Periodic Reporting for period 2 - VirFree (Virus free Fruit Nurseries)
Okres sprawozdawczy: 2019-01-01 do 2022-10-31
Fruit trees and grapevine are propagated vegetatively, therefore they suffer from pathogens such as viruses and viroids, several of which cause 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. 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. The VirFree consortium was formed in this direction and brought 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 plant material of fruit trees and grapevine. In this project diagnostic tools that are currently used in certification programs have been combined with cutting edge technologies such as High Throughput Sequencing (HTS) and Nanobodies. Overall, new knowledge was obtained, and pipelines have been optimised for the identification of woody plant infecting viruses through HTS. Moreover, new virus-specific diagnostic assays were developed, and emphasis was given on the adjustment of the micropropagation process towards fruit tree species of interest to the consortium, and the production of a best-practices protocol. The knowledge obtained has been shared among the partners of VirFree and further communicated to the broader scientific community, nurseries and elsewhere through various dissemination processes. Most importantly training of a new generation of researchers has been done through their secondments in close contact to the needs of both industrial and academic sectors. In this direction, also the three training schools that took place throughout the course of VirFree have significantly fostered the skills of the participating young researchers thus strengthening their potential.
Within VirFree the optimization of HTS for the identification of known and new fruit tree and grapevine viruses or viroids was addressed. Pipelines using the software CLC Genomics Workbench 10, Geneious Prime® 2022.2.1 and MacVector, Inc. were developed and optimized for the detection of plant viruses in woody plants. These pipelines were used for screening of samples resulting in the detection of more than 60 viruses/viroids, including five new virus species and a new viroid-like RNA in fruit trees as well as several divergent strains of known viral pathogens of woody plants. On several occasions the viruses and viroids identified were reported for the first time in a country, thus expanding the list of endemic pathogens, and in new hosts which directly affect their epidemiology. The data obtained was used to update detection assays and determine the previously unknown, etiological agents of diseases. The high number of new sequences from different isolates obtained from woody plants were incorporated in alignments to assess the specificity of the existing RT-PCR assays and redesign the primers where needed. RT-PCR assays were developed for the detection of CiVA, PrVI and ctaHVd-LR1 to establish their spread and assist further studies. RT-PCR and RT-qPCR assays were also developed for several viral pathogens (CVTR, GVE, GVF, GVH, GVT, GPGV, GRLDaV and PeSV), and they will become useful tools in the production of high-quality plant material. LAMP assays were designed for the sensitive and rapid detection of grapevine viruses (GVA, GPGV, GRLDaV) and a fruit tree viroid (ASBVd) however further experimentation will be needed before their use for diagnosis. In addition, nanobodies were raised against a grapevine infecting pathogen (RpRSV) and incorporated in an ELISA assay thus improving its diagnostic efficiency. The method is being exploited by the involved consortium participants. Moreover, the phytosanitary improvement of nursery material and optimization of the micropropagation methods for fruit trees through training and exchange of knowledge on virus elimination and micropropagation techniques was attempted. Fruit tree species of interest (Prunus species, pomegranate etc.) were micropropagated and, in some cases, attempts were made for sanitation. Also, the stage of acclimatization/hardening of young plants was optimized and a best practices protocol was prepared, to produce high-quality plant material of fruit trees. The proposed protocol is currently being used by the Greek nursery that participated in VirFree. Finally, the results of VirFree were disseminated to the scientific community, stakeholders and the general public through national and international conferences, press releases, presentations in open public events, social media and the project’s website –www.virfree.eu. Moreover, 18 scientific articles related to VirFree activities, detection methods and new virus species were published, and the sequences obtained in this project have or will be submitted to public databases, making the results readily available to everyone.
The results of VirFree have enriched the knowledge on viruses/viroids associated with different diseases, improved the sensitivity of their detection methods, led to new detection products, and are anticipated to further contribute to the improvement of the disseminated propagative material of fruit trees and grapevine.
More specifically, VirFree has generated optimized pipelines for virus and viroid detection through HTS data. The use of HTS has unveiled the virome of several fruit tree species and grapevine. On some occasions this has led, or will lead, in combination with further biological characterization, to the association of the identified pathogens with diseases of unknown etiology. The data collected will be an important component of the development of serological or molecular tools for the detection of novel or commercially important viral/viroid threats. The knowledge obtained and the experience of partners have been integrated in a best practices protocol to produce virus tested nursery material.
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 many people in the countries involved in VirFree live on such crops. Moreover, within the frame of the project Early-Stage Researchers have had 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 all companies involved have directly benefited 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. Likewise, the developed serological and molecular tools for new threats, or for threats with increased commercial importance directly interest the two SMEs that produce diagnostic kits. Finally, the company involved with HTS can further promote their activities and gain insight from the cutting edge developments in the plant virus field.
More specifically, VirFree has generated optimized pipelines for virus and viroid detection through HTS data. The use of HTS has unveiled the virome of several fruit tree species and grapevine. On some occasions this has led, or will lead, in combination with further biological characterization, to the association of the identified pathogens with diseases of unknown etiology. The data collected will be an important component of the development of serological or molecular tools for the detection of novel or commercially important viral/viroid threats. The knowledge obtained and the experience of partners have been integrated in a best practices protocol to produce virus tested nursery material.
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 many people in the countries involved in VirFree live on such crops. Moreover, within the frame of the project Early-Stage Researchers have had 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 all companies involved have directly benefited 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. Likewise, the developed serological and molecular tools for new threats, or for threats with increased commercial importance directly interest the two SMEs that produce diagnostic kits. Finally, the company involved with HTS can further promote their activities and gain insight from the cutting edge developments in the plant virus field.