Direct and specific assessment of colonisation of wheat rhizoplane by Pseudomonas fluorescens Pf29A

The efficacy of fluorescent pseudomonads as suppressors of soil-borne diseases is linked to their ability to colonise plant roots. Monitoring the dynamics of biocontrol agents in the rhizosphere should improve the irreliability. We designed a pair of Sequenced Characterised Amplified Region (SCAR) primers specific to Pseudomonas fluorescens Pf29A, based on a specific 700 bp RAPD product selected in a previous work. Primer specificity was tested with DNA samples extracted from rhizospheric soil and rhizoplane of wheat plants grown in two different non-sterile soils. We assessed the total population of Pf29A by PCR and the culturable population by counting a tetracycline-resistant Pf29A transformant producing Green Fluorescent Protein (GFP), on selective medium 5 days after inoculation of non-sterile soil. SCAR primers were specific for Pf29A in both soils. We evaluated the limit of detection to 14.2 fg of target DNA, equivalent to 242 Pf29A cells per cm of wheat root. Culturable populations of Pf29A transformant accounted for 13% and 4% of the total populations 5 days after treatment with 10(3) and 10(7) CFU of transformed Pf29A per gram of soil. The SCAR derived sequence is a good candidate to develop a strain specific and sensitive PCR-quantification of Pf29A available for population dynamic studies in fields. We confirm that only a small proportion of the total Pf29A rhizosphere population is culturable.