Abstract
TaqMan-based quantitative PCR (qPCR) assays were developed to study the persistence of two well-characterized strains of plant growth-promoting rhizobacteria (PGPR), Pseudomonas fluorescens Pf153 and Pseudomonas sp. DSMZ 13134, in the root and rhizoplane of inoculated maize plants. This was performed in pot experiments with three contrasting field soils (Buus, Le Caron and DOK-M). Potential cross-reactivity of the qPCR assays was assessed with indigenous Pseudomonas and related bacterial species, which had been isolated from the rhizoplane of maize roots grown in the three soils and then characterized by Matrix-Assisted Laser Desorption Ionization (MALDI) Time-of-Flight (TOF) mass spectrometry (MS). Sensitivity of the qPCR expressed as detection limit of bacterial cells spiked into a rhizoplane matrix was 1.4 × 102 CFU and 1.3 × 104 CFU per gram root fresh weight for strain Pf153 and DSMZ 13134, respectively. Four weeks after planting and inoculation, both strains could readily be detected in root and rhizoplane, whereas only Pf153 could be detected after 8 weeks. The colonization rate of maize roots by strain Pf153 was significantly influenced by the soil type, with a higher colonization rate in the well fertile and organic soil of Buus. Inoculation with strain DSMZ 13134, which colonized roots and rhizoplane to the same degree, independently of the soil type, increased yield of maize, in terms of biomass accumulation, only in the acidic soil of Le Caron, whereas inoculation with strain Pf153 reduced yield in the soil Buus, despite of its high colonization rate and persistence. These results indicate that the colonization rate and persistence of inoculated Pseudomonas strains can be quantitatively assessed by the TaqMan-based qPCR technique, but that it cannot be taken for granted that inoculation with a well-colonizing and persistent Pseudomonas strain has a positive effect on yield of maize.
Highlights
Plant growth-promoting rhizobacteria (PGPR) are able to facilitate plant nutrient acquisition and can act as biocontrol agents by suppressing soil-borne diseases (Lucy et al, 2004)
As expected from the Matrix-Assisted Laser Desorption Ionization (MALDI)-TOF cluster diagram (Figure 1) and the in silico sequence comparison of the dnaX gene, the tracing tool for strain DSMZ 13134 showed a strong cross-reactivity with Pseudomonas protegens CHA0, Pseudomonas protegens Pf-5 and Pseudomonas chlororaphis LMG 1245
The quantitative PCR (qPCR) tools developed for this study have been successfully applied for quantitative tracing of the two bacterial strains in environmental samples
Summary
Plant growth-promoting rhizobacteria (PGPR) are able to facilitate plant nutrient acquisition and can act as biocontrol agents by suppressing soil-borne diseases (Lucy et al, 2004). The improved plant biomass and increased phosphorus acquisition by a plant after inoculation of a competent strain were found to be higher in soils which have a low microbial biomass and activity (Fliessbach et al, 2009; Mäder et al, 2011) and low levels of plant available phosphorus (Egamberdiyeva, 2007) Another reason explaining the inconsistency of these effects is explained by the poor survival of the inoculant in the soil (Khan et al, 2007). Survival ability of PGPR is influenced by the abundance and the composition of the indigenous microbiota (Strigul and Kravchenko, 2006) and by soil chemical and physical properties, such as pH, clay and soil organic matter content (Hartel et al, 1994; Bashan et al, 1995) In this context, the objective of the presented study was to develop a quantitative qPCR assay allowing the specific assessment of the colonization rate by two PGPR inoculants and enabling multiplex reactions. A secondary objective was to study the plant responses to PGPR application
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