Accurate identification of fungal phytopathogens is essential for virtually all aspects of plant pathology, from fundamental research on the biology of pathogens to the control of the diseases they cause. Although molecular methods, such as polymerase chain reaction (PCR), are routinely used in the diagnosis of human diseases, they are not yet widely used to detect and identify plant pathogens. Here we review some of the diagnostic tools currently used for fungal plant pathogens and describe some novel applications. Technological advances in PCR-based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens and are now being applied to practical problems. Molecular methods have been used to detect several pathogens simultaneously in wheat, and to study the development of fungicide resistance in wheat pathogens. Information resulting from such work could be used to improve disease control by allowing more rational decisions to be made about the choice and use of fungicides and resistant cultivars. Molecular methods have also been applied to the study of variation in plant pathogen populations, for example detection of different mating types or virulence types. PCR-based methods can provide new tools to monitor the exposure of a crop to pathogen inoculum that are more reliable and faster than conventional methods. This information can be used to improve disease control decision making. The development and application of molecular diagnostic methods in the future is discussed and we expect that new developments will increase the adoption of these new technologies for the diagnosis and study of plant disease.

http://www.rothamsted.bbsrc.ac.uk/ppi/links/pplinks/plasmod/index.html, http://www.dpvweb.net/, http://www.rothamsted.bbsrc.ac.uk/ppi/Iwgpvfv/index.html, http://www.rothamsted.bbsrc.ac.uk/ppi/links/pplinks/bymoviruses/index.html, http://oak.cats.ohiou.edu/~braselto/plasmos/

Accurate disease diagnosis and precise identification of any pathogens involved is an essential prerequisite for understanding plant diseases and controlling them effectively. Traditional methods of identifying plant pathogens can be slow and inconclusive, and this has prompted the search for alternative diagnostic techniques. Here, we review recent developments in antibody-based and nucleic acid-based diagnostic methods, and their applications in plant pathology. We also highlight some new developments in diagnostics that are likely to make an impact in the future.

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.Conventional methods for identifying and enumerating airborne fungi and other microorganisms rely on microscopic or cultural techniques and, as a consequence, are time-consuming and laborious. Additionally, microscopy is unreliable for detection of the small, nondescript spores produced by many fungi, while cultural techniques are unsuitable for detection of spores that are slow growing, or nonculturable in vitro and the choice of medium may influence which species can grow. These difficulties have restricted the use of routine air sampling in the study of plant, animal, and human diseases.Recently, however, molecular methods have been used in the development of diagnostic tests for a variety of fungi involved in plant diseases (8,25,26,28,30). While the potential of these techniques for detection of airborne spores has been recognized for some time (12,14), there have been few reports on progress in this area. The use of immunoassay in the detection of airborne plant pathogenic fungi has been investigated (10,20,21). However, the application of these methods is restricted by the difficulties of developing antibodies showing the required specificity. DNA-based detection methods offer greater potential for sensitive and specific detection, and some progress has been made in the detection of airborne bacteria using these techniques (1, 2, 9, 15, 17). Progress with fungi has been slower, although dete...

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