Advances in molecular phytodiagnostics - new solutions for old problems

Abstract

In the last decade, developments in molecular (nucleic acid-based) diagnostic methods have made significant improvements in the detection of plant pathogens. By using methods such as the polymerase chain reaction (PCR), the range of targets that can now be reliably diagnosed has grown to the extent that there are now extremely few, known pathogens that cannot be identified accurately by using laboratory-based diagnostics. However, while the detection of pathogens in individual, infected samples is becoming simpler, there are still many scenarios that present a major challenge to diagnosticians and plant pathologists. Amongst these are the detection of pathogens in soil or viruses in their vectors, high throughput testing and the development of generic methods, that allow samples to be simultaneously screened for large numbers of pathogens. Another major challenge is to develop robust technologies that avoid the reliance on well-equipped central laboratories and making reliable diagnostics available to pathologists in the field or in less-developed countries. In recent years, much of the research carried out on phytodiagnostics has focussed in these areas and as a result many novel, routine diagnostic tests are becoming available. This has been possible due to the introduction of new molecular technologies such real-time PCR and microarrays. These advances have been complemented by the development of new nucleic acid extraction methods, increased automation, reliable internal controls, assay multiplexing and generic amplification methods. With developments in new hardware, field-portable real-time PCR is now also a reality and offers the prospect of ultra-rapid, on-site molecular diagnostics for the first time. In this paper, the development and implementation of new diagnostic methods based upon novel molecular techniques is presented, with specific examples given to demonstrate how these new methods can be used to overcome some long-standing problems.

Keywords: TaqMan; microarrays; molecular diagnostics; on-site testing; phytodiagnostics; real-time PCR.