Green Fluorescent Protein-Marked Pseudomonas fluorescens : Localization, Viability, and Activity in the Natural Barley Rhizosphere

Abstract

The gfp-tagged Pseudomonas fluorescens biocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3-to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.

Colonization pattern of primary tomato roots by Pseudomonas fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence, confocal and scanning electron microscopy

“…The non-culturable state has been proposed as being a survival strategy [34], although this has been subject to controversy [6,35]. In agreement with previous works [20,36,37], the demonstration, made in the present study, that most bacteria in the rhizosphere are non-culturable, stresses the importance of taking this physiological state in account when assessing the autoecology of fluorescent pseudomonads introduced into the rhizosphere.…”

“…In most studies, bacterial densities were quantified by dilution plating [23], in others the distribution patterns were characterized by electronic [19] or confocal [18,21,22] microscopical observations. However, a combination of bacterial quantification and colonization pattern description has rarely been achieved [20,24,25] and, even in these studies, data were not related to morphologically different root zones.…”

“…Attention has been given to patterns of root colonization by various fluorescent pseudomonad strains in the rhizosphere of different host-plants [18][19][20][21][22]. In most studies, bacterial densities were quantified by dilution plating [23], in others the distribution patterns were characterized by electronic [19] or confocal [18,21,22] microscopical observations.…”

Microbial succession in the rhizosphere of live and decomposing barley roots as affected by the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil

“…The fate of DR54-BN14 is similar to an earlier report of a 100-fold reduction in CFU in the upper (near seed) rhizosphere during the ¢rst 14 days followed by a slower decline [14]. The poor colonisation of root tips by DR54-BN14 is in agreement with [14,41].…”

Secondary metabolite production facilitates establishment of rhizobacteria by reducing both protozoan predation and the competitive effects of indigenous bacteria

“…The roots were gently removed by shaking off the soil and rhizosphere bacteria were subsequently extracted as described in Normander, Hendriksen & Nybroe (1999). Briefly, the roots were gently rinsed with sterile PBS to remove soil aggregates and placed in 8 mL PBS buffer.…”