REVIEW ARTICLE Innovative tools for detection of plant pathogenic viruses and bacteria Received: 6 June 2003 / Accepted: 15 July 2003 / Published online: 11 September 2003 Ó Springer-Verlag and SEM 2003 Abstract Detection of harmful viruses and bacteria in plant material, vectors or natural reservoirs is essential to ensure safe and sustainable agriculture. The tech- niques available have evolved significantly in the last few years to achieve rapid and reliable detection of patho- gens, extraction of the target from the sample being important for optimising detection. For viruses, sample preparation has been simplified by imprinting or squashing plant material or insect vectors onto mem- branes. To improve the sensitivity of techniques for bacterial detection, a prior enrichment step in liquid or solid medium is advised. Serological and molecular techniques are currently the most appropriate when high numbers of samples need to be analysed. Specific monoclonal and/or recombinant antibodies are avail- able for many plant pathogens and have contributed to the specificity of serological detection. Molecular detection can be optimised through the automatic purification of nucleic acids from pathogens by columns or robotics. New variants of PCR, such as simple or multiplex nested PCR in a single closed tube, co-oper- ative-PCR and real-time monitoring of amplicons or quantitative PCR, allow high sensitivity in the detection of one or several pathogens in a single assay. The latest development in the analysis of nucleic acids is micro- array technology, but it requires generic DNA/RNA extraction and pre-amplification methods to increase detection sensitivity. The advances in research that will result from the sequencing of many plant pathogen ge- nomes, especially now in the era of proteomics, repre- sent a new source of information for the future development of sensitive and specific detection tech- niques for these microorganisms. Keywords Antibodies Æ Co-operational PCR Æ DNA microarrays Æ ELISA Æ Enrichment Æ FISH Æ Multiplex PCR Æ Nested-multiplex PCR Æ Real time PCR Introduction Plant pathogenic viruses and bacteria are responsible for increasing economic losses worldwide. They can cause a large range of symptoms in most cultivated plants, which can be affected in their different parts with various agronomic impact. Viruses and bacteria cause plant diseases that are difficult to control because of the lack of efficient products for chemical treatment under field conditions. Consequently, preventive measures to avoid planting of contaminated material are of the highest importance in the context of an integrated approach to control. Among such measures, testing of planting material for pathogen-free status is an important, al- though not exclusive, method for controlling bacterial and viral diseases of plants [26, 36]. As many pathogenic viruses and bacteria remain latent in the planting material, and in very low numbers, methods of high sensitivity, specificity and reliability are required. Public institutions and the agro-food industry used to control the sanitary quality of seeds, fruits and plant material by microbiological testing for bacteria and biological indexing for viruses. These methods were often expen- sive and time-consuming and some of them were not sensitive and specific enough. In addition, biological indexing cannot be applied on the large scale required. Detection deals with establishing the presence of a particular target organism within a sample, with special emphasis on symptomless individuals. Diagnosis relates to the identification of the nature and cause of the dis- ease problem, thus concerning plants showing symptoms [35]. Accurate routine disease detection requires high levels of specificity, sensitivity and speed. In this context, specificity is defined as the capability to detect the Int Microbiol (2003) 6: 233–243 DOI 10.1007/s10123-003-0143-y Marı´a M. Lo´pez Æ Edson Bertolini Antonio Olmos Æ Paola Caruso Marı´a Teresa Gorris Æ Pablo Llop Ramo´n Penyalver Æ Mariano Cambra M. M. Lo´pez (&) Æ E. Bertolini Æ A. Olmos Æ P. Caruso M. T. Gorris Æ P. Llop Æ R. Penyalver Æ M. Cambra Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial. 46113, Moncada, Valencia, Spain E-mail: mlopez@ivia.es Tel.: +34-96-3424000 Fax: +34-96-3424001