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

Contract nr: FAIR-CT97-3889
Project nr: 3889
Project type: SC
Starting date: 01/02/1998
Duration: 39 months
Total cost: 1,842,500 EUR
EC Contribution: 1,500,000 EUR
Scientific Officer: Richard HARDWICK
Research topic: Plant health

Large scale micropropagation of Rosaceous fruit plant species is routine, but solving important challenges concerning evaluation of plant health could considerably improve commercial exploitation of the technology. In the certification of fruit plants, there is an urgent need for the development of rapid, reliable, sensitive and user-friendly methods for detection and identification of harmful organisms. The release of fruit plant cultivars to fruit plant growers takes several years until all diseases - of both known and unknown etiology - affecting a plant species are checked by indexing methods currently in use ("base line" tests are carried out in the field with woody indicators). The combination of eliminating and indexing diseases attacking in vitro plants, using reliable laboratory diagnostics, would considerably reduce the effort and contribute to savings of time, money and labour.

The aim of this project is to develop and assess broad spectrum and specific assays for the detection of filamentous, bacilliform and nematode-transmitted viruses and phytoplasmas, to be used in tissue culture laboratories involved in the production of certified elite propagation material. The availability of broad spectrum assays - which imply a single test able to check for the presence of a range of pathogens, including previously uncharacterised ones - are required in certification programmes, as the primary testing purpose is to check for freedom from pathogens. The methodology to be used for broad-spectrum and/or specific detection tests (RT-PCR, PCR-ELISA, NASBA) is based on the amplification of the pathogen genome. The combination of efficient protocols for pathogen elimination and highly sensitive diagnostics will constitute a considerable step forward in avoiding the spread of disease, and will allow safe commercial transactions of propagating plant material.

Efforts are being made to develop broad specificity and specific assays using molecular biological techniques for detection of the ubiquitous group of filamentous viruses in fruit trees, as well as for the genus of nematode-transmitted viruses and isometric or bacilliform viruses and phytoplasma, including options for pathogen identification. Using similar methodology, this issue will be extended to investigate diseases of - to date - unknown etiology in fruit trees.
From traditional sanitation programmes, it is well known that elimination measures may suppress the pathogen titer below the threshold of detection. Serodiagnostics currently in use are of little value for early screening.
New detection techniques are being developed for in vivo application. However, validation is necessary for in vitro material. An important aspect of this validation is to determine the influence of in vitro media and environmental conditions on pathogen titre in tissue cultures. Pathogen-infected in vitro germplasm collections of fruit tree species, strawberry and related soft crops have been established. Virus-host interactions, such as 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. Elimination procedures, such as meristem culture, heat therapy, chemotherapy, tetracycline treatment and combinations thereof, will be compared, to evaluate their efficiency in eliminating recalcitrant pathogens. 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 of shortening the indexing period and the number of tests. All results obtained from the proposal will be consolidated into guidelines to be proposed for the in vitro certification of Rosaceous species.
Key words: In vitro culture, broad spectrum and specific assays, disease-indexing in vitro, pathogen elimination in vitro.

Current situation/results:
A collection of filamentous fruit tree viruses - such as Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV), or the still poorly investigated Little cherry virus (LChV), Cherry mottle leaf virus (CMLV), Cherry twisted leaf virus (ChTLV), Cherry virus A (CVA) and diseases of currently-unknown etiology - has been established in the field and in the greenhouse. The knowledge gained from research on the molecular biology of the disease agents resulted in the production of detection reagents, which are currently being validated by various partners.
Concerning isometric or bacilliform fruit tree viruses, an IC-RT PCR allowing the simultaneous detection of Prunus necrotic ringspot virus and Apple mosaic virus (PNRSV + ApMV) and a specific PCR assay for detection of Prune dwarf virus (PDV), have been developed.
For Nematode-transmitted fruit crop viruses - such as Arabis mosaic (ArMV), Rasberry Ringspot (RRSV), Strawberry Latent Ringspot (SLRSV), Tomato Ringspot Virus (TomRV) and Tomato Black Ring Virus (TBRV) - a standardised NASBA system for broad spectrum and specific detection was established.
Phytoplasmas cause fruit tree diseases like apple proliferation, pear decline, European stone fruit yellows (including apricot chlorotic leaf roll, leptonecrosis of Japanese plum (Prunus salicina) and decline disorders of peach, European plum and almond), and virescence and green petals symptoms in strawberries. Polymerase chain reaction (PCR) is employed using primers from ribosomal and non-ribosomal fragments of the phytoplasma chromosome. Both universal and group-specific primers are now available for detection of phytoplasmas.
A representative collection of pome-, stonefruit and strawberry pathogens is being established, propagated and stored in vitro. The influence of tissue culture medium, culture environment and plant growth regulators on pathogen titer in plant tissues in vitro are being investigated to determine the conditions under which maximum pathogen titer can be achieved to optimise the pathogen assay, and to determine any possible pathogen suppressive effects of environmental factors. ELISA screening for ASGV, ACLSV, PPV, PNRSV is being compared to the new, most suitable and sensitive approaches of broad spectrum and specific assays and diagnosis is extended to other pathogens listed above. The characterisation of virus-host interactions will provide information on the choice of optimum sample material, optimum developmental stage for sample selection and pathogen spread in host plants.
New protocols for elimination of recalcitrant pathogens and selection of pathogen-free plants are urgently needed. Meristem culture, a combination of heat therapy and meristem culture, a combination of cold treatment and meristem culture, a combination of chemotherapy and meristem culture, a combination of Ribavirin and heat therapy as well as tetracycline treatment are applied in accordance with the pathogen-host combination under investigation. The protocols are being validated for high survival rates of plants and on their effectiveness for pathogen elimination. Success rates in elimination of pathogens show high variation and depend on plant species, pathogen and the type of infection (single or mixed) under investigation.


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