Svetlana Vinogradova

Ampelographic collections play an important role in the conservation of grapevine genetic resources and therefore require continuous phytosanitary monitoring. In this study, the virome of grapevines from the Magarach ampelographic collection in Russia was analyzed using total RNA high-throughput sequencing. A total of twenty-seven grapevine viruses and four viroids were identified. Two viruses were characterized as putative novel species: (+) ssRNA grapevine umbra-like virus 5 (GULV-5) and the bipartite (+) ssRNA grapevine Magarach secovirus (GMSV), which, together with related viruses, may represent a novel genus within the family Secoviridae. Among the economically important viruses, the most prevalent were grapevine fanleaf virus (76%), grapevine leafroll-associated virus 1 (39%), and grapevine virus A (33%). Mixed infections involving two or three of these viruses were detected in 50% of the analyzed grapevines. Grapevine virus D was detected in Russia for the first time. Phylogenetic analysis of 222 assembled virus and viroid genome sequences revealed high genetic diversity. The obtained results were summarized and compared with previous virome studies conducted on four Russian ampelographic collections.

The metagenomic approach based on high-throughput sequencing is becoming increasingly prevalent for the detection of viral infections in plants. This method allows us to study the species composition of viruses associated with the plant, including novel species, describe their population genetic structure, and develop genetic test systems for routine diagnostics. A metagenomic approach to phytosanitary monitoring can help to determine the cause of unknown plant diseases, which is particularly important for preventing the spread of pathogens, such as viruses. Furthermore, as it is impossible to eliminate plant viruses in field conditions, comprehensive diagnostics using high-throughput sequencing is becoming an effective tool for complying with quarantine regulations on the import of foreign material, as well as for producing high-quality local planting material. High-throughput sequencing is becoming more affordable every year, with both the instrumentation and analytical capacity improving. This review summarizes key approaches to analyzing plant virome using high-throughput sequencing. The analysis process, from sample collection to bioinformatic data processing, validation and interpretation, is described in detail. The features of sequencing platforms and the factors affecting sequencing quality, including contamination, are discussed. Three complementary approaches to processing bioinformatic data are described: mapping reads to reference viral sequences; assembling and annotating contigs; taxonomic classification of reads without assembly. The importance of carefully interpreting the results is emphasized, considering the bioinformatic analysis and the validation by molecular genetic methods. This review will be useful for both researchers and specialists who have no experience with high-throughput sequencing, and those who have used this method for other applications.

This study analyzed the microbiome of three varieties differing in genotype and technical purpose: Cristal, Riesling, and Avgustin, all exhibiting decline symptoms of unknown etiology. A total of 92 symptomatic and asymptomatic grapevines were analyzed using ITS and 16S rRNA amplicon sequencing and molecular genetic methods. Phytoplasmas and the pathogenic bacteria Xylella fastidiosa and Xylophilus ampelinus were not present in the samples. The decline symptoms were associated with a cocktail of fungal pathogens that cause grapevine trunk diseases. In particular, the analysis revealed the causative agents of Botryosphaeria dieback (Sphaeropsis spp. and Botryosphaeria spp.), fungi associated with the Esca complex (Phaeomoniella spp., Phaeoacremonium spp., Inonotus spp., Seimatosporium spp., Stereum spp., and Cadophora spp.), and the causative agents of Phomopsis dieback (Diaporthe spp.). The symptoms of decline may be increased by several facultative grapevine pathogens that have been identified in microbiome (genera Stemphylium, Alternaria, Aspergillus, Penicillium, Talaromyces, and Fusarium). The metagenomic data of the grapevine microbiome provides opportunities for developing disease control strategies, which is important for the sustainable management of vineyards.

The spread of cultivated grapevine from primary centers of origin is inevitably accompanied by the range expansion of its pathogens, including viruses. A limited number of wild Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi populations have survived in the centers of grapevine domestication and can be used for comprehensive studies. We analyzed 50 grapevines collected in protected areas of the Black Sea region, which belong to the Caucasian domestication center. Based on genotyping of grapevines using simple sequence repeats as DNA markers, we determined the phylogenetic placement of V. vinifera ssp. sylvestris from the Black Sea region compared to cultivated and wild grapevines of the world. Using high-throughput sequencing of total RNA, we obtained the viromes of these grapevines. Ten viruses and one viroid were identified. The most common viruses detected were Vitis cryptic virus, grapevine rupestris stem pitting–associated virus, grapevine Pinot gris virus, and grapevine virus T. Among the economically significant viruses, we identified grapevine leafroll-associated virus 1 and grapevine virus A. A total of 91 complete or nearly complete virus genomes and one viroid genome were assembled, and phylogenetic analysis was performed. Two novel (+) ssRNA viruses were discovered, tentatively named Abrau grapevine-associated virus in the order Hepelivirales and Taurida grapevine-associated virus in the order Picornavirales. It is important to comprehensively consider the phylogeography of both viruses and their plant hosts. This is the first study that simultaneously addresses the population genetics of V. vinifera ssp. sylvestris from the Caucasian domestication center and its viruses.

Forest trees significantly affect human life. The spread of pathogens, including bacterial ones, poses a serious threat to their health. Despite this, however, the species composition and distribution of pathogenic bacteria, as well as the etiology of common diseases affecting forest trees, remain virtually unstudied. In this study, we, for the first time, describe different species of Pseudomonas and Pantoea as new etiological agents associated with the symptoms of leaf spotting and wood darkening on Acer tataricum L., Fraxinus pennsylvanica L., Ulmus minor Mill. Ulmus laevis Pallas. and Populus tremula L. For the identification of bacteria species, we used an integrated approach based on the characterization of their morphology, biochemistry, physiology and genetics. Phylogenetic analysis was performed using multilocus typing for five genes for Pseudomonas and six genes for Pantoea. Leaf spotting on A. tataricum, F. pennsylvanica, U. minor and U. laevis was shown to be caused by Pseudomonas cerasi, Pseudomonas congelans, Pseudomonas graminis, Pseudomonas syringae and Pantoea agglomerans both in monoinfection and coinfection. Wood darkening in U. minor U. laevis and P. tremula was found to be associated with the presence of Pantoea sp. and P. agglomerans. The coinfection of forest trees with bacteria of the genera Pseudomonas and Pantoea indicates a complex mechanism of interaction between the two populations, which will be the subject of future studies.

In this study, we carried out large-scale leaf spot symptom observation on vineyards in the Krasnodar Krai of Russia and determined their distribution. The incidence and severity of leaf spot were higher on the Euro-American grapevine hybrids (Bianka, Levokumskij, Avgustin, Moldova, Pervenets Magaracha, Dunavski lazur). A total of 433 isolates that belonged to the genus Alternaria were isolated from samples with leaf spot. Pathogenicity testing confirmed the ability of the representative isolates to cause necrosis on the grapevine. The isolates of Alternaria sp. were typed by the loci of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (gapdh), Alternaria allergen a1 (Alt a1), β-tubulin (tub), and translation elongation factor (tef1). Isolates from grapevine causing leaf spot were shown to cluster with isolates of Alternaria. alternata (Fr.) Keissl. and Alternaria. arborescens E.G. Simmons species complex. Of the fungicides tested to inhibit Alternaria growth, the most effective were mixtures, such as pyrimethanil and fluopyram, cyprodinil and fludioxonil, and those that included difenoconazole. The results of the study expand our knowledge of the biodiversity of Alternaria sp. fungi and can be used to limit the spread of Alternaria leaf spot of the grapevine.

In this study, an analysis of the virome of 51 grapevines from the Don ampelographic collection named after Ya. I. Potapenko (Russia) was performed using high-throughput sequencing of total RNA. A total of 20 previously described grapevine viruses and 4 viroids were identified. The most detected were grapevine rupestris stem pitting-associated virus (98%), hop stunt viroid (98%), grapevine Pinot gris virus (96%), grapevine yellow speckle viroid 1 (94%), and grapevine fleck virus (GFkV, 80%). Among the economically significant viruses, the most present were grapevine leafroll-associated virus 3 (37%), grapevine virus A (24%), and grapevine leafroll-associated virus 1 (16%). For the first time in Russia, a grapevine-associated tymo-like virus (78%) was detected. After a bioinformatics analysis, 123 complete or nearly complete viral genomes and 64 complete viroid genomes were assembled. An analysis of the phylogenetic relationships with reported global isolates was performed. We discovered and characterized the genomes of five novel grapevine viruses: bipartite dsRNA grapevine alphapartitivirus (genus Alphapartitivirus, family Partitiviridae), bipartite (+) ssRNA grapevine secovirus (genus Fabavirus, family Secoviridae) and three (+) ssRNA grapevine umbra-like viruses 2, -3, -4 (which phylogenetically occupy an intermediate position between representatives of the genus Umbravirus and umbravirus-like associated RNAs).

Among other pathogens, more than 80 viruses infect grapevine. The aim of this work was to study the virome diversity of grapevine viruses and mycoviruses of a vineyard using high-throughput sequencing technologies. The grapevine virome was studied in symptomatic vines of the Rkatsiteli cultivar (V. vinifera) collected at the vineyards of the Krasnodar Krai in Russia. Ribosomal-depleted total RNA and isolated small RNAs were used for library preparation and high-throughput sequencing. Six grapevine-infecting viruses and two viroids were validated by RT-PCR and analyzed phylogenetically. We identified the presence of grapevine leafroll-associated virus 3, grapevine Pinot gris virus, grapevine virus T, grapevine rupestris stem-pitting-associated virus, grapevine fleck virus, and grapevine rupestris vein feathering virus, as well as two viroids, grapevine yellow speckle viroid 1 and hop stunt viroid. We also studied the mycovirome of the vineyard and identified nine viruses with single-stranded positive-sense RNA genomes: alternaria arborescens mitovirus 1, botrytis cinerea mitovirus 1, botrytis cinerea mitovirus 2, botrytis cinerea mitovirus 3, botrytis cinerea mitovirus 4, sclerotinia sclerotiorum mitovirus 3, botrytis cinerea hypovirus 1, grapevine-associated narnavirus 1, and botrytis virus F. In addition, we identified botrytis cinerea hypovirus 1 satellite-like RNA and two single-stranded negative-sense RNA viruses. This is the first study of grapevine mycoviruses in Russia. The obtained result will contribute to the development of biocontrol strategies in the future.

In this study, we analyzed the virome of 73 grape samples from two Dagestan ampelographic collections in Russia using high-throughput sequencing of total RNAs. Fourteen viruses and four viroids were identified, with one to eleven of them detected in each plant. For the first time in Russia, we identified grapevine leafroll-associated virus 7 and grapevine Kizil Sapak virus. A total of 206 genomes of viruses and viroids were obtained, and their phylogenetic analysis was carried out. The de novo assembly and tblastx analysis allowed us to obtain contigs of a novel (+) ssRNA genome of a plant virus from the genus Umbravirus, which was tentatively named grapevine umbra-like virus (GULV), as well as contigs of a novel dsDNA pararetrovirus from the genus Caulimovirus, which was tentatively named grapevine pararetrovirus (GPRV). Complete genomes of these viruses were obtained and used for Sequence Demarcation Tool (SDT) analysis and phylogeny studies. GULV and GPRV were detected in 16 and 33 germplasm samples from the Dagestan collections, respectively.

Grapevine germplasm collections are unique repositories of grape cultivars; therefore, it is necessary to minimize their infection with pathogens, including viruses, and develop various programs to maintain them in a virus-free state. In our study, we examined the virome of the largest Russian grapevine germplasm collection, the Anapa Ampelographic Collection, using high-throughput sequencing of total RNAs. As a result of bioinformatics analysis and validation of its results by reverse transcription PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR), we identified 20 viruses and 3 viroids in 47 libraries. All samples were infected with 2 to 12 viruses and viroids, including those that cause economically significant diseases: leafroll, fleck, and rugose wood complex. For the first time in Russia, we detected Grapevine virus B (GVB), Grapevine virus F (GVF), Grapevine asteroid mosaic-associated virus (GAMaV), Grapevine Red Globe virus (GRGV), Grapevine satellite virus (GV-Sat), Grapevine virga-like virus (GVLV), Grapevine-associated jivivirus 1 (GaJV-1) and Vitis cryptic virus (VCV). A new putative representative of the genus Umbravirus with the provisional name Grapevine umbra-like virus (GULV) was also identified in Russian grape samples.