A Rapid, Sensitive Assay for Ralstonia solanacearum Race 3 Biovar 2 in Plant and Soil Samples Using Magnetic Beads and Real-Time PCR.

Abstract

The Ralstonia solanacearum species complex causes economically significant diseases in many plant families worldwide. Although generally limited to the tropics and subtropics, strains designated race 3 biovar 2 (R3Bv2) cause disease in cooler tropical highlands and temperate regions. R3Bv2 has not become established in North America but, due to concerns that it could devastate the U.S. potato industry, it has been designated a Select Agent, and is subject to strict quarantine regulations. Quarantine screening for R3Bv2 requires rapid and robust assays applicable to small populations present in plant tissues or soil, and must distinguish R3Bv2 from the multiple other R. solanacearum subgroups. We developed a 100%-accurate real-time polymerase chain reaction (RT-PCR) assay that can detect R3Bv2 populations >1,000 cells ml-1. However, detection by RT-PCR was inhibited by compounds present in some plant and soil samples. Therefore, we developed simple immunomagnetic separation (IMS) and magnetic capture hybridization (MCH) methods to purify R. solanacearum cells or DNA from PCR inhibitors. When coupled with RT-PCR, these tools permitted detection of R3Bv2 at levels >500 cells ml-1 in stem, tuber, and soil samples when direct RT-PCR failed, and reduced detection time from days to hours. IMS-RT-PCR was usually more sensitive than MCH-RT-PCR, especially at lower population levels.