Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight

• Published in: FEMS microbiology letters 2005-08, Vol.249 (2), p.343-352
• Main Authors: Pujol, Marta, Badosa, Esther, Cabrefiga, Jordi, Montesinos, Emilio
• Format: Article
• Language: English
• Subjects: Bacteriology; Base Sequence; Biocontrol; Biological and medical sciences; Biological control; Biomonitoring; Blight; DNA Primers; Erwinia amylovora; Fire blight; Flowers; Fundamental and applied biological sciences. Psychology; Gene Amplification; Genetics; Greenhouses; Microbiology; Monitoring; Monitoring methods; Most probable number; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; PCR; Plant Diseases - microbiology; Plants (botany); Polymerase Chain Reaction; Population levels; Pseudomonas fluorescens; Pseudomonas fluorescens - classification; Pseudomonas fluorescens - genetics; Pseudomonas fluorescens - growth & development; Pseudomonas fluorescens - isolation & purification; Pseudomonas fluorescens EPS62e; Random amplified polymorphic DNA; RAPD; SCAR
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/j.femsle.2005.06.029

Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU (g f.w.) −1, which in turn decreased progressively to 4–5 log CFU (g f.w.) −1 after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU (g f.w.) −1, thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.