Monitoring the biocontrol agent of fire blight Pseudomonas fluorescens EPS62e, by means of Real-Time PCR

摘要

Real-Time PCR has been developed in order to detect and quantify the biological control agent of fireWight Pseudomonas fluorescens EPS62e after field release. RAPD and U-PCR were used to find naturalgenomic markers within the EPS62e genome which differentiate the biocontrol strain against other P.fluorescens strains. Two differential amplified fragments were characterized as SCAR markers, and nosimilarities against known sequences from the GenBank database were found. Two SCAR primer pairswere designed, the SCAR 450 primer pair which amplified a 177 bp fragment and the SCAR 900 primerpair which amplified a 392 bp fragment. Both primers were selected for their specificity against EPS62e,as they amplified neither the 162 strains of P. fluorescens tested, nor the 71 strains of other closelyrelated species analysed. Then, a Real-Time PCR was designed within each SCAR sequence developed,minimising the length of the amplification product and designing a TaqMan~R probe for each fragment.The specificity of both designs was verified. Finally, the new molecular monitoring method wasvalidated against a classical microbiological monitoring method based on dilution plating on selectivemedia and colony forming units counting. The experiment was carried out on blossoms of Golden appletrees in the field and on detached branches in the greenhouse. Blossoms were sprayed with a EPS62eNalmutant suspension and were periodically sampled. Each sample was doubly evaluated by means of Real-Time PCR and dilution plating methods. There were no significant differences between both techniquesregarding to the EPS62e population level estimated. Moreover, the biocontrol agent colonised andsurvived well on apple flowers in field conditions, reaching population values between 10~7 to 10~8cfu/blossom and remaining stable at this level in immature fruit 55 days after inoculation.