rev. 4/9/99

Supported by EC

Project FAIR5 PL97-3889

HEALTH CERTIFICATION OF ROSACEOUS SPECIES
BASED ON DISEASE-INDEXING OF IN VITRO PLANTS:
VALIDATION OF DIAGNOSTICS AND DIAGNOSTIC STRATEGIES

Participants

Background

Objectives

TASKS AND EXPECTED RESULTS



Descriptions and background information such as symptomatology, classification and molecular biology on the viruses and phytoplasma diseases concerned with FAIR 3889 can be obtained from the following websites.

Task 1.
Development and assessment of broad spectrum assays for specific detection of pathogen genera/groups and specific assays for identification of the pathogen

Broad spectrum assays and specific assays for detection of filamentous fruit tree viruses

Many of the viruses affecting pome and stone fruit species have filamentous particles, such as the already better characterized Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV) and 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 possibly also diseases of so far unknown etiology.
To achieve the major aim of producing detection reagents and establishing diagnostic assays, the applied methodology includes the collecting of isolates, the isolation of dsRNA from symptomatic trees, cDNA synthesis from dsRNA and molecular cloning in E. coli, sequence analysis and characterization of the genome. Probes, appropriate primer pairs and antisera to be used as detection reagents result from the investigations. Broad spectrum assays using degenerate primers for detection of a range of pathogens including previously uncharacterized ones in a single test and specific assays for pathogen identification are developed based on RT-PCR and IC-RT-PCR.
The best PCR protocols in terms of specificity and polyvalence will be further simplified to reduce hands-on-time (one tube assays, simple sample extraction, non-hazardous product visualisation) and optimized for large-scale screening.
The diagnostics will be evaluated on germplasm cultured in vitro and the protocols then transferred to the tissue culture level for verification on large scale.

Broad spectrum and specific assays for detection of isometric or bacilliform fruit tree viruses

An IC-RT PCR allowing the simultaneous detection of Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV) and a specific PCR assay for detection of prune Dwarf Virus (PDV) have been developed. The multiplex PCR for detection of PNRSV and ApMV and the PCR for PDV detection are validated for polyvalence and transferred for testing in vitro propagated material.

Broad spectrum and specific assays for detection of nematode-transmitted fruit crop viruses

Arabis Mosaic Virus (ArMV), Rasberry Ringspot Virus (RRSV), Strawberry Latent Ringspot Virus (SLRSV), Tomato Ringspot Virus (TomRV) (and http://biology.anu.edu.au/research-groups/MES/vide/descr836.htm) and Tomato Black Ring Virus (TBRV) infect strawberries and related soft crop species as well as many Prunus species and apple.
A standardized NASBA system for broad spectrum and specific detection of nepoviruses is established for use in routine virus-indexing of in vitro plants. This amplification system including a non-radioactive hybridization assay will allow direct detection of nepoviruses: Arabis Mosaic Virus (ArMV), Raspberry Ringspot Virus (RRV) Strawberry Latent Ringspot Virus (SLRV), Tomato Black Ring Virus (TBRV) and Tomato Ringspot Virus (ToRSV) in strawberry in vitro material. The NASBA reaction is based on the primer-dependent, specific amplification of RNA by the concurrent activity of the enzymes AMV RT, RNase H and T7 RNA polymerase at one single temperature (41°C) and without the need of intermediate addition of reagents or thermocycling. The reactions can be performed in any common laboratory incubator. Specific primer sets will be designed, on the basis of known sequences, for virus-specific or group-specific NASBA protocols. A simple and fast method will be developed for sampling and extraction of viral RNA from in vitro material. Virus-specific hybridization assays using non-radioactive labelled probes will be developed for the detection of the NASBA-amplified product. The effectiveness of the NASBA-based systems will be evaluated, then optimized and standardized for routine detection.

Broad spectrum and specific assays for detection of phytoplasma diseases

Fruit tree phytoplasmas (http://www.uniud.it/phytoplasma/index.html) cause apple proliferation, pear decline, and European stone fruit yellows. The latter disease includes apricot chlorotic leaf roll, leptonecrosis of Japanese plum (Prunus salicina) and decline disorders of peach, European plum, and almond. The presence of phytoplasma in strawberry with virescence and green petals symptoms was recently discovered. Polymerase chain reaction (PCR) technology 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. Although PCR is the method of choice in phytoplasma detection, this technology is not widely employed in routine diagnosis. This is mainly because the method is still relatively complicated and not sufficiently efficient for large-scale work. A PCR procedure that is based on either sequence capturing or immuno capturing will avoid laborious DNA extraction. Existing diagnostic techniques for phytoplasma detection will be optimized and simplified for routine use, especially for testing in vitro plants.

Task 2.
In vitro culture and disease-indexing of in vitro pome-, stonefruits and strawberries

Establishment, propagation and storage of pome-, stonefruit and strawberry cultures in vitro.

The establishment and maintenance of in vitro collections of Malus, Pyrus, Prunus (cherry, apricot, plum) and Fragaria and related soft fruit crops is carried out following the experiences gained over several years. In this collection a certain number of cultivars testing positive for the selected target pathogens are maintained, tested and subjected to different elimination treatments. Genotypes carrying pathogens not yet available in tissue culture, will also be established in vitro to serve as models for host-pathogen relationships (see also 3). Pathogen-infected donor material has been provided by the partners focussed on pathogen detection from their institutional pathogen isolate collections either as dormant budwood or grafted plants. The influence of pathogen infection on success rate during establishment will be statistically evaluated.

Disease-indexing of in vitro pome-, stonefruits and strawberries

Research is necessary to validate the application of diagnostics developed for plant application in vivo to in vitro material. The influence of: (a) tissue culture medium, (b) culture environment and (c) plant growth regulators on pathogen titre in plant tissues in vitro will be investigated. The objectives are to determine the conditions under which maximum pathogen titre can be achieved to optimize the pathogen assay and to determine any possible pathogen suppressive effects of environmental factors. The Irish Partners will undertake the proposed factorial experiments with all the pathogen-host model cultures as the necessary facilities and equipment (heterotrophic and (photo) autotrophic plant growth rooms) are available at their institution.
The disease-indexing of in vitro culture collections will also be carried out under conditions optimized for rapid plant micropropagation. ASGV, ACLSV, PPV, PNRSV and PDV have been so far reoutinely detected in in vitro cultures by ELISA and this procedure will in future be extended for screening for all the pathogens listed in 1, as far as antisera are available. The assays described in 1. will be transferred to the tissue culture laboratories through of short term scientists exchanges from both sides. The most suitable and sensitive approaches of broad spectrum and specific assays will be combined for final testing of selected pathogen-free in vitro plants.

Task 3.
Characterization of virus-host interactions in specific virus/plant combinations

Virus distribution throughout in vitro host plants under heterotrophic in vitro conditions

The characterization of virus-host interactions will provide information on (a) choice of optimum sample material, (b) optimum developmental stage for sample selection and (c) pathogen spread in host plants. The distribution patterns of PPV, PNRSV, ASGV and ACLSV in their respective in vitro host plants (Malus domestica and Prunus sp.) will be followed in selected fruit tree viruses during subculture cycles in cases where elimination proved extremely troublesome focussing on the temporal and spatial expression. Novel information will be provided on distribution patterns of selected strawberry viruses (SLRV, ArMV, TBRV and RRV). The methodology used will be immuno-tissue printing and non-isotopic tissue printing hybridization. In addition in-situ RT-PCR will be established for the localisation of the generally low-titred ASGV in tissues.

Comparison of virus distribution throughout in vitro host plants under heterotrophic and autotrophic conditions

Micropropagated strawberry plantlets when cultured autotrophically (growing photosynthetically) develop higher weights and are considerable taller than in vitro plantlets grown on sucrose containing media. Their growth rate and survival rate may be increased when transferred from in vitro to autotrophic conditions. As virus long-distance movement and expression is influenced by the growth habit and tissue organisation the temporal and spatial location and expression of strawberry viruses will be compared when grown under heterotrophic and autotrophic conditions using the methods of 3.1. with the excemption of in-situ RT-PCR.

Task 4.
Elimination of pathogens

New protocols for elimination of recalcitrant pathogen

Heat treatment, chemotherapy and tissue culture techniques have been used either alone or in combination to eliminate viral pathogens from plants. Among these three techniques chemotherapy is the newest and least utilized. Experience from traditional elimination programmes (treatment of potted plants) correlate with recent results from in vitro elimination procedures: 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.
New protocols for elimination of recalcitrant pathogen are validated in accordance with results from selecting pathogen-free plants. 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 will be applied in accordance with the pathogen-host combination under investigation. The protocols will be validated for high survival rates of plants and on their effectiveness for pathogen elimination.
To clarify the mechanisms of pathogen elimination, hybridisation assays and in-situ RT-PCR to investigate, whether viral replication of ASGV takes place in Malus domestica during elimination treatment, analyses on the interaction between Ribavirin and plant growth regulators in vitro in relation to the antiviral activity of Ribavirin and the incorporation of tetracycline step(s) during micropropagation for phytoplasma elimination are foreseen.

Task 5.
Selection of pathogen-free in vitro plants

In sanitation programmes in vitro therapy and in vivo disease-indexing is common practice, resulting in a lack of knowledge on the speed of pathogen recovery, in case that the elimination treatment failed. Disease-indexing of in vitro plants as soon as possible after therapy would allow resources to be concentrated on multiplication and reestablishment of healthy plants. Serodiagnostics are of little value in that respect, as therapy alters or destroys viral protein and therefore molecular biological diagnostics will have to be evaluated. The minimum number of subcultures following pathogen elimination for statistically reliable screening will be determined. The data recorded will provide information on the efficiency of the elimination method applied (see 4). Novel information on the characteristics of virus-host combinations (see 3) and validation of diagnostics (see 1 and 2) will be achieved by then to allow the best sampling and testing strategy. In a second and third ulterior screening of selected negative plants the diagnostic assay that proved to be the most sensitive will be used to confirm the former indexing. Broad spectrum and specific assays will be compared with the aim to shorten the indexing period and the number of tests.
All the partners will be involved in the final selection of methodology. The partners will consolidate all the results obtained from the project into guidelines to be proposed for the certification of rosaceous species in vitro.

Task 6.
Coordination and diffusion

All results obtained from the project will be collected in a booklet to be delivered to the Commission and to be distributed to the interested public as well as to European organizations concerned with the certification of rosaceous species.
The group will contribute information to the elaboration of a booklet containing 2-3 pages per pathogen, covering aspects like biological and molecular characteristics, methods recommended for detection as well as for elimination.
1/14/99 by Siegfried Huss