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Development of a Magnetic Capture Hybridization Real-Time PCR Assay for Detection of Tumorigenic Agrobacterium vitis in Grapevines

June 2013 , Volume 103 , Number  6
Pages  633 - 640

Kameka L. Johnson, Desen Zheng, Supaporn Kaewnum, Cheryl Lynn Reid, and Thomas Burr

Plant Pathology and Plant-Microbe Biology, Cornell University, Geneva, NY 14456.

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Accepted for publication 8 January 2013.

Agrobacterium vitis, the causal agent of grape crown gall, can have severe economic effects on grape production. The bacterium survives systemically in vines and, therefore, is disseminated in propagation material. We developed an assay for use in indexing programs that is efficient and sensitive for detecting A. vitis in grape tissue. Initially, real-time polymerase chain reaction (PCR) primers specific for diverse tumorigenic strains of A. vitis were developed using the virD2 gene sequence. To overcome the effects of PCR inhibitors present in plant tissue, DNA extraction methods that included magnetic capture hybridization (MCH), immunomagnetic separation (IMS), and extraction with the Mo Bio Powerfood kit were compared. The assays incorporating MCH or IMS followed by real-time PCR were 10,000-fold more sensitive than direct real-time PCR when tested using boiled bacterial cell suspensions, with detection thresholds of 101 CFU/ml compared with 105 CFU/ml. DNA extraction with the Powerfood DNA extraction kit was 10-fold more sensitive than direct real-time PCR, with a detection threshold of 104 CFU/ml. All three assays were able to detect A. vitis in healthy-appearing grapevine cuttings taken from infected vines.

This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2013.