Development of a qPCR assay for detection and quantification of rhizogenic Agrobacterium biovar 1 strains

Agrobacterium biovar 1 strains harbouring a Ri-plasmid cause extensive root proliferation (so-called "hairy roots") on hydroponically grown Cucurbitaceae and Solanaceae crops, resulting in severe economic losses. The aim of this study was to develop a SYBR Green-based quantitative real-time PCR (qPCR) assay based on the rol locus of the Ri-plasmid for detection and quantification of these rhizogenic agrobacteria in hydroponic systems. The assay is designed based on all rolB sequences currently available in GenBank and developed using a collection of both target and non-target strains, and was specific for rhizogenic Agrobacterium biovar 1 strains. Based on a calibration with artificially contaminated water samples mimicking hydroponic conditions, unknown bacterial concentrations could be accurately quantified in water samples from surveys carried out in different greenhouses. Further, a detection limit was obtained of less than one cell per mL water, following filtration of a volume of 500 mL. Altogether, our results illustrate the power of this qPCR assay for routine detection of rhizogenic Agrobacterium biovar 1 strains in aqueous samples. Importantly, the assay can be used for pathogen assessment at the presymptomatic stage of infection, enabling adequate control of the disease in hydroponics before plants become infected.