6.1, 3.7 and 4.9 log CFU.g−1, respectively, 7 days after inoculation. These results showed that PMA-qPCR is a powerful approach for quantifying viable and viable but nonculturable cells in inoculants and maize roots. PMA-qPCR allowed reliable obtained results much faster than culture-dependent methods. It is the first PMA-qPCR developed for this important nitrogen-fixing and plant-growth promoting bacteria.