Molecular diagnosis of plant pathogenic bacteria

Abstract AR

-Real-time, fluorogenic, PCR assays have recently shown great promise in the diagnosis of many plant pathogenic bacteria.

In such assays repeated PCR cycles result in exponential amplification of the PCR product and a corresponding increase in fluorescence intensity, providing “real-time” analysis of the reaction kinetics and allowing quantification of specific DNA targets.

As no post-PCR processing steps (such as gel electrophoresis) are required such assays also lend themselves to high throughput screening of samples.

Assays that detect Ralstonia solanacearum, Agrobacterium spp., and Xanthomonas fragariae have been developed at CSL, and assays for Clavibacter michiganensis subsp.

sepedonicus and Erwinia amylovora have been developed elsewhere.

The key in the development of any test is the selection of an appropriate target DNA sequence and the development of a suitable DNA extraction protocol, directly from plant material.

A real-time PCR assay (recently developed at CSL) which detects the strawberry angular leaf spot pathogen Xanthomonas fragariae (Xf) was designed using sequence data obtained from the housekeeping gyraseB gene.

Although this gene is found in all bacteria, unique sequences were found for use as Xf specific PCR primers and probe, when compared to gyraseB sequence data obtained from closely related bacteria.

In conjunction with a rapid and sensitive DNA extraction protocol this assay can detect the pathogen at 103 cells per reaction – a population level associated with latent infections by Xanthomonas fragariae.