Partners and International Organizations
(English)
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AT, BE, BG, CH, CY, CZ, DE, DK, ES, FI, FR, GR, HU, IT, LT, NL, NO, PL, PT, SI, UK
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Abstract
(English)
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Oomycetes, such as Phytophthora, downy mildew causal agents, and Pythium, form a unique branch of eukaryotic-plant pathogens with an independent evolutionary history (Kamoun, 2003). The Genus Phytophthora comprises a devastating group of plant pathogens existing in a wide range of ecological niches, causing severe losses of cultivated crops or forestry and having a serious impact on native vegetation. Although more than 80 species have been formally identified, their classification is in constant evolution. Species identification is a specialised and time consuming task and the true extent of diversity in the genus remains unknown. The principal reasons for such difficulties are: - Phytophthora is often associated with stem bases and roots in which more than one species may be associated as a disease complex. Diagnosis and detection are therefore troublesome. - Even when isolated there are few reliable morphological markers to discriminate species. Many isolates are therefore misidentified or remain as 'unknown Phytophthora sp.' Such inadequacy may result in delaying identification of new threats and failures to rapidly evaluate current problems. The development of an accurate and reliable detection and identification system is particularly relevant for Phytophthora diseases. Commercial kits based on serological methods are rapid, but insufficiently discriminatory. To date, genotypic methods are time-consuming and sometimes labor-intensive, but they provide better results in terms of reproducibility and species discrimination. Several molecular targets have been successfully explored, including ITS rRNA (Cooke et al., 2000), Cox I and Cox II (Martin and Tooley, 2003) and more recently Translation Elongation Factor 1?, ?-Tubulin, Cytochrome c Oxidase subunit 1, P4, NADH dehydrogenase subunit 1 and P2 genes and regions (Kroon et al., 2004). Microarray technology is increasingly used for assessing genomic diversity, gene expression and genotyping. This technology has the potential to combine species identification, speciation, typing, detection of virulence factors and pesticide resistance profiling in a single experiment. Microarrays dedicated to identification and testing of plant pathogens have been successfully used such as for viruses (Boonham et al., 2003; Lee et al., 2003; Bystricka et al., 2003, Deyong et al., 2005). A first DNA array designed and tested by authors, though successful for the detection of many Phytophthora species, suffered from serious drawbacks due to the use of single loci detection units (Belbahri et al, unpublished). We showed in this first array that identification at the subspecies level was not possible using ITS rDNA for some species, similar to what had been previously described for other taxonomic markers in other organisms. In the framework of this project a Phytophthora identification high-density microarray containing a high number of oligonucleotide probes complementary to seven diagnostic units recently described in Phytophthora (Kroon et al., 2004) have been successfully designed. The microarray allowed detection based on the sequence-specific hybridization of regions characteristic of each Phytophthora species. Initial testing of the array showed promising results as expected. However, through testing of this array is necessary and is actually being developped. The ability to reliably identify these taxa diversity in complex substrates such as soil and water is also implemented.
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