Grapevine Leaf Research Articles

Correlative Analysis of Fluorescent Phytoalexins by Mass Spectrometry Imaging and Fluorescence Microscopy in Grapevine Leaves.

Plant response to their environment stresses is a complex mechanism involving secondary metabolites. Stilbene phytoalexins, namely resveratrol, pterostilbene, piceids and viniferins play a key role in grapevine (Vitis vinifera) leaf defense. Despite their well-established qualities, conventional analyses such as HPLC-DAD or LC-MS lose valuable information on metabolite localization during the extraction process. To overcome this issue, a correlative analysis combining mass spectroscopy imaging (MSI) and fluorescence imaging was developed to localize in situ stilbenes on the same stressed grapevine leaves. High-resolution images of the stilbene fluorescence provided by macroscopy were supplemented by specific distributions and structural information concerning resveratrol, pterostilbene, and piceids obtained by MSI. The two imaging techniques led to consistent and complementary data on the stilbene spatial distribution for the two stresses addressed: UV-C irradiation and infection by Plasmopara viticola. Results emphasize that grapevine leaves react differently depending on the stress. A rather uniform synthesis of stilbenes is induced after UV-C irradiation, whereas a more localized synthesis of stilbenes in stomata guard cells and cell walls is induced by P. viticola infection. Finally, this combined imaging approach could be extended to map phytoalexins of various plant tissues with resolution approaching the cellular level.

Occurrence of Asian grapevine leaf rust disease caused by Phakopsora meliosmae-myrianthae in Mexico

During the falls of 2015 and 2016, rust symptoms were observed on grapevine leaves in a backyard garden located in Tlaxiaco, Oaxaca, Mexico. Based on morphology, analysis of the variable D1/D2 region of the large subunit rDNA, and the fulfilment of Koch’s postulates on grapevine leaves, the causal agent was identified as Phakopsora meliosmae-myrianthae. This is the first report of Asian grapevine leaf rust disease caused by Phakopsora meliosmae-myrianthae in Mexico.

Starch Concentrations in Grapevine Leaves, Berries and Roots and the Effect of Canopy Management

Diurnal and seasonal starch changes in leaves, berries and roots of Vitis vinifera L. cv. Cabernet Sauvignon/99 Richter and the effect of canopy management (a combination of suckering, shoot positioning, and 33% defoliation) on these processes were investigated under field conditions. An increase in starch concentration of basal leaves occurred from the morning to the afternoon during the most active vegetative growth period (up to veraison), indicating a proportional change in storage or export of assimilates between day and night. During later developmental stages diurnal starch levels slightly declined or remained the same. Seasonally, leaf starch remained relatively stable until veraison, whereafter it increased, reaching highest concentrations at the post-harvest stage. Canopy management generally increased leaf starch concentrations. Berries contained no significant amounts of starch. Root starch concentrations were usually higher than those of leaves. Diurnally as well as seasonally, root starch accumulation patterns coincided with those of leaves, indicating a close relationship between source and sink tissue. Canopy management resulted in stable diurnal root starch concentrations without affecting the daily mean starch level; afternoon values of treated vines were, however, generally lower. The results imply that carbohydrate supply and starch-synthesising enzyme systems were not limited by manipulating the canopy and decreasing foliage. It seems, however, that particularly late in the season starch accumulation in roots may be delayed by canopy manipulation as applied. This indicates a shift in sucrose partitioning and its utilisation in roots and other sink areas, e.g. the berries, when the canopy is manipulated.

Grapevine Leafroll-Associated Virus 2 (GLRaV-2)- Mechanical Transmission, Purification, Production and Properties of Antisera, Detection by ELISA

Grapevine leafroll-associated virus 2 (GLRaV-2) was transmitted from Vitis vinifera cv. Muscat d'Alexandrie to Nicotiana benthamiana following inoculation with grapevine leaf petioles extract concentrated by ultracentrifugation. Virus-infected N. benthamiana showed symptoms of chlorotic local lesions, systemic vein clearing followed by yellowing, stem necrosis and death of the plant. The plants were shown to be infected only with GLRa V-2. Antisera produced to GLRa V-2 strongly decorated particles of the virus in immunoelectron microscopy (IEM) and clearly and specifically detected GLRaV-2 in concentrated extracts of infected grapevines in Western Blots. The antisera were used with success for the specific and sensitive detection of GLRaV-2 by ELISA. Treatment of purified GLRaV-2 preparations with glutaraldehyde before immunisation markedly improved the quality of the antisera.

Application of the Enzyme-linked Immunosorbent Assay Procedure to the Detection of Grapevine Fanleaf Virus

Enzyme-linked immunosorbent assay was used to detect grapevine fanleaf virus (GFV) directly in grapevine leaf tissue. The addition of nicotine or nicotine and sodium diethyldithiocarbamate to the extracting buffer solution greatly enhanced the sensitivity of the procedure and enabled detection of GFV concentrations as low as 12 ng/m t The conjugated GFV gamma-globulin detected the GFV strains, grapevine vein banding and grapevine yellow mosaic but failed to detect the GFV serotype arabis mosaic in grapevine leaf tissue. The technique makes possible the collection and processing of numerous samples throughout the growing season and is, therefore, particularly suitable for studies on the incidence and spread of GFV in the field. It also facilitates the resolution of disease syndromes with which GFV is associated.

Grapevine (Vitis viniferaL. ‘Pinotage’) leaf stomatal size and density as modulated by different rootstocks and scion water status

Grapevine (<i>Vitis vinifera</i>L. ‘Pinotage’) leaf stomatal size and density as modulated by different rootstocks and scion water status

The Effects of Preservation Methods of Grapevine Leaves on Total Phenols, Total Flavonoids and Antioxidant Activity

Preservation methods may affect total phenols, flavonoids contents, and antioxidant capacity of many vegetables and fruits. These effects may cause degradation of antioxidant compounds, formation of new products, or destruction of the active metabolites. This study investigated the effects of different preservation methods, such as canning and freezing, on total flavonoids, total phenols, and antioxidant activity on grapevine leaves, which can be consumed either as medicine or food. Phytochemical screening was performed by using standard analytical methods; antioxidant activity was assayed by using 2, 2-diphenyl-1-picryl-hydrazyl-hydrate reagent method; and total flavonoids and total phenols content were determined by using the rutin reference standard method and by Folin-Ciocalteu’s method, respectively. The phytochemical content of all the studied grapevine leaf extracts were same. Fresh leaf extract showed the highest antioxidant capacity as well as total phenols and flavonoids contents. This was followed by the frozen leaf extract, while the canned leaf extract showed lower antioxidant capacity and reduced phenolic and flavonoids contents. Canning and freezing preservation methods of these leaves had no deleterious effects on total antioxidant capacity as well as total phenols and flavonoid contents. Therefore these methods can be used for preparing nutraceutical, cosmeceutical, and pharmaceutical supplements. However, the preservation of V. vinifera leaves by canning is economically and environmentally favored over freezing. In addition the storing, handling, and maintenance of canned leaves is easier than fresh and frozen leaves.

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.

Lipid, phenol and carotenoid changes in 'Bianca' grapevine leaves after mechanical wounding: a case study.

Metabolic changes can occur in plants in response to abiotic stress. Extensive use of leaf discs (mechanical wounding) in studies regarding the effect on the biochemical response of the grapevine to different types of biotic stress makes it necessary to understand metabolic perturbation after injury. In this study, we investigate how mechanical wounding can affect the metabolism of grapevine leaf tissue using Bianca variety as case study. Two sizes of leaf discs (1.1 and 2.8cm in diameter) were excised from leaves, and phenol, lipid and carotenoid perturbation were investigated 0, 6, 12, 24, 48, 96 and 120h post cutting. In our study, we found an accumulation of molecules belonging to stilbenoid and stilbene classes such as trans-resveratrol, trans-piceide, Z-miyabenol C, E-cis-miyabenol C and ampelopsin D + quadrangularin A after abiotic stress. The increase in fatty acids such as linoleic acid, linolenic acid and oleic + cis-vaccenic acid during the first 12h after injury, followed by a return to basal level, allowed us to surmise their role in response to abiotic stress, in particular to mechanical wounding in grapevine leaves. Different-sized discs caused a different response to the tissue, with a higher accumulation in 1.1-cm-diameter discs, especially of phenol compounds. The results of this work can be used to better understand metabolic changes due to biotic stress, having previous knowledge about the perturbation caused by abiotic stress.

Digital Morphometrics of Two North American Grapevines (Vitis: Vitaceae) Quantifies Leaf Variation between Species, within Species, and among Individuals.

Recent studies have demonstrated that grapevine (Vitis spp.) leaf shape can be quantified using digital approaches which indicate phylogenetic signal in leaf shape, discernible patterns of developmental context within single leaves, and signatures of local environmental conditions. Here, we extend this work by quantifying intra-individual, intraspecific, and interspecific variation in leaf morphology in accessions of North American Vitis riparia and V. rupestris in a common environment. For each species at least four clonal replicates of multiple genotypes were grown in the Missouri Botanical Garden Kemper Center for Home Gardening. All leaves from a single shoot were harvested and scanned leaf images were used to conduct generalized Procrustes analysis, linear discriminant analysis, and elliptical Fourier analysis. Leaf shapes displayed genotype-specific signatures and species distinctions consistent with taxonomic classifications. Leaf shape variation within genotypes and among clones was the result of pest and pathogen-induced leaf damage that alters leaf morphology. Significant trends in leaf damage caused by disease and infestation were non-random with respect to leaf position on the shoot. Digital morphometrics is a powerful tool for assessing leaf shape variation among species, genotypes, and clones under common conditions and suggests biotic factors such as pests and pathogens as important drivers influencing leaf shape.

Efficacy of reduced copper dosages against Plasmopara viticola in organic agriculture

The grapevine is the most widely cultivated and economically important fruit species worldwide. Downy mildew produced by Plasmopara viticola, which occurs throughout the wide, is one of the most destructive of all grapevine diseases. In organic farming, copper is the only product effective against this pathogen accepted under European organic farming regulation EC 889/2008. Recently, due to its high environmental impact, the use of copper in organic farming has been limited by legislation to a yearly maximum of 6 kg/ha and its use will probably be even more restricted in the future. For this reason, the average dose recommended by pesticide producers of about 1 kg/ha per treatment should be revised. The aim of this work was to provide scientific support to effectively use reduced copper concentrations. A leaf disk assay was used to determine the minimum copper dosage that provided effective control. Dosages higher than 5 mg Cu/m2 (of grapevine leaf material) did not prove to be more effective than the dosage of 5 mg Cu/m2 itself. With a post-infection disk assay, copper treatments made 1 h after a simulated infectious rain, were also confirmed to give the same efficacy as preventive copper treatments. The efficacy of preventative low copper doses was validated in field trials. In the field, the rates of 200 and 400 g Cu/ha (equivalent to 5 and 10 mg Cu/m2, respectively) was able to significantly reduce downy mildew (72–89% efficacy), confirming results obtained from leaf disks assays. Adjusting from higher copper spray rates, to those between 200 and 400 g Cu/ha should provide sufficient control depending on the infection pressure. This spraying regime should also enable viticulturists to remain under the current annual limit of 6 kg/ha and likely under lower limits that might be proposed in future regulations.

Asian grapevine leaf rust caused by Phakopsora euvitis: an important disease in Brazil

Anthropogenic influences such as tourism and climate change affect ecosystems. One consequence is that invasive species can spread to and colonize novel areas. A recent example of this scenario is the appearance, in the early 2000s in Brazil and Australia, of the plant pathogenic fungus Phakopsora euvitis, which causes Asian grapevine leaf rust. It is speculated that the disease was introduced either by long distance transport of spores in the atmosphere or the import of contaminated plant material. While the disease could be eradicated from the Australian continent it is still present in Brazil. The pathogen has spread successfully throughout most Brazilian grapevine‐growing regions, most probably because of the lack of resistant plant cultivars. Today, the disease is endemic in Brazil. This review was written to highlight the increasing impact of the disease in Brazil and summarizes the current knowledge on the disease monocycle under different environmental conditions and the consequences for grapevine management. Gaps in the present state of knowledge are highlighted with the purpose of stimulating further research aimed at identifying efficient strategies to combat the disease.

Patterns of phytoalexins in the grapevine leaf stripe disease (esca complex)/grapevine pathosystem

Vineyards containing vines affected with grapevine leaf stripe disease (GLSD), one of the diseases of the esca complex, suffer losses in grape yield and quality every growing season. To examine the relation between GLSD foliar symptoms and levels of phytoalexins in grapevine, phytoalexin levels were monitored in the leaves of symptomatic, asymptomatic/diseased, and healthy grapevine leaves, at various growth stages, in two vineyards in Italy, over four growing seasons. At the same time, the leaf symptoms of the vines at some of those growth stages were recorded in each vineyard and in each growing season. The compounds extracted and identified were: trans -resveratrol, trans -pterostilbene, trans-e-viniferin and trans-δ-viniferin. The most common phytoalexin found was resveratrol. Amounts of all the phytoalexins were generally greater in symptomatic leaves than in asymptomatic/diseased or healthy leaves. In symptomatic leaves, resveratrol levels were greatest at pre-bunch closure, and peaks in pterostilbene occurred at the same time. Leaves of each category (symptomatic, asymptomatic/diseased, healthy) had lower amounts of these compounds at veraison and generally higher amounts at the stages of harvesting and/or the softening of berries. It seems therefore that the formation and pattern over time of the phytoalexins was linked to the growth stage of the vines. Leaf symptoms never occurred before pre-bunch closure, but became much more common from veraison to harvest. This study provides evidence of a relationship between the levels of phytoalexins, grapevine growth stage, and the seasonal pattern of development of GLSD symptoms.

Ultrastructural leave features of grapevine cultivars (&lt;em&gt;Vitis vinifera&lt;/em&gt; L. ssp. &lt;em&gt;vinifera&lt;/em&gt;)

&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Aim&lt;/strong&gt;: To investigate and compare of &lt;em&gt;Vitis vinifera&lt;/em&gt; Linné subsp. &lt;em&gt;vinifera&lt;/em&gt; leaves of cultivars important for the Austrian wine producers in order to learn more about their surface architectures in the microand nanoscale.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Methods and results&lt;/strong&gt;: Atomic force microscopy, binocular fluorescence microscopy, contact angle measurements, and environmental scanning electron microscopy were employed in order to assess physicochemical features of fresh plant material. Erect and prostrate trichomes are the most characteristic features present on the epidermal surfaces of grapevine leaves. These hairs occur in rather different densities from none to densely covering the whole surface. Contact angels are highly affected by these hairs, resulting in individual cases in values &amp;gt;150° in the presence of a high reclining hair density. On nanoscopic scale blades of the varieties differ with respect to their wax structures, in orientation, shape and size. Cuticular striae and epicuticular waxes, mostly granules and platelets, are the most conspicuous characteristics of grapevine leaf ultrastructure.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Conclusion&lt;/strong&gt;: The microscopy techniques applied are complementary, enabling morphological analysis at different scales. They are not only efficient tools for descriptive botanics and finding morphological adaptions to the environmental conditions, they provide also an insight into the habitat of leaf colonizing microbes, pathogenic as well as beneficial ones and may add to the understanding of the conditions they find on leaf surfaces.&lt;/p&gt;&lt;p style="text-align: justify;"&gt;&lt;strong&gt;Significance and impact of the study&lt;/strong&gt;: Leaf surface structures and chemicals are part of the defence system of the plant. Water-repellency can be advantageous for the plant as it creates unfavourable conditions for the successful colonization of pathogens. The knowledge of wetting properties of leaf surfaces will advance the insight in the interaction with additives, promoting the secure and optimal use of plant protection agents applied by spray deposition, especially under difficult weather conditions. The application of such research will be better contact and or penetration, better adhesion of pesticides and other plant protecting agents but also improved adhesion of plant promoting bacteria in biocontrol applications.&lt;/p&gt;

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.

Method validation for determination of metals in Vitis labrusca L. grapevine leaf extracts by inductively coupled plasma mass spectrometry (ICP-MS).

Vitis labrusca L. is the main species used for wine and juice production in Brazil. The grapevine leaves can be used both as functional foods and as cheapest sources for the extraction of phenolic compounds. Besides the antioxidant activity, grapevine leaves exhibited significant anti-inflammatory activity. Therefore, the aim of this study was to develop and validate an analytical methodology to determine the metals selenium (96Se), chromium (53Cr), nickel (62Ni), cadmium (111Cd) and lead (206Pb) in 30 samples of grapevine leaf extracts (Vitis labrusca, Bordo cultivar) using inductively coupled plasma mass spectrometry (ICP-MS). To obtain the grapevine leaf extracts the samples were milled, weighed and digested in microwave oven with nitric acid. The method showed linearity, precision, accuracy and limits of quantification and detection acceptable for INMETRO protocol validation of analytical methods. Therefore, the method using ICP-MS was developed and validated to determine metals concentrations in grapevine leaves of Vitis labrusca L. and the proposed method could be applied in routine analytical laboratory.

Association between genetic variability and titre of Grapevine Pinot gris virus with disease symptoms

The present work was carried out in order to verify the possible association between a new grapevine disease, characterized by leaf mottling and deformation, and the genetic variability and concentration of Grapevine Pinot gris virus (GPGV), a recently identified virus tentatively associated with the pathology. After vineyard surveys and the establishment of real‐time qPCR assays, characterization of GPGV isolates and evaluation of GPGV titre were assessed in more than 100 samples of grapevine Glera, collected from plants regardless of whether or not they showed the symptomatology. Results showed that there was an important association between the GPGV variants and manifestation of the symptoms, and that grapevines with symptoms harboured significantly higher GPGV titre than symptomless vines. Moreover, an interesting relationship among the phylogenetic clustering of the isolates originating from plants with symptoms and some epidemiological characteristics of the disease was found. The current study confirmed the role of GPGV in the emergent disease characterized by grapevine leaf mottling and deformation.

Stability of Ampelometric Characteristics of Vitis vinifera L. cv. ‘Syrah’ and ‘Sauvignon blanc’ Leaves: Impact of Within-vineyard Variability and Pruning Method/Bud Load

Historically, grapevine (Vitis vinifera L.) leaf characterisation has been a driving force in the identification of cultivars. In this study, ampelometric (foliometric) analysis was done on leaf samples collected from hand-pruned, mechanically pruned and minimally pruned ‘Sauvignon blanc’ and ‘Syrah’ vines to estimate the impact of within-vineyard variability and a change in bud load on the stability of leaf properties. The results showed that within-vineyard variability of ampelometric characteristics was high within a cultivar, irrespective of bud load. In terms of the O.I.V. coding system, zero to four class differences were observed between minimum and maximum values of each characteristic. The value of variability of each characteristic was different between the three levels of bud load and the two cultivars. With respect to bud load, the number of shoots per vine had a significant effect on the characteristics of the leaf laminae. Single leaf area and lengths of veins changed significantly for both cultivars, irrespective of treatment, while angle between veins proved to be a stable characteristic. A large number of biometric data can be recorded on a single leaf; the data measured on several leaves, however, are not necessarily unique for a specific cultivar. The leaf characteristics analysed in this study can be divided into two groups according to the response to a change in bud load, i.e. stable (angles between the veins, depths of sinuses) and variable (length of the veins, length of the petiole, single leaf area). The variable characteristics are not recommended to be used in cultivar identification, unless the pruning method/bud load is known.

Specific adjustments in grapevine leaf proteome discriminating resistant and susceptible grapevine genotypes to Plasmopara viticola

Grapevine downy mildew is an important disease affecting crop production leading to severe yield losses. This study aims to identify the grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). Leaf proteome analysis was performed using 2D difference gel electrophoresis followed by protein identification via mass spectrometry. In addition, we analysed ROS production, antioxidant capacity, lipid peroxidation and gene expression. Proteins related to photosynthesis and metabolism allowed the discrimination of resistant and susceptible grapevine cultivars prior to P. viticola inoculation. Following inoculation increase of hydrogen peroxide levels, cellular redox regulation, establishment of ROS signalling and plant cell death seem to be key points differentiating the resistant genotype. Lipid associated signalling events, particularly related to jasmonates appear also to play a major role in the establishment of resistance. The findings from this study contribute to a better understanding of genotype-specific differences that account for a successful establishment of a defence response to the downy mildew pathogen. Biological significanceHere, we present for the first time grapevine cultivar-specific adjustments of leaf proteome that allow the discrimination between resistance and susceptibility towards P. viticola (constitutive (0h) and in after inoculation (6, 12 and 24h). We have highlighted that, following inoculation, the major factors differentiating the resistant from the susceptible grapevine cultivars are the establishment of effective ROS signalling together with lipid-associated signalling events, particularly related to jasmonates. It is believed that plants infected with biotrophic pathogens suppress JA-mediated responses, however recent evidences shown that jasmonic acid signalling pathway in grapevine resistance against Plasmopara viticola. Our results corroborate those evidences and highlight the importance of lipid- signalling for an effective resistance response against the downy mildew pathogen.

Rapid loop-mediated isothermal amplification assays for grapevine yellows phytoplasmas on crude leaf-vein homogenate has the same performance as qPCR

A fluorescence-based real-time loop-mediated isothermal amplification (LAMP) assay for ‘Candidatus Phytoplasama solani’ (Bois noir phytoplasma; BNp) detection was developed and optimised for rapid laboratory and on-site BNp detection. This assay is highly specific, rapid and as sensitive as qPCR. It was validated according to European and Mediterranean Plant Protection Organisation recommendations. In addition, 286 grapevine leaf samples from the 2015 growing season were tested with this new real-time LAMP assay and an assay previously developed for detection of Flavescence dorée phytoplasma (FDp). These LAMP assays for detection of both BNp and FDp used without any DNA extraction step, which is a required step for qPCR analysis, were comparably effective to qPCR, and positive results were obtained in less than 35 min.