are now becoming available. This has been possible due to the introduction of new molecular technologies including real-time PCR, microarrays and direct sequencing. 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 e.g. 'Padlock' probes. 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. Further advances include the development of low-density conventional and real-time PCR arrays, which offer a combination of high sensitivity and simultaneous multi-target detection. Potential future developments include the use of micro-fluidic card technology. 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 overcoming some long-stand problems.
Rapid detection of brown rot and ring rot in potato tubers based on sensors for volatile organic compounds
Ben de Lacy Costello1, Katy Garner1, Norman Ratcliffe1, Tanja Schleicher2 & Peter Spencer-Phillips2
1Centre for Research in Analytical, Materials & Sensor Science, 2Centre for Research in Plant Science, University of the West of England, Coldharbour Lane, Bristol BS16 1 QY, UK.
A sensor system for the early detection of Erwinia soft rot in stored potato tubers has been developed recently (de Lacy Costello et al., In: Pests & Diseases 2002, Volume 1, pp. 425-432. British Crop Protection Council, Brighton, UK. 2002). We are now using a similar approach, based on detecting volatile organic compounds (VOCs), to design a portable system for use by the Plant Health Inspectorate. The specific aim is to develop a device that will enable exclusion from the UK of the statutory organisms Ralstonia solanacearum and Clavibacter michiganensis ssp. sepedonicus by screening all imported consignments of potato tubers. Protocols were developed for inoculating tubers of the potato cv. Maris Piper with R. solanacearum (brown rot), C. michiganensis ssp. sepedonicus (ring rot) and Erwinia carotovora subsp. carotovora (soft rot). Inoculated tubers developed the symptoms characteristic for each disease. The VOCs produced were collected over a 3 week period post-inoculation using solid phase micro-extraction (SPME) fibres. A sampling procedure was established whereby air from 2-litre jars containing circa 900 g of inoculated tubers was circulated (at 400 ml min-1) via a pump over the SPME fibre for 1 h at 20oC. The compounds were thermally desorbed from the fibres and then analysed using Gas Chromatography Mass Spectrometry (GCMS) to identify VOCs and establish markers for the infections. Distinct patterns of VOCs were obtained for ring rot, brown rot and soft rot infected tubers when compared with each other and with VOCs from other common storage pathogens (Fusarium coeruleum and Phytophthora infestans; for references, see de Lacy Costello et al., 2002). The major VOCs changed over the three week period, with the largest number present at 3 weeks post-inoculation (59, 78 and 62 VOCs for brown rot, ring rot and soft rot, respectively). Each pathogen produced a unique profile of VOCs, as well as specific marker compounds. Controls of both sound tubers and sterile distilled water inoculated tubers produced 21 major VOCs that remained constant throughout the 3 week period. Comparison of the laboratory data with VOCs collected from tubers of cv. Provento, obtained from the recent UK outbreak of ring rot, indicated that a detection system based on specific markers would be problematic in the field. Recent work has focused instead on methods for distinguishing the metabolomic profiles of the most abundant VOCs generated by brown rot and ring rot infections of tubers.
Plant cultivars, plant pathogens and plant pathologists: Managing the interactions
UMR INRA-Agrocampus Rennes BiO3P, Domaine de la Motte, BP 35327, F-35653 Le Rheu cedex, France. firstname.lastname@example.org
Cultivar resistance is an essential asset to develop durable control strategies of most major plant pathogens. However, despite its obvious advantages in terms of cost, environmental impact, and performance, resistance remains largely under-exploited in current agriculture, both in developed and developing countries. Based on examples from recent work on the management of potato diseases, this paper offers a reflection on possible reasons for this situation, along three main lines:
? There is a lot of information available now on host pathogen interactions, the genetics of resistance, and pathogenicity, but a lot of this information either is not or cannot be used directly for the development of applicable control methods, while we still know very little about the adaptive processes in pathogen populations which condition the efficacy and durability of resistance;