Real-time PCR Detection of Rhodococcus fascians and Discovery of New Plants Associated with R. fascians in Pennsylvania

Rhodococcus fascians is a gram-positive bacterium that causes bacterial fasciation on a wide range of ornamental plants. To address the need for a reliable, sensitive, and specific method for detecting R. fascians in infected plant materials, a real-time (RT) PCR assay was developed. The target for detection was fas-1, a plasmid-borne gene that is essential for virulence. DNAs from all confirmed pathogenic strains of R. fascians consistently tested positive, with detection limit of 30 fg of R. fascians DNA. In repeated PCR experiments with R. fascians pure culture, as few as 2.5 CFU were tested positive. Direct detection of R. fascians from clinical samples of Coreopsis was successful, whereas detection of R. fascians in Chrysanthemum, Pelargonium, Phlox, and Veronica required enrichment on modified D2 (mD2) medium. In the case of geraniums, as few as 10 2 CFU/100 mg plant tissues were successfully detected after 72 h enrichment on mD2. In total, 115 strains, isolated from 41 different kinds of flowering crops in Pennsylvania greenhouses during 1984-2010, were confirmed to be R. fascians by RT PCR. Geraniums and speedwell were the most frequently submitted clinical samples to Pennsylvania Department of Agriculture. The following are the first report of plants associated with R. fascians in PA: Ajania pacifica, Anemone sp., Aruncus sp., Baptisia sp., Eutrochium maculatum, Helianthemum sp., Lewisia sp., Monarda sp., Osteospermum ecklonis, Rudbeckia nitida, and Saponaria ocymoides. Accepted for publication 30 November 2011. Published 27 February 2012.