Quantification of Pseudomonas fluorescens strains F113, CHA0 and Pf153 in the rhizosphere of maize by strain-specific real-time PCR unaffected by the variability of DNA extraction efficiency.
Pseudomonas fluorescens strains F113 and CHA0 are well-known plant growth-promoting rhizobacteria (PGPR) often used as model strains in biocontrol experiments. To monitor their persistence in large scale field experiments, culture-independent methods are needed. In this study, a strain-specific real-time PCR quantification tool was developed based on sequence-characterized amplified regions (SCAR) for P. fluorescens strains F113, CHA0 and Pf153. Differences in DNA extraction efficiencies from rhizosphere samples were circumvented using plasmid APA9 as internal standard to normalize C(T) values after real-time amplification. The detection limits of the real-time PCR assays for all three strains were approximately 10 cells for genomic DNA and 10(4)cells/g rhizosphere for maize samples grown in different natural soils. Population sizes of the three strains in the rhizosphere of maize measured by the new real-time PCR approaches were similar to those measured by most probable number (MPN)-PCR. A persistence study of the three strains indicated that the strains persisted differently over a period of 5weeks. In conclusion the newly developed real-time PCR approach is a fast and resource efficient method for monitoring individual biocontrol strains in natural soil, which makes it an apt quantification tool for future large-scale field experiments.
Von Felten
Andreas
A
Institute of Integrative Biology, Plant Pathology, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland.
Défago
Geneviève
G
Maurhofer
Monika
M
D015169:Colony Count, Microbial / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D018517:Plant Roots / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011551:Pseudomonas fluorescens / Q000235:genetics* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D013997:Time Factors; D003313:Zea mays / Q000382:microbiology*
D004269:DNA, Bacterial