Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - VirFree (Virus free Fruit Nurseries)

Teaser

Fruit trees and grapevine are propagated vegetatively and are often grafted. Therefore they suffer from pathogens such as viruses and viroids, a number of which are causing severe yield losses and reduce the productive life of the affected plants. In the countries involved in...

Summary

Fruit trees and grapevine are propagated vegetatively and are often grafted. Therefore they suffer from pathogens such as viruses and viroids, a number of which are causing severe yield losses and reduce the productive life of the affected plants. In the countries involved in Virfree the agricultural sector has significant contribution to the national income and agriculture affects a significant portion of their society activities. Fruit trees and grapevine occupy large areas of the respective participants’ countries. Therefore addressing the phytosanitary issue of these crops is of paramount importance. The most efficient way to combat viruses/viroids is the use of high quality pathogen-tested propagative material. Nevertheless, this procedure is not simple and both academia and private sectors are working towards its improvement. Virfree was built towards this direction and brings together academia and private companies to collaborate through their expertise on the following objectives: 1) to identify new viral and viroid strains or species affecting fruit trees and grapevine, 2) to optimise existing and develop novel detection methods and 3) to improve propagation and sanitation methods for producing high quality (virus-tested) plant material of fruit trees and grapevine. In this project diagnostic tools that are currently used in certification programs will be combined with cutting edge technologies such as High Throughput Sequencing (HTS) and Nanobodies. The project has eight academic partners within the EU and associated countries, four within Third Countries and five non-academic partners. The knowledge obtained will be shared among the partners of the proposal and further disseminated to academic institutions, nurseries and other private sectors. Most importantly training of a new generation of researchers will be done in close contact to the needs of both industrial and academic sectors.

Work performed

VirFree consists of four work packages (WP). WP1 is oriented towards the optimization of the use of HTS for the identification and characterization of known and new viruses and viroids in commercial woody plant crops. The performance of different RNA extraction methods in terms of template suitability for the subsequent HTS was assessed. The obtained results showed that the best protocol varied according to the plant species used.
HTS was performed by different partners using a selection of samples. In the runs, samples from woody plants of interest were sequenced on Illumina platforms. Raw data was subjected to analysis using CLC Genomics Workbench v.10.1.1 and Geneious software, while optimizing the pipeline for woody plants. The selected pipeline was able to produce full genome sequences for the high-titer viruses present in the samples, and large contigs that represent partial genomes for the rest of the viruses. So far pipelines for both CLC and Geneious have been developed. The screening of samples revealed the presence of many viruses and viroids in the crops of interest to the beneficiaries of the action. Even though most of the viruses detected represent isolates of already characterized virus species, a number of divergent isolates, known species in new hosts and possibly new species have been detected as well. WP2 is focused on the development of detection methods for the new pathogens found by HTS, as well as the improvement of the existing RT-PCR assays. The acquired sequences of different virus isolates detected in woody plants were incorporated in alignments in order to assess the specificity of the existing RT-PCR assays and redesign the primers where needed. Moreover, RT-PCR assays were developed for the newly detected pathogens in order to establish the spread of these pathogens. LAMP primers were also designed for the sensitive and rapid detection of two emerging viruses and a well characterized one. Two of the assays developed exhibited promising results in terms of sensitivity whereas the third one could not produce reliable results. One of the assays was optimized and its detection range was assessed however, both of them will be further optimized in future secondments. Another goal was the development of novel methodologies based on Nanobodies (Nb) for the detection of viruses. Currently, a nanobody based detection assay is under development for an emerging pathogen and will be assessed in the following months. WP3’s goal is to improve the phytosanitary status of nursery material and optimize the micropropagation methods for fruit trees. During the first two years, in addition to the exchange of knowledge on virus elimination and micropopagation, tree species of interest were micropropagated and attempts were made for their sanitation. Finally, under the scope of WP4 the communication strategy of VirFree was set. Social media outreach was established. The project’s website –www.virfree.eu - was made available to the public. VirFree was communicated to specialists (Conferences) and non-specialists through press releases in the local press and presentations in open public events. So far several articles related to Virfree activities were published.

Final results

The results of VirFree will enrich the knowledge on viruses/viroids associated with different diseases, improve the sensitivity of their detection methods, lead to new detection products and further contribute to the improvement of the disseminated propagative material of fruit trees and grapevine.
More specifically, VirFree will provide an optimized pipeline for the analysis of HTS data in order to detect viruses and viroids in grapevine and woody plants. The use of HTS will identify the virome of the selected crops which will be subsequently characterized in order to provide data regarding the association of the identified pathogens with diseases of unknown etiology. The data collected will be also used on the development of serological or molecular tools for the detection of novel viral/viroid threats or for threats with increased market importance.
The knowledge obtained and the sensitive detection methods developed will be integrated in a best practices protocol in order to produce virus tested nursery material in combination to protocols for the micropropagation and sanitation of stone and pome fruits.
The production of virus tested propagative material is essential for the viability of the production of the crop systems studied in VirFree. This is important at a societal level given that a large number of people in the countries involved in Virfree live on such crops. Moreover within the frame of the project Early Stage Researchers will have the chance to spend time with industrial leaders in their field and interact with other groups of Academic excellence. From a financial point of view it is anticipated that all 5 SMEs will directly benefit from the activities of VirFree. Protocols for the detection of novel viral/viroid threats are of immediate and high interest to the two nurseries involved but also eventually to all nurseries working with similar plant species. Equally, the development of serological and molecular tools for new threats, or for threats with increased market importance directly interests the two SMEs producing diagnostic kits. Finally, the HTS company will have the opportunity to promote their activities and gain insight from cutting edge developments in the plant virus field.

Website & more info

More info: http://www.virfree.eu.