Project description:Meristem culture and somatic embryogenesis is an effective tool for virus elimination of vegetatively propagated crops including grapevine. While they both are proved to be useful to eliminate the main grapevine viruses their efficiency differs according to the virus and the variety. In our work we investigated their efficiency using small RNA high-throughput sequencing as virus diagnostic method. Field grown mother plants of four clones representing three cultivars, infected with different viruses and viroids were selected for sanitation via somatic embryogenesis and meristem culture. Our results show that the sanitation with SE was efficient against all of the presenting viruses, including grapevine Pinot gris virus, grapevine rupestris vein feathering virus and grapevine Syrah virus 1, having no data using somatic embryogenesis for their elimination. In case of other viruses and viroids such as GFkV, GRSPaV, GYSVd-1, HSVd this study confirms the findings of earlier researches, that SE is a possible way for elimination. While the efficiency of the elimination of different viruses was high, in case of viroids this ratio was lower. Our work demonstrated that efficiency of SE is comparable to the technically difficult meristem culture technique, and show promising way for the high demand of the production of virus-free grapevine in the future.
Project description:Our experiments investigated the effects of various known chemotherapeutic agents (2-thiouracil, ribavirin and zidovudine) on grapevine for the elimination of several different viruses and viroids. Viruses and viroids were identified by small RNA HTS in the mother plants of different grapevine cultivars, and RT-PCR verified the results. Among the tested agents, ribavirin had the broadest elimination effect on most viruses. It proved to be particularly effective against GFkV, GLRaV-4, GPGV and GRSPaV, however, as a single treatment it was not able to eliminate GFLV. In our experiments, ribavirin also showed very low efficacy against GLRaV-1 and GVA viruses. The application of 2-TU caused high phytotoxicity, with only 25% efficacy against GLRaV-4 among the viruses tested. Zidovudine failed to eliminate any of the viruses we tested. However, the combination of ribavirin and zidovudine was able to eliminate GFLV, though with low efficacy (12.5%). In contrast, the combination of ribavirin and 2-TU has been shown to be very effective against GRSPaV and GPGV. Compared to ribavirin, zidovudine and 2-thiouracil did not demonstrate significant virus elimination efficacy in the case of tested viruses. In our experiments ribavirin could not remove HSVd and GYSVd-1 viroids. To the best of our knowledge, no prior studies have been published the successful use of ribavirin or other chemotherapeutic agents against GLRaV-4, and this is the first publication of a successful application of 2-TU and zidovudine against grape viruses .
Project description:Several systemic diseases affect Vitis vinifera worldwide with important consequent management costs. Phytoplasma and viruses represent the most detrimental pathogens inducing symptoms and metabolic alterations that modify quantitatively the crop production. In the aim to investigate the plant/pathogen interactions, different grapevine samples, naturally affected (in mixed or single infections) by Stolbur phytoplasma (agent of Bois Noir disease) and viruses, in comparison to healthy and recovered controls, to identify the plant response to systemic pathogen infection. The preliminary results showed that expression levels of thousands of genes were altered in infected plants, involving various metabolic pathways.
Project description:Several pathogens infect grapevine, including viruses and viroids. Considering that there are no effective plant protection treatments against these pathogens and vineyards are cultivated through decades usage of high quality and pathogen-free propagation material (rootstocks and scions) is essential. Although presence of regulated pests is routinely checked using ELISA or rarely RT-PCR, these diagnostics methods can detect only particular pathogens moreover can fail to detect variant strains. High-throughput sequencing of small RNAs can be an effective, alternative method to avoid these disadvantages. Since for production of grafts, pathogen free cultivars and rootstocks must be used, 17 grapevine rootstock plantations and 2 rootstock variety collections were selected for characterisation of their virom by high throughput sequencing of virus derived small RNAs.
Project description:MicroRNAs (miRNAs) are a class of non-coding RNA molecules which have significant gene regulation roles in organisms. The advent of new high throughput sequencing technologies has enabled the revelation of novel miRNAs. Although there are two recent reports on high throughput sequencing analysis of small RNA libraries from different organs of two grapevine wine varieties, there were significant divergence in the number and kinds of miRNAs sequenced in these studies. More sequencing of small RNA libraries is still important for the discovery of novel miRNAs in grapevine. Here, we initially constructed a small RNA library of flower and fruit tissues of a table grapevine cultivar ‘Summer Black’ and performed sequencing and analysis of sRNAs using the Illumina Solexa platform, expecting to discover more miRNAs related to the development of grapevine flowers and berries and the formation of dessert quality in grapevine berries. Totally, 130 conserved grapevine miRNA (Vv-miRNA) belonging to 28 Vv-miRNA families were validated, and 92 novel potential grapevine-specific ones representing 80 unique ones were first discovered. Forty-two (48.84%) of the novel miRNAs possessed differential semi-quantitative PCR expression profiles in various grapevine tissues that could further confirm their existence in the grapevine, among which twenty were expressed only in grapevine berries, indicating some fruit-specificity. 130 target genes for 46 novel miRNAs could be predicted. The locations of these potential target genes on grapevine chromosomes and their complementary levels with the corresponding miRNAs were also analyzed.
Project description:As virus diseases cannot be controlled by traditional plant protection methods the risk of their spread have to be minimized on vegetatively propagated plants, such as grapevine. Metagenomics approaches used for virus diagnostics, offer a unique opportunity to reveal the presence of all viral pathogens in the investigated plant, why their usage can reduce the risk of using infected material for a new plantation. Here we used a special field, deep sequencing of virus derived small RNAs, of this high throughput method for virus diagnostics and determined viromes of vineyards in Hungary. With NGS of virus derived small RNAs we could detect not only the viruses tested routinely, but also new ones, which have never been described in Hungary before. Virus presence didn’t correlated with the age of the plantation, moreover phylogenetic analysis of the identified virus isolates suggests that infections mostly caused by the usage of infected propagating material. Our results, validated by other molecular methods, highlighted further questions to be answered before these method can be introduced as a routine, reliable test for grapevine virus diagnostics.
Project description:Several systemic diseases affect Vitis vinifera worldwide with important consequent management costs. Phytoplasma and viruses represent the most detrimental pathogens inducing symptoms and metabolic alterations that modify quantitatively the crop production. In the aim to investigate the plant/pathogen interactions, different grapevine samples, naturally affected (in mixed or single infections) by Stolbur phytoplasma (agent of Bois Noir disease) and viruses, in comparison to healthy and recovered controls, to identify the plant response to systemic pathogen infection. The preliminary results showed that expression levels of thousands of genes were altered in infected plants, involving various metabolic pathways. Total RNA was extracted from central leaf midribs and petioles from different V. vinifera cultivars in different conditions (healthy, infected and recovered). Microarray analyses were conducted using different biological replicates for treatment. The submitter of this dataset can no longer locate the raw data