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Contenu archivé le 2024-04-30

Health certification of rosaceous species based on disease-indexing of in vitro plants : validation of diagnostics and diagnostic strategies

Objectif

In vitro culture techniques are used for mass clonal propagation and disease elimination from germplasm of fruit tree and strawberry cultivars. However disease-indexing is carried out on the donor plants prior to culture or is based on the field progeny. On the one hand this situation results from the fact that many diseases affecting rosaceous species are of unknown etiology, thus are only detectable in lengthy and cumbersome field tests using indicator plants. On the other hand even for better investigated pathogens (viruses, phytoplasma) there exists a lack of rapid, reliable, sensitive and user-friendly laboratory methods for routine large-scale application to detect the harmful organisms.

It is within the objectives of the proposed project to develop and assess broad spectrum and specific assays for detection of filamentous, bacilliform and nematode-transmitted viruses. Broad spectrum assays, which means a single test, that is able to check for the presence of a range of pathogens, including previously uncharacterized ones, are an ultimate request in certification programmes, as the primary testing purpose is to check for freedom from pathogens. Phytoplasma detection is largely based on universal and group specific primer thus the aim is to optimize and simplify existing diagnostic techniques. The methodology to be used (RT-PCR, PCR-ELISA, NASBA) is based on the amplification of the pathogen genome. Research is necessary to validate the application of diagnostics developed for plant application in vivo to in vitro material. An important aspect of this validation is to determine the influence of in vitro media and culture environment on pathogen titre in in vitro tissues. In vitro plant growth conditions that maximise titre to facilitate detection and to avoid pathogen suppression will be defined. In vitro germplasm collections of fruit tree species, strawberry and related soft crops have been established in the partners laboratories. Virus-host interactions, that is, tissue specific accumulation of virus and long-distance spread, will be compared under autotrophic and heterotrophic in vitro conditions to optimise sampling from in vitro plants. Elimiation procedures, such as meristem culture, heat therapy, chemotherapy, tretracycline treatment and combinations thereof, will be compared to evaluate their effectivity to eliminate recalcitrant pathogens. From traditional sanitation programmes it is well known that elimination measures may suppress the pathogen titer below the threshold of detection. The number of subcultures following therapy that allows the pathogen to recover (in case that the elimination treatment failed) will be determined. Serodiagnostics currently in use are of little value for early screening as viral proteins are affected through elimination treatment. The diagnostic assay developed, that proved to be the most sensitive will be used to confirm the indexing. Broad spectrum and specific assays will be compared with the aim to shorten the indexing period and the number of tests. All results obtained from the proposed proposal will be consolidated into guidelines to be proposed for the certification of rosaceous species in vitro. In vitro culture, broad spectrum and specific assays, disease-indexing in vitro, pathogen elimination in vitro.

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UNIVERSITY OF AGRICULTURE
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Adresse
Muthgasse 18
1190 WIEN
Autriche

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