Downy mildew is a severe disease that leads to significant losses in grape yields worldwide. It is caused by the oomycete Plasmopara viticola. The study of the distribution of this agent and the search for endophytic organisms that inhibit the growth of P. viticola are essential objectives to facilitate the transition to sustainable and high-yield agriculture, while respecting the environment. In this study, high-throughput sequencing of the ITS (ITS1f/ITS2 region) and 16S (V4 region) amplicons was employed to analyze 80 samples of leaves and stems from different grapevine species and cultivars grown in the Russian Far East (Vitis amurensis Rupr., Vitis coignetiae Pulliat, and several grapevine cultivars). The analysis revealed the presence of P. viticola in 53.75% of the grape samples. The pathogen P. viticola was not detected in V. amurensis samples collected near Vladivostok and Russky Island. Among the P. viticola-affected samples, only two (out of the eighty analyzed grape samples) from the Makarevich vineyard in Primorsky Krai exhibited disease symptoms, while the majority appeared visually healthy. We also found six distinct P. viticola ASVs in our metagenomic data. Based on phylogenetic analysis, we hypothesize that the P. viticola population in the Russian Far East may have originated from the invasive P. viticola clade aestivalis, which has spread around the world from North America. To identify putative microbial antagonists of P. viticola, a differential analysis of high-throughput sequencing data was conducted using the DESeq2 method to compare healthy and P. viticola-affected samples. The in silico analysis revealed an increased representation of certain taxa in healthy samples compared to P. viticola-affected ones: fungi—Kabatina sp., Aureobasidium sp., and Vishniacozyma sp.; bacteria—Hymenobacter spp., Sphingomonas spp., Massilia spp., Methylobacterium-Methylorubrum spp., and Chryseobacterium spp. This in-silico-obtained information on the potential microbial antagonists of P. viticola serves as a theoretical basis for the development of biocontrol agents for grapevine downy mildew.

Polygonum cuspidatum, alternatively known as Fallopia japonica or Reynoutria japonica, is a perennial herb belonging to the Polygonaceae family. Commonly called Japanese knotweed or Asian knotweed, this plant is native to East Asia, particularly in regions such as Korea, China, and Japan. It has successfully adapted to a wide range of habitats, resulting in it being listed as a pest and invasive species in several countries in North America and Europe. This study focuses on analysing the composition of the bacterial and fungal endophytic communities associated with Japanese knotweed growing in the Russian Far East, employing next-generation sequencing (NGS) and a cultivation-based method (microbiological sowing). The NGS analysis showed that the dominant classes of endophytic bacteria were Alphaproteobacteria (28%) and Gammaproteobacteria (28%), Actinobacteria (20%), Bacteroidia (15%), and Bacilli (4%), and fungal classes were Agaricomycetes (40%), Dothideomycetes (24%), Leotiomycetes (10%), Tremellomycetes (9%), Pezizomycetes (5%), Sordariomycetes (3%), and Exobasidiomycetes (3%). The most common genera of endophytic bacteria were Burkholderia-Caballeronia-Parabukholderia, Sphingomonas, Hydrotalea, Methylobacterium-Metylorubrum, Cutibacterium, and Comamonadaceae, and genera of fungal endophytes were Marasmius, Tuber, Microcyclosporella, Schizothyrium, Alternaria, Parastagonospora, Vishniacozyma, and Cladosporium. The present data showed that the roots, leaves, and stems of P. cuspidatum have a greater number and diversity of endophytic bacteria and fungi compared to the flowers and seeds. Thus, the biodiversity of endophytic bacteria and fungi of P. cuspidatum was described and analysed for the first time in this study.

Many grape endophytic microorganisms exhibit high potential for suppressing the development of grape diseases and stimulating grapevine growth and fitness, as well as beneficial properties of the crop. The microbiome of wild grapevines is a promising source of biocontrol agents, which can be beneficial for domesticated grapevines. Using next-generation sequencing (NGS) and classical microbiology techniques, we performed an analysis of bacterial and fungal endophytic communities of wild grapevines Vitis amurensis Rupr. and Vitis coignetiae Pulliat growing in the Russian Far East. According to the NGS analysis, 24 and 18 bacterial taxa from the class level were present in V. amurensis and V. coignetiae grapevines, respectively. Gammaproteobacteria (35%) was the predominant class of endophytic bacteria in V. amurensis and Alphaproteobacteria (46%) in V. coignetiae. Three taxa, namely Sphingomonas, Methylobacterium, and Hymenobacter, were the most common bacterial genera for V. amurensis and V. coignetiae. Metagenomic analysis showed the presence of 23 and 22 fungi and fungus-like taxa of class level in V. amurensis and V. coignetiae, respectively. The predominant fungal classes were Dothideomycetes (61-65%) and Tremellomycetes (10-11%), while Cladosporium and Aureobasidium were the most common fungal genera in V. amurensis and V. coignetiae, respectively. A comparative analysis of the endophytic communities of V. amurensis and V. coignetiae with the previously reported endophytic communities of V. vinifera revealed that the bacterial biodiversity of V. amurensis and V. coignetiae was similar in alpha diversity to V. vinifera's bacterial biodiversity. The fungal alpha diversity of V. amurensis and V. coignetiae was statistically different from that of V. vinifera. The beta diversity analysis of bacterial and fungal endophytes showed that samples of V. vinifera formed separate clusters, while V. amurensis samples formed a separate cluster including V. coignetiae samples. The data revealed that the endophytic community of bacteria and fungi from wild V. amurensis was richer than that from V. coignetiae grapes and cultivated V. vinifera grapes. Therefore, the data obtained in this work could be of high value in the search for potentially useful microorganisms for viticulture.

Grapes and wines represent the most important source of edible stilbenes and other phenolic metabolites, which demonstrate a wide range of valuable biological activities. However, there is no information about the profile and content of phenolic compounds in Russian wines. We firstly analyzed phenolics (stilbenes, phenolic acids, and flavonols) in some representatives of Russian wines, including eleven red and seven white Russian wines from Fanagoria, Krasnodarsky Territory. The Russian red wines contained six stilbenes (trans-resveratrol, cis-resveratrol, trans-, cis-piceid, trans-piceatannol, δ-viniferin), while the white wines contained only five stilbenes (cis-resveratrol, trans-, cis-piceid, trans-piceatannol, trans-resveratrol). More than a half of the total stilbenes in the wines (65% of all stilbenes) were presented by trans-piceid and cis-piceid, while trans-resveratrol reached 16% of all the stilbenes. The red wines also contained six phenolic acids and six flavonols, while the white wines contained six phenolic acids and only three flavonols. Myrecitin-3-O-glucoside, quercetin-3-O-glucoside, and myricetin were the major flavonols in the red wines, while dihydroquercetin-3-O-rhamnoside was the major flavonol in the white wines. The red wines contained markedly higher amounts of stilbenes, phenolic acids, and flavonols than the white wines. Thus, the data showed that young red Russian Fanagoria wines represent a rich source of phenolic compounds. The study also revealed that younger wines were more abundant in phenolics, and wine storage for six months in the dark at +10 °C led to a decrease in the total content of phenolics, primarily monomeric stilbenes and quercetin-3-O-glucoside and quercetin flavonols.

The complete mitochondrial (mt) genomes of five subspecies of the Eurasian (Common) magpie Pica pica were determined for the first time. Lengths of the circular genomes comprise 13 protein-coding genes, two rRNA genes (for 12S rRNA and 16S rRNA), 22 tRNA genes, and the non-coding control region (CR). Gene content and lengths of the genomes (16,936–16,945 bp) are similar to typical vertebrate mt genomes. The subspecies studied differs by several single substitutions and indels, especially in the CR. The phylogenetic tree based on complete mt genomes shows a deep divergence of the two groups of subspecies which supports the proposed division into two distinct species: P. pica and P. serica.

Two East Asian grape species Vitis amurensis and Vitis coignetiae have been investigated for their increased cold and drought tolerance and high level of resistance to infections. Nowadays, the grape species attract attention due to their capability to produce a group of valuable plant secondary metabolites named stilbenes. The basic stilbene, t-resveratrol, is known to demonstrate a high bioactivity and is synthesized by stilbene synthase (STS) in grape cells. In order to find the suitable biological model for t-resveratrol production, 12 cell culture lines derived from the leaf, petiole and stem explants of the East Asian grape species were tested for their ability to produce t-resveratrol and other stilbenes. The comparative analysis showed that the V. coignetiae cell culture lines were the most productive in terms of stilbene content. This observation was confirmed by the increased level of STS expression in V. coignetiae cells in comparison to V. amurensis. The expression analysis of the genes encoding transcription factors potentially involved in regulation of STS genes revealed the significant species-specific differences, suggesting the molecular basis underlying the observed differences in stilbene biosynthetic potential of V. amurensis and V. coignetiae. The comparative analysis revealed the greater stilbene production potential for cells of V. coignetiae due to enhanced STS expression determinated by Myb1/15, Myb40 and Myb60 transcription factors.

Plant cell and tissue cultures are considered as a source of valuable secondary metabolites but usually produce insufficient level of the compounds, which is the limiting factor for their application in biotechnology. We obtained 18 callus cell cultures from different organs of wild grape Vitis amurensis Rupr. collected at different seasons and analyzed stilbene accumulation in combination with calli growth parameters. This analysis showed that temporal and tissue origin of the calli affected the rate of stilbene biosynthesis. Stem-derived calli accumulated higher stilbene levels and exhibited a higher expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes than calli derived from the leaves and petioles. The highest content of stilbenes was detected in the calli initiated from grapevine stems collected in the autumn. In general, all “autumn” cell cultures contained more than 2 mg g− 1 dry wt (up to 11 mg g− 1 dry wt) and exhibited high PAL and STS genes expression in comparison with the calli initiated in the summer. The content of stilbenes in the “autumn” cell cultures were comparable to the highest stilbene contents detected in other plant sources described in the literature. Thus, selecting the most optimal explant source for cell culture establishment could be an effective approach towards developing plant cell cultures producing high stilbene levels.

Objective Studies concerning 2b protein from Cucumovirus showed 2b to effectively repress functioning of the plant silencing complex, current study aimed whether retransformation with 2b gene able to restore silenced transgene expression in plant cells. Results A rolB -transgenic cell culture of Vitis amurensis Rupr. that was continuously subcultured during more than 10 years and exhibited decreased transcription of the rolB transgene was retransformed with the 2b gene of Cucumovirus-NK. Three cell lines retransformed with 2b showed a significant up-regulation of rolB expression accompanied with enhancements in their stilbenes content level in more than 2,7-fold compared to parental rolB-transgenic cell line. The mentioned increase in the level of stilbenes content was due to activation of certain stilbene synthase genes expression responsible for stilbenes biosynthesis in V. amurensis cells. Restoration of rolB expression upon 2b -retransformation led to increase in the expression levels of VaSTS2 - VaSTS5 and VaSTS7 isoforms. Conclusions 2b from CMV-NK can reactivate a silenced transgene expression, even after 10 years of subcultivation, nevertheless, optimization of the methods concerning 2b introduction in plant genomes is necessary to avoid undesirable silencing effects.

It is known that somatic mutations arising during animal growth and ageing contribute to the development of neurodegenerative and other animal diseases. For plants, several studies showed that small-scale somatic DNA mutations accumulated during Arabidopsis life cycle. However, there is a lack of data on the influence of environmental stresses on somatic DNA mutagenesis in plants. In this study, we analyzed the effects of ultraviolet C (UV-C) irradiation, high soil salinity, and cadmium (CdI3) stresses on the level of small-scale somatic DNA mutations in Arabidopsis thaliana. The number of DNA mutations was examined in the Actin2 3′UTR (Actin-U1), ITS1-5.8rRNA-ITS2 (ITS), and ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) DNA regions. We found that somatic mutation levels considerably increased in CdI3-treated Arabidopsis plants, while the mutation levels declined in the UV-C- and NaCl-treated A. thaliana. Cadmium is a mutagen that is known to inhibit DNA repair processes. The detected stress-induced alterations in somatic DNA mutation levels were accompanied by markedly increased expression of base excision repair genes (AtARP, AtDME, AtDML2, AtDML3, AtMBD4, AtROS, AtUNG, and AtZDP), nucleotide excision repair genes (AtDDB1a, AtRad4, and AtRad23a), mismatch repair genes (AtMSH2, AtMSH3, and AtMSH7), and photoreactivation genes (AtUVR2, AtUVR3). Thus, the results demonstrated that UV-C, high soil salinity, and cadmium stresses influence both the level of DNA mutations and expression of DNA repair genes. Salt- and UV-induced activation of DNA repair genes could contribute to the stress-induced decrease in somatic mutation level.

The taxonomic composition of bacteria associated with two species of tetrodotoxin-bearing (TTX-bearing) ( Hubrechtella juliae and Lineus alborostratus ) and two species of non-TTX-bearing ( Quasitetrastemma stimpsoni and Malacobdella grossa ) ribbon worms collected from the Peter the Great Bay of Sea of Japan was studied. Bacterial isolates were identified using 16S rRNA gene sequencing and phenotypic characteristics. Thirty-eight strains of heterotrophic bacteria from the eight genera: Pseudoalteromonas , Shewanella , Ruegeria , Pseudomonas , Defluviicoccus , Vibrio , Alteromonas , and Bacillus , were isolated and characterized. γ-Proteobacteria dominated among the associated microflora of nemerteans (76.3% of the total number of isolates). Sensitivity analysis of 38 strains to antibiotics of various classes revealed multiple resistance to three or more antibiotics in all of the studied isolates. The 15 bacterial strains isolated in the study exhibited antimicrobial activities against at least one of five indicator microorganisms, most of which corresponded to the Pseudoalteromonas genus. Screening of the TTX-producing bacteria was performed using confocal laserscanning microscopy and polyclonal antibodies. A TTX-producing strain, Pseudoalteromonas sp., was found in the nemertean H. juliae . A correlation between the presence of TTX-positive microflora and the toxicity of nemerteans was determined.

It has previously been shown that exogenous application of p‐ coumaric acid (CA), a precursor of phenolic compounds, improved stilbene production in cell cultures of Vitis amurensis . This study examines the effect of cinnamic (Cin) and caffeic (Caf) acids, which are also phenolic precursors, on stilbene biosynthesis in the cell cultures. Five stilbenes, t‐ resveratrol diglucoside, t‐ piceid ( t‐ resveratrol glucoside), t‐ resveratrol, t‐ε‐ viniferin, and t‐δ‐ viniferin, were found in the treated and untreated cells. Cin acid increased the total stilbene production in the grape cell cultures 2.3–3.5 times in comparison with that in the untreated cells. Caf acid increased the total stilbene production by 1.8‐ to 1.9‐fold, but this increase was not considerably different from stilbene production in the untreated cells. Cin acid affected the total stilbene production via a marked increase in the content of t‐ resveratrol diglucoside (up to 2.2 times), t‐ piceid (up to three times), t‐ resveratrol (up to 5.1 times), t‐ε‐ viniferin (up to eight times), and t‐δ‐ viniferin (up to 9.2 times). Transcription levels of VaSTS5 , 6 , 7 , 8 , and 10 genes considerably increased under 0.1, 0.25, and 0.5 mM Cin acid. These results indicate that Cin acid increased stilbene production in V . amurensis calli via a selective enhancement of STS gene expression.

Grapevine Vitis amurensis Rupr. accumulates several valuable secondary metabolites; the most known is resveratrol—a key compound in biosynthesis of stilbenes. There is lack in the information of the regulation of the resveratrol biosynthesis in grapevin. For example, the grapevine MYB R2R3 (MYB) transcription factors (TFs) were found to modulate several branches of the flavonoid pathway. The flavonoid pathway is close to the stilbene pathway; therefore, we decided to investigate expression of the 22 MYB genes in the grape V. amurensis cell cultures and plant parts with different resveratrol content. Using real-time PCR, we showed that MYB9, 14, 15а, 60, 40, and 107 expression increased in several probes with the highest resveratrol content; therefore, probably, those genes are positive regulators of the stilbene biosynthesis. Also, we discussed the participation of the MYB transcription factors in the regulation of the stilbene biosynthesis in the grape cells.

Among the many secondary plant metabolites, resveratrol attracts the attention of researchers because of its unique biological and pharmacological properties. Amur grape (Vitis amurensis Rupr.), including resveratrol-producing plant, has a high level of content of phenolic compounds. Despite a number of activities aimed at the study of the regulation of the biosynthesis of resveratrol, an understanding of the mechanism of transcriptional regulation of genes of key enzymes of this metabolic pathway—the STS gene—it has a lot of white spots. This study aims to examine the role in the biosynthesis of resveratrol transcription factor VaMyb1 belonging to a large group R2R3-Myb transcription factors plant. During the study of the transcription factor gene expression in the cells of V. amurensis and the impact of its increased expression in the content of resveratrol and the expression of STS genes, evidence of its negative role in the biosynthesis of resveratrol was obtained.

To investigate the role of cytosine methylation in regulation of polyketide compounds biosynthesis in larvae of Strongylocentrotus intermedius.

Ultraviolet (UV) emerging with the sun light plays crucial role in plants ontogenesis as the one of the main environmental stresses. Prolonged exposure to UV causes damage to DNA, proteins and membranes, and the inhibition of protein synthesis and photosynthetic reactions. Moreover, UV radiance is known to induce metabolic modifications in plants, particularly to secondary metabolite biosynthesis. Multitude of the studies published to date considering exposure of grape cells to UV revealed enhancement in stilbene compounds production, and many of which possess unique biological and pharmacological properties. However, the epigenetic mechanisms regulating expression of the genes involved in stilbene biosynthesis in response to UV exposure were not studied. In this study as model objects were used grape Vitis amurensis plant cell cultures capable to produce trans-resveratrol (t-resveratrol). T-resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. T-resveratrol synthesized via the phenylpropanoid pathway, where phenylalanine ammonia-lyase (PAL) is the first enzyme in this pathway and stilbene synthase (STS) is the enzyme that directly catalyzes the reaction of t-resveratrol formation. Exposure of V. amurensis cells to UV of C type significantly increased t-resveratrol production and certain STS and PAL expression. Using bisulfite sequencing, we demonstrated that methylation level of the VaSTS2, VaSTS6 and VaSTS10 genes significantly decreased, while methylation level of constitutively expressed VaSTS1 gene did not changed significantly.

During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs.

Salicylic acid (SA) treatment selectively reduced the cytosine DNA methylation of stilbene synthase ( STS ) genes and stimulated resveratrol production in cell cultures of Vitis amurensis.

Age-related accumulation of somatic mutations has been implicated as an important mechanism contributing to ageing and various diseases in animals. However, it is unclear whether the somatic mutation frequency changes as plants age and to what extent somatic mutagenesis contributes to plant ageing or senescence. The contribution of somatic mutagenesis to plant ageing and senescence was not investigated. We used the species Arabidopsis thaliana to study whether an increasing chronological age of an annual plant species influences the total amount and molecular spectrum of somatic DNA mutations. The number of small-scale somatic mutations was studied in nine randomly chosen A. thaliana control DNA regions, including those in the nuclear genome (Actin2 coding region, Actin2 3′-UTR region, CMT3 methyltrasferase gene, tRNAPro, and ITS1-5.8rRNA-ITS2), mitochondrial DNA (cytochrome oxidase C, a gene encoding an unnamed protein, and a noncoding mitochondrial DNA region), and chloroplast DNA (rbcL gene). We found that the frequency of single nucleotide substitutions, insertions, and deletions increased considerably during the A. thaliana growth and development from the seeds to the 12-week-old plants. The vast majority of nucleotide substitutions (~86 %) were transitions with A:T→G:C transitions being the most frequent type of substitution. The data indicate that at least some plant DNA regions accumulate point mutations during plant aging.

Resveratrol is a plant-derived phenol but the mechanism that regulates its biosynthesis remains unidentified. Stilbene synthase (STS) catalyzes resveratrol formation in vivo and we have proposed that inducers of resveratrol production affect STS expression through an unidentified epigenetic mechanism. To investigate the role of DNA methylation in resveratrol biosynthesis, we treated both rolB transgenic and empty vector control Vitis amurensis cell cultures with the DNA demethylation agent, 5-azacytidine. Treated cells had increased resveratrol production through activation of VaSTS10 expression. The lowest levels of cytosine methylation were at the 5′- and 3′-ends of the VaSTS1 protein-coding sequence. Cytosine methylation decreased mostly at the 5′- and 3′-ends of VaSTS10 after azaC treatment with an intriguing regularity in the number of cytosine nucleotides within the 5′- and 3′- ends of the protein-coding sequences. Thus, cytosine methylation is crucial for the regulation of the resveratrol biosynthetic pathway.

The numbers of chromosomes and nucleoli in cultured cells of Vitis amurensis transformed with the rolB oncogene from A. rhizogenes have been studied. In general, the integration of the rolB gene in grape DNA mostly caused the elevation of the level of the chromosome variability, as well as higher numbers of nucleoli in the cultured cells. The possible influence of the observed chromosomal modifications on the productivity parameters of the grape cell cultures is discussed.