Grapevine Tissues Research Articles

Corrigendum

EPPO BulletinVolume 47, Issue 2 p. 288-288 CorrigendumFree Access Corrigendum This article corrects the following: PM 7/079 (2) Grapevine flavescence dorée phytoplasma Volume 46Issue 1EPPO Bulletin pages: 78-93 First Published online: March 8, 2016 First published: 06 June 2017 https://doi.org/10.1111/epp.12393AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Bulletin OEPP⁄EPPO Bulletin 46 (2016), 78–93. Since the publication of the EPPO Standard PM 7/079 (2) Grapevine flavescence dorée phytoplasma (EPPO, 2016) the authors have brought to our attention that the CTAB buffer used in routine analysis, and that was evaluated by the authors, is not the same as the one in the publication quoted. The authors considered an erratum should be prepared to describe the concentration that they used and evaluated. Therefore the following paragraph in Appendix 1 should be replaced: Old version CTAB procedure for Nucleic acids extraction (Boudon-Padieu et al., 2003 modified from Doyle & Doyle (1990). Nucleic acids can be extracted from fresh or frozen (−20 or −80°C) grapevine tissues (preferably veins or petioles). Grind 1 g of tissue in 10 mL of 3% CTAB buffer (3% CTAB or cethyl-trimethyl-ammonium bromide in 1 M Tris–HCl pH8, 10 mM EDTA, 1.4 M NaCl) at room temperature. Transfer into a 2 mL clean Eppendorf tube and centrifuge at 1000 g for 10 min. Transfer 1 mL of the suspension to an Eppendorf tube, add 2 μL of 2-marcaptoethanol (for a final concentration of 0.2%), vortex briefly and incubate for 20 min at 65°C. Then, add an equal volume of chloroform:isoamyl alcohol (24:1). Vortex and centrifuge at 10 000 g for 10 min. Recover the aqueous phase and precipitate the nucleic acids with an equal volume of cold isopropanol. Shake by inversion and centrifuge at 10 000 g for 15 min to recover the precipitate. Wash the pellet with 70% ethanol, dry and dissolve in 400 μL of TE buffer or nuclease-free water. New version CTAB procedure for Nucleic acids extraction (adapted from Boudon-Padieu et al., 2003 modified and Doyle & Doyle (1990). Nucleic acids can be extracted from fresh or frozen (−20 or −80°C) grapevine tissues (preferably veins or petioles). Grind 1 g of tissue in 10 mL of 3% CTAB buffer (3% CTAB or cethyl-trimethyl-ammonium bromide in 0.1 M Tris–HCl pH8, 25 mM EDTA, 1.4 M NaCl) at room temperature. Transfer into a 2 mL clean Eppendorf tube and centrifuge at 1000 g for 10 min. Transfer 1 mL of the suspension to an Eppendorf tube, add 2 μL of 2-marcaptoethanol (for a final concentration of 0.2%), vortex briefly and incubate for 20 min at 65°C. Then, add an equal volume of chloroform:isoamyl alcohol (24:1). Vortex and centrifuge at 10 000 g for 10 min. Recover the aqueous phase and precipitate the nucleic acids with an equal volume of cold isopropanol. Shake by inversion and centrifuge at 10 000 g for 15 min to recover the precipitate. Wash the pellet with 70% ethanol, dry and dissolve in 400 μL of TE buffer or nuclease-free water. Reference EPPO (2016) PM 7/079 (2) Grapevine flavescence dorée phytoplasma, Bulletin OEPP⁄EPPO Bulletin, 46, 78– 93. Volume47, Issue2August 2017Pages 288-288 ReferencesRelatedInformation

ABA Represses the Expression of Cell Cycle Genes and May Modulate the Development of Endodormancy in Grapevine Buds.

Recently, the plant hormone abscisic acid (ABA) has been implicated as a key player in the regulation of endodormancy (ED) in grapevine buds (Vitis vinifera L). In this study, we show that in the vine, the expression of genes related to the biosynthesis of ABA (VvNCED1; VvNCED2) and the content of ABA are significantly higher in the latent bud than at the shoot apex, while the expression of an ABA catabolic gene (VvA8H3) showed no significant difference between either organ. A negative correlation between the content of ABA and transcript levels of cell cycle genes (CCG) was found in both tissues. This result suggested that ABA may negatively regulate the expression of CCG in meristematic tissues of grapevines. To test this proposition, the effect of ABA on the expression of CCG was analyzed in two meristematic tissues of the vine: somatic embryos and shoot apexes. The results indicated that cell cycle progression is repressed by ABA in both organs, since it down-regulated the expression of genes encoding cyclin-dependent kinases (VvCDKB1, VvCDKB2) and genes encoding cyclins of type A (VvCYCA1, VvCYCA2, VvCYCA3), B (VvCYCB), and D (VvCYCD3.2a) and up-regulated the expression of VvICK5, a gene encoding an inhibitor of CDKs. During ED, the content of ABA increased, and the expression of CCG decreased. Moreover, the dormancy-breaking compound hydrogen cyanamide (HC) reduced the content of ABA and up-regulated the expression of CCG, this last effect was abolished when HC and ABA were co-applied. Taken together, these results suggest that ABA-mediated repression of CCG transcription may be part of the mechanism through which ABA modulates the development of ED in grapevine buds.

Association of Botryosphaeriaceae grapevine trunk disease fungi with the reproductive structures of Vitis vinifera

Several species belonging to the Botryosphaeriaceae were isolated from grapevine ( Vitis vinifera ) tissue other than wood during a survey of two vineyards planted to cultivars ‘Chardonnay’ and ‘Shiraz’ in the Hunter Valley, New South Wales, Australia over the 2007/08 and 2008/09 growing seasons. A total of 188 isolates corresponding to nine different species of Diplodia, Dothiorella and Neofusicoccum anamorphs were isolated from dormant buds, flowers, pea-sized berries and mature berries prior to harvest in addition to 142 isolates from the trunks of the same vines. Furthermore, the occurrence of Dothiorella viticola, Diplodia mutila and Neofusicoccum australe is reported here for the first time from grapevines in the Hunter Valley. These findings may provide important information for the management and spread of Botryosphaeriaceae in vineyards where they are considered serious wood-invading pathogens. Botryosphaeriaceae are occasionally found on bunches, however, until now they have not directly been related to bunch rots. Control strategies for trunk diseases caused by Botryosphaeriaceae are currently limited to remedial surgery and wound protection. These strategies do not consider other grapevine tissue as potential inoculum sources for infection of Botryosphaeriaceae in the vineyard.

Cultivar-specific gene modulation in Vitis vinifera: analysis of the promoters regulating the expression of WOX transcription factors

The family of Wuschel-related Homeobox (WOX) genes is a class of transcription factors involved in the early stages of embryogenesis and organ development in plants. Some of these genes have shown different transcription levels in embryogenic tissues and mature organs in two different cultivars of Vitis vinifera: ‘Chardonnay’ (CH) and ‘Cabernet Sauvignon’ (CS). Therefore, we investigated the genetic basis responsible for these differences by cloning and sequencing in both the cultivars the promoter regions (~2000 bp) proximal to the transcription start site of five VvWOX genes. We then introduced these promoters into Arabidopsis thaliana for expression pattern characterisation using the GUS reporter gene. In the transgenic Arabidopsis, two promoters isolated from CS (pVvWOX13C_CS and pVvWOX6_CS) induced increased expression compared to the sequence isolated in CH, confirming the data obtained in grapevine tissues. These results were corroborated by transient expression assays using the agroinfiltration approach in grapevine somatic embryos. Truncated versions of pVvWOX13C demonstrated that few nucleotide differences between the sequences isolated from CH and CS are pivotal for the transcriptional regulation of VvWOX13C. Analysis of promoters using heterologous and homologous systems appear to be effective for exploring gene modulation linked with intervarietal sequence variation in grapevine.

Rapid measurement of total non-structural carbohydrate concentration in grapevine trunk and leaf tissues using near infrared spectroscopy

Carbohydrate assays are commonly used in crops and plant research to understand the way in which carbohydrates are allocated within the vine and to assess its influence on the physiology and phenology of the plant. Total non-structural carbohydrate (TNC) concentration is normally assessed by wet chemistry methods which are time consuming and costly, especially when studying carbohydrate dynamics over seasons. Near infrared (NIR) spectroscopy is a fast and easy technique that has lately gained wide acceptance for the analysis of the chemical composition of grain, food, wine, pharmaceutical products, among others. Near infrared is the region of the electromagnetic spectrum between 750nm and 2500nm and it is used to gather information on the relative proportions of CH, NH and OH bonds of the organic molecules. This study collected NIR spectra from grapevine trunk and leaf tissues, measured TNC concentration of the same samples using a wet chemical method and compared the results using multivariate data analysis to develop a rapid procedure for the estimation of TNC concentration in grapevine tissues. Results showed that NIR spectroscopy could be used to predict starch and TNC concentration in freeze-dried and ground grapevine trunk and leaf tissues. Moreover, it has been demonstrated that a robust universal model could be applied to the prediction of TNC in both leaves and trunks. Therefore, this method could be used as a practical tool for a rapid screening of TNC concentration for high temporal and spatial assessment of grapevine tissues at given phenological stages. The main advantages of this technique over traditional methods are the rapidity and the ease-of-use protocol in routine analysis, which allows a considerable reduction of costs and time.

The MADS-box gene Agamous-like 11 is essential for seed morphogenesis in grapevine.

Despite the wide appreciation of seedless grapes, little is known about the molecular mechanisms that drive the stenospermocarpic seedless-type phenotype in grapevine. In order to address the molecular mechanisms that control seedlessness in grapevine, our study aimed to characterize VviAGL11, a class D MADS-box transcription factor gene that has been proposed as the major candidate gene involved in Vitis vinifera seed morphogenesis. VviAGL11 allelic variations in seeded and seedless grapevine cultivars were determined, and its correlations with allele-specific steady-state mRNA levels were investigated. VviAGL11 relative expression was significantly higher in seeds at 2, 4, and 6 weeks after fruit set, whereas in the seedless grape its transcript levels were extremely low in all stages analyzed. In situ hybridization revealed transcript accumulation specifically in the dual endotesta layer of the seeds, which is responsible for elongation and an increase of cell number, a necessary step to determine the lignification and the final seed size. No hybridization signals were visible in the seedless grapevine tissues, and a morphoanatomical analysis showed an apparent loss of identity of the endotesta layer of the seed traces. Ectopic expression of VviAGL11 in the Arabidopsis SEEDSTICK mutant background restored the wild-type phenotype and confirmed the direct role of VviAGL11 in seed morphogenesis, suggesting that depletion of its expression is responsible for the erroneous development of a highly essential seed layer, therefore culminating in the typical apirenic phenotype.

Determination of the Activity Signature of Key Carbohydrate Metabolism Enzymes in Phenolic-rich Grapevine Tissues.

Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenols and polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and interfere with enzyme detection. The following protocol for activity assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf. As a case study we applied the protocol to grapevine leaf samples infected with plant pathogenic bacteria 'Candidatus Phytoplasma solani', known to alter carbohydrate metabolism in grapevine. The described adaptations may be useful for determination of metabolic fingerprints for physiological phenotyping of other plant species with inherently high levels of phenolic compounds.

In vitro and in vivo screening of antagonistic bacterial strains from vineyards to controlBotrytis cinereain grapevine tissues

Botrytis bunch rot, caused by Botrytis cinerea, is an important fungal disease of grapevine with high economic importance in wine grape production and postharvest storage of table grapes. Biological control by antagonistic bacteria is a promising strategy for reducing the common use of synthetic fungicides to control this pathogen. A total of 45 bacterial strains, isolated from grapevine tissues in 'Bordeaux' vineyards were screened in vitro for their potential antifungal activity against two major vineyard subpopulations of B. cinerea, i.e., transposa and vacuma. These two transposon genotypes differ significantly in virulence on grape berries. The inhibitory effects of the bacterial strains on the mycelial growth were tested in vitro for detecting the potential production of diffusible metabolites and volatile organic compounds. Furthermore, ten strains among the most effective ones were selected and evaluated in vivo on detached grapevine host organs: leaf discs and grape berries. We showed that some of the bacterial strains strongly inhibited B. cinerea mycelial growth under in vitro conditions, and also significantly reduced rot severity in vivo. The results suggest the potential to control postharvest gray mold by some of the bacterial strains tested.

Spray Deposition and Control of Botrytis cinerea on Grape Leaves and Bunches: Part 1 (Table Grapes)

Insufficient quantity but also quality of spray deposition on susceptible grapevine tissue (i.e. target sites) and favourable conditions for pathogens could lead to control failure during high disease pressure situations. To determine deposition quantity and quality benchmarks for biologically effective spray deposits, bunches and leaves of table grapes (Waltham Cross) were sprayed at various growth stages, using different application volumes of a mixture of fenhexamid and a fluorescent tracer pigment and subsequently dusted with dry conidia of Botrytis cinerea where after infection levels on pedicels, receptacles and leaves were determined. Pigment deposition quantity and quality were determined from photos of sprayed parts taken with a digital camera under a stereo microscope and black light illumination at ×30 or ×10 magnifications and assessed with digital image and Hoerl regression analyses. The deposition quantity resulting in 75% control of B. cinerea infection (FPC75 values) was calculated from biological efficacy curves (sigmoidal regression analyses) for leaves and for each growth stage, for pedicels and receptacles. Deposition quantity and quality measurements correlated favourably with Botrytis infection. An optimal deposition value for the control of B. cinerea was determined by increasing spray volume, however by increasing spray volume and deposition quantity or quality levels past this optimum will not significantly improve disease control further. It was indicated that efficacy of agricultural chemicals could be influenced by improving both deposition quantity and quality, quantifiable by digital image analyses of fluorescent pigment deposition. FPC75 values obtained in this study can be used as benchmarks to evaluate future spray application in vineyards.

Study on Expression Modes and Cleavage Role of miR156b/c/d and its Target Gene Vv-SPL9 During the Whole Growth Stage of Grapevine.

miR156 regulates the expression of its target SPL (PROMOTER BINDING-LIKE) genes during flower and fruit development, diverse developmental stage transitions, especially from vegetative to reproductive growth phases, by cleaving the target mRNA SPL of one plant-specific transcription factor. However, systematic reports on grapevine have yet to be presented. Here, the precise sequence of miR156 (vvi-miR156b/c/d) in grapevine "Takatsuma" was cloned with a previously cloned grapevine SPL (Vv-SPL9). Expression profiles in 18 grapevine tissues were identified through stem-loop RT-PCR. The interaction mode between vvi-miR156b/c/d and Vv-SPL9 was further validated by detecting the cleavage site and cleavage products of 3'- and 5'-ends via an integrated approach of 5'-RLM-RACE (RNA ligase-mediated 5'-rapid amplification of cDNA ends), 3'-PPM-RACE (poly(A) polymerase-mediated 3'-rapid amplification of cDNA ends), and qRT-PCR (real time reverse transcriptase-polymerase chain reaction). The variation in their cleavage roles in the whole growth stage of grapevine was also systematically investigated. Results showed that vvi-miR156b/c/d exhibited typical temporal-spatial-specific expression levels. The expression levels were higher in vegetative organs, such as leaf, than in reproductive organs, such as tendrils, flowers, and berries. A significant variation was observed during vegetative-to-reproductive transition. The expression patterns of Vv-SPL9 showed the opposite trends with those of vvi-miR156b. We confirmed that the cleavage site was at the 10th site of vvi-miR156b/c/d complementary to Vv-SPL9 in "Takatsuma" grapevine. We also identified the temporal-spatial variation of the cleavage products. This variation can indicate the regulatory function of miR156 on SPL in grapevines. Our findings provide further insights into the functions of vvi-miR156b/c/d and its target Vv-SPL9, and also help enrich our knowledge of small RNA-mediated regulation in grapevine.

Transcriptome analyses of the Dof-like gene family in grapevine reveal its involvement in berry, flower and seed development.

The Dof (DNA-binding with one finger) protein family spans a group of plant transcription factors involved in the regulation of several functions, such as plant responses to stress, hormones and light, phytochrome signaling and seed germination. Here we describe the Dof-like gene family in grapevine (Vitis vinifera L.), which consists of 25 genes coding for Dof. An extensive in silico characterization of the VviDofL gene family was performed. Additionally, the expression of the entire gene family was assessed in 54 grapevine tissues and organs using an integrated approach with microarray (cv Corvina) and real-time PCR (cv Pinot Noir) analyses. The phylogenetic analysis comparing grapevine sequences with those of Arabidopsis, tomato, poplar and already described Dof genes in other species allowed us to identify several duplicated genes. The diversification of grapevine DofL genes during evolution likely resulted in a broader range of biological roles. Furthermore, distinct expression patterns were identified between samples analyzed, corroborating such hypothesis. Our expression results indicate that several VviDofL genes perform their functional roles mainly during flower, berry and seed development, highlighting their importance for grapevine growth and production. The identification of similar expression profiles between both approaches strongly suggests that these genes have important regulatory roles that are evolutionally conserved between grapevine cvs Corvina and Pinot Noir.

Root-associated bacteria promote grapevine growth: from the laboratory to the field

Background and AimsLaboratory and greenhouse experiments have shown that root-associated bacteria have beneficial effects on grapevine growth; however, these effects have not been tested in the field. Here, we aimed to demonstrate whether bacteria of different geographical origins derived from different crop plants can colonize grapevine to gain a beneficial outcome for the plant leading to promote growth at the field scale.MethodsTo link the ecological functions of bacteria to the promotion of plant growth, we sorted fifteen bacterial strains from a larger isolate collection to study in vitro Plant Growth Promoting (PGP) traits. We analysed the ability of these strains to colonise the root tissues of grapevine and Arabidopsis using green-fluorescent-protein-labelled strain derivatives and a cultivation independent approach. We assessed the ability of two subsets randomly chosen from the 15 selected strains to promote grapevine growth in two field-scale experiments in north and central Italy over two years. Parameters of plant vigour were measured during the vegetative season in de novo grafted vine cuttings and adult productive plants inoculated with the bacterial strains.ResultsBeneficial bacteria rapidly and intimately colonized the rhizoplane and the root system of grapevine. In the field, plants inoculated with bacteria isolated from grapevine roots out-performed untreated plants. In both the tested vineyards, bacteria-promotion effects largely rely in the formation of an extended epigeal system endowed of longer shoots with larger diameters and more nodes than non-inoculated plants.ConclusionsPGP bacteria isolated in the laboratory can be successfully used to promote growth of grapevines in the field. The resulting larger canopy potentially increased the photosynthetic surface of the grapevine, promoting growth.

Distribution of Rotundone and Possible Translocation of Related Compounds Amongst Grapevine Tissues in Vitis vinifera L. cv. Shiraz.

Rotundone is an attractive wine aroma compound, especially important for cool climate Shiraz. Its presence in wine is mainly from the grape skin, but can also be found in non-grape tissues, such as leaves and stems. Whether rotundone is produced independently within different grapevine tissues or transported amongst non-grape tissues and grape berries remains unclear. The current study investigated the distribution of this compound in different vine tissues during development and studied the most likely mode of rotundone translocation—via phloem—using stable isotope feeding. In addition, local production of rotundone induced by herbivore feeding was assessed. Results showed that rotundone was firstly detected in the petioles and peduncles/rachises within the development of Vitis vinifera L. cv. Shiraz. Different grapevine tissues had a similar pattern of rotundone production at different grape developmental stages. In the individual vine shoots, non-grape tissues contained higher concentrations and amounts of rotundone compared to berries, which showed that non-grape tissues were the larger pool of rotundone within the plant. This study confirmed the local production of rotundone in individual tissues and ruled out the possibility of phloem translocation of rotundone between different tissues. In addition, other terpenes, including one monoterpenoid (geraniol) and six sesquiterpenes (clovene, α-ylangene, β-copaene, α-muurolene, δ-cadinene, and cis/trans-calamenene) were, for the first time, detected in the ethylenediaminetetraacetic acid-facilitated petiole phloem exudates, with their originality unconfirmed. Unlike other herbivore-induced terpenes, herbivorous activity had limited influences on the concentration of rotundone in grapevine leaves.

Structural Changes Induced in Grapevine (Vitis vinifera L.) DNA by Femtosecond IR Laser Pulses: A Surface-Enhanced Raman Spectroscopic Study.

In this work, surface-enhanced Raman spectra of ten genomic DNAs extracted from leaf tissues of different grapevine (Vitis vinifera L.) varieties, respectively, are analyzed in the wavenumber range 300–1800 cm−1. Furthermore, structural changes induced in grapevine genomic nucleic acids upon femtosecond (170 fs) infrared (IR) laser pulse irradiation (λ = 1100 nm) are discussed in detail for seven genomic DNAs, respectively. Surface-enhanced Raman spectroscopy (SERS) signatures, vibrational band assignments and structural characterization of genomic DNAs are reported for each case. As a general observation, the wavenumber range between 1500 and 1660 cm−1 of the spectra seems to be modified upon laser treatment. This finding could reflect changes in the base-stacking interactions in DNA. Spectral shifts are mainly attributed to purines (dA, dG) and deoxyribose. Pyrimidine residues seem to be less affected by IR femtosecond laser pulse irradiation. Furthermore, changes in the conformational properties of nucleic acid segments are observed after laser treatment. We have found that DNA isolated from Feteasca Neagra grapevine leaf tissues is the most structurally-responsive system to the femtosecond IR laser irradiation process. In addition, using unbiased computational resources by means of principal component analysis (PCA), eight different grapevine varieties were discriminated.

The accumulation and localization of chalcone synthase in grapevine (Vitis vinifera L.)

Chalcone synthase (CHS, E.C.2.3.1.74) is the first committed enzyme in the flavonoid pathway. Previous studies have primarily focused on the cloning, expression and regulation of the gene at the transcriptional level. Little is yet known about the enzyme accumulation, regulation at protein level, as well as its localization in grapevine. In present study, the accumulation, tissue and subcellular localization of CHS in different grapevine tissues (Vitis vinifera L. Cabernet Sauvignon) were investigated via the techniques of Western blotting, immunohistochemical localization, immunoelectron microscopy and confocal microscopy. The results showed that CHS were mainly accumulated in the grape berry skin, leaves, stem tips and stem phloem, correlated with flavonoids accumulation. The accumulation of CHS is developmental dependent in grape berry skin and flesh. Immunohistochemical analysis revealed that CHS were primarily localized in the exocarp and vascular bundles of the fruits during berry development; in palisade, spongy tissues and vascular bundles of the leaves; in the primary phloem and pith ray in the stems; in the growth point, leaf primordium, and young leaves of leaf buds; and in the endoderm and primary phloem of grapevine roots. Furthermore, at the subcellular level, the cell wall, cytoplasm and nucleus localized patterns of CHS were observed in the grapevine vegetative tissue cells. Results above indicated that distribution of CHS in grapevine was organ-specific and tissue-specific. This work will provide new insight for the biosynthesis and regulation of diverse flavonoid compounds in grapevine.

Endophytic bacteria with antagonistic traits inhabit the wood tissues of grapevines from Tunisian vineyards

Vineyards throughout the world, including Tunisia, are being attacked by Grapevine Trunk Diseases (GTDs) such as Esca and Botryosphaeriae dieback. In this study, the bacterial microflora colonizing the non-necrotic and necrotic wood tissues of Tunisian mature grapevines (cv Muscat d’Italie) was investigated. Both types of tissues were studied in order to decipher microbial communities associated with them and to find a suitable BCA that can be applied to the Tunisian terroir. Single-Strand Conformation Polymorphism (SSCP) analyses showed that complex bacterial communities specifically colonized both types of wood tissues. The 19 most abundant cultivable strains, selected on their morphology, were isolated from plant samples and assigned to Pantoea, Pseudomonas, Curtobacterium and Bacillus species based on the 16S rRNA and rpoB genes. Biochemical and microbiological screenings revealed that those 19 strains (i) metabolized differently carbon sources, even within the same species, (ii) possessed antibiotic genes, (iii) produced siderophores and solubilized phosphates and (iv) had an in vitro antagonistic effect against 3 fungal pathogens (Lasidiodiplodia pseudotheobromae, Neofusicoccum parvum and Schizophyllum commune) involved in GTDs. One strain, Bacillus subtilis “B6”, had a positive effect on young vines of a cultivar, Muscat d’Italie, frequently planted in Tunisia, by reducing the size of the wood necrosis caused by N. parvum, showing its potential to counteract infection caused by this GTDs agent.

Impact of nickel on grapevine (Vitis vinifera L.) root plasma membrane, ROS generation, and cell viability

Abstract The present study investigated the impact of nickel (Ni2+) on trans-membrane electrical potential (EM) and permeability properties of plasma membrane (PM) in epidermal cells of adventitious grapevine roots. The relationship between disturbances of membrane functionality and the production of superoxide anion, hydrogen peroxide and cell viability after the exposure of roots to Ni2+ was also studied. Treatments with 0.1-5 mmol L-1 NiCl2 induced a concentration-dependent transient PM depolarization, which was recovered to the initial resting potential within 50-70 min in the presence of Ni2+. Longer (up to 24 h) exposure of roots to 1 mmol L-1 of Ni2+ hyperpolarized the EM by approximately 17 mV. Application of the highest 5 mmol L-1 concentration of Ni2+ during longer treatments (up to 48 h) resulted in the increase of membrane permeability; however the EM, cell viability, and superoxide content remained unaffected. The increase in the formation of hydrogen peroxide was time- and concentration- dependent and maximum production was recorded after 180 min of Ni2+ treatment. We can conclude that oxidative stress resulting from an imbalance in the generation and/ or removal of hydrogen peroxide in the root tissues of grapevine was the major cause of Ni2+ toxicity.

Various fungal communities colonise the functional wood tissues of old grapevines externally free from grapevine trunk disease symptoms

Background and Aims The objective of this study was to analyse the various fungal communities that colonise functional wood tissues of old vines that did not express symptoms of grapevine trunk diseases (i.e. Esca and Eutypa dieback) in the year of sampling. Plants of the cultivar Baco Blanc a grapevine hybrid of Folle Blanche and Noah used to produce Armagnac in France were sampled. Methods and Results Forty-two and 58-year-old vines, planted in the same vineyard, were uprooted, cut longitudinally and their functional wood tissues sampled. Culture-dependent and single-strand conformational polymorphism methods were used to compare the fungal communities colonising these wood tissues. It was shown that the fungal communities were significantly different depending on the age of the grapevines. A total of 421 fungal strains were isolated and identified by internal transcribed spacer region sequencing. Conclusions Many grapevine trunk diseases fungal pathogens, particularly the causal agents of Esca (42-year-old vines) and Eutypa dieback (58-year-old vines), as well as numerous potentially plant-beneficial mycoparasites (e.g. Trichoderma spp.), were isolated from the functional wood tissues of old grapevines. Significance of the Study The lack of foliar symptoms among older grapevines may reflect an ‘equilibrium’ among trunk fungal pathogens, mycoparasites and saprobes in the functional wood tissues of trunks.

The usefulness of apricot gum as an organic additive in grapevine tissue culture media

The growth and morphogenesis of cultured plant tissues can be improved by small amounts of some organic elements. In addition to being a natural source of carbon, organic additives may contain natural vitamins, phenols, fiber, hormones and also proteins. Hence, the physiological effects of apricot gum on the regeneration capacity and growth rate of three different plant species i.e. carrot (as a model plant), stevia (as an herbaceous plant), and grapevine (as a woody plant) were examined. The proliferated callus cultures of carrot and in vitro-derived microcuttings of stevia and grapevine were inoculated on their respective standardized proliferation media supplemented with 2.0-6.0 g/l apricot gum. The growth parameters of treated samples were measured and compared to gum-free medium. Earlier callus initiations with greater fresh weight, volume, as well as improved pigmentation were recorded in media fortified with apricot gum. The usefulness of gum application was also obvious in both stevia and grapevine with respect to better shoot multiplication and rooting parameters. Due to positive effects of apricot gum, longer vines with a higher number of lateral shoots, internodes and leaf area were achieved. Overall, the gum at the rate of 4.0 g/L was found to be a logical concentration with respect to encouraging response in all three species. Owing to promising results evolved in the present research, the application of gum in commercial tissue culture protocols is highly recommended. However, further studies are needed to exploit plant derived gums as an alternative carbon source in plant tissue culture media.

Bacteria in a wood fungal disease: characterization of bacterial communities in wood tissues of esca-foliar symptomatic and asymptomatic grapevines.

Esca is a grapevine trunk disease (GTD) associated with different pathogenic fungi inhabiting the woody tissues. Bacteria can also be found in such tissues and they may interact with these fungal colonizers. Although such types of microbial interactions have been observed for wood diseases in many trees, this has never been studied for grapevine. In this study, the bacterial microflora of different vine status (esca-symptomatic and asymptomatic), different anatomical part (trunk and cordon) and different type of tissues (necrotic or not) have been studied. Based on Single Strand Conformation Polymorphism (SSCP) analyses, data showed that (i) specific complexes of bacterial microflora colonize the wood of both necrotic and non-necrotic tissues of esca-foliar symptomatic and asymptomatic vines, and also that (ii) depending on the anatomical part of the plant, cordon or trunk, differences could be observed between the bacterial communities. Such differences were also revealed through the community-level physiological profiling (CLPP) with Biolog EcoplatesTM. Two hundred seventeen bacterial strains were also isolated from plant samples and then assigned to bacterial species based on the 16S rRNA genes. Although Bacillus sp. and Pantoea agglomerans were the two most commonly isolated species from all kinds of tissues, various other taxa were also isolated. Inoculation of vine cuttings with 14 different bacterial species, and one GTD fungus, Neofusicoccum parvum, showed no impact of these bacteria on the size of the wood necroses caused by N. parvum. This study showed, therefore, that bacterial communities differ according to the anatomical part (trunk or cordon) and/or the type of tissue (necrotic or non-necrotic) of wood of grapevine plants showing external symptoms of esca disease. However, research into bacteria having a role in GTD development needs further studies.