Detection and Sequence Analysis of Grapevine Leafroll‐Associated Virus 2 Isolates from China

Several grapevine leafroll‐associated viruses (GLRaVs) have been found frequently in grapevines behaving GLD. Among them, GLRaV‐2 is the only one belonging to Closterovirus, and mainly induces leafroll symptoms and graft incompatibility. In this study, new degenerate primer pairs designed against the HSP70 gene were applied in polymerase chain reaction (PCR) and nested PCR (nPCR) to detect GLRaV‐2 in 132 samples collected from 14 provinces and regions of China. Of the samples, 51.5% were infected with GLRaV‐2, and most did not exhibit GLD symptoms. Some popular grape cultivars had a high incidence of GLRaV‐2 infection, such as Cabernet Sauvignon (92.3%), Chardonnay (80%), Red Globe (75%) and Italian Riesling (73.7%). ‘Beta’ rootstocks, previously identified as negative samples, were also found to be highly infected with GLRaV‐2 (50%). GLRaV‐2 isolates obtained in this study showed identities ranging from 68.9% to 100% and 76.47% to 100.0% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the HSP70 gene showed that all GLRaV‐2 isolates in China belong to three of five reported phylogenetic groups. Different variants belonging to the PN and RG groups were present in a single isolate. The results showed that the new degenerate primer pairs could detect more GLRaV‐2 isolates than the previously reported primers. This is the first detailed report on the prevalence and gene diversity of GLRaV‐2 in China and also provides an nPCR method to improve the sensitivity of PCR as an alternative method when no real‐time PCR device is available.     

基于温度-伤害度关系分析酿酒葡萄根系及芽抗寒性

采用差热分析系统(DTA)对8个主栽酿酒葡萄品种的芽和根系进行低温放热分析(LTE),建立各品种芽、根系韧皮部及木质部的温度-伤害度(LT-I)回归方程,评估不同品种的根系及芽抗寒性.结果表明： 8个品种的根系韧皮部伤害度50%的温度从高到低为马瑟兰>品丽珠>赤霞珠>小芒森>霞多丽>蛇龙珠>贵人香>熊岳白;不同品种木质部伤害度50%的温度从高到低为马瑟兰>霞多丽>赤霞珠>小芒森>品丽珠>蛇龙珠>贵人香>熊岳白;芽伤害度50%的温度从高到低为赤霞珠>小芒森>蛇龙珠>品丽珠>霞多丽>贵人香>马瑟兰>熊岳白.利用模糊隶属函数值法综合评价根系及芽的抗寒性,马瑟兰根系的抗寒性最差,熊岳白根系的抗寒性最好;赤霞珠、品丽珠、小芒森和蛇龙珠芽的抗寒性最差,贵人香和熊岳白芽的抗寒性最好.     

Transmission of Grapevine virus A and Grapevine leafroll‐associated viruses 1 and 3 by Planococcus ficus and Planococcus citri fed on mixed‐infected plants

The Grapevine virus A (GVA) and Grapevine leafroll‐associated viruses 1 and 3 (GLRaV‐1 and GLRaV‐3) are associated with grapevine diseases that induce severe reductions in yield and berry quality. These three viruses are known to coexist in both grapevine and insect vectors, but their cotransmission has been poorly characterised so far. This study investigates the acquisition and transmission of GLRaV‐1, GLRaV‐3 and GVA by Planococcus ficus and Planococcus citri (Hemiptera: Pseudococcidae) following feeding on multiple‐infected plants. The retention and load of the three viruses in the two insect species were analysed. After feeding onto GVA, GLRaV‐1 and GLRaV‐3 mixed‐infected grapevines, nymphs of P. ficus and P. citri showed similar virus acquisition rates and retained low quantities of viruses until the third post‐acquisition day. Despite the similar acquisition patterns, the two vectors differed in transmission efficiency: P. ficus showed a higher efficiency in transmitting GVA and GLRaV‐3, whereas P. citri transmitted GLRaV‐1 more efficiently. When focusing on the virus cotransmission, it appears that GVA could be transmitted to grapevine without GLRaV‐1 and/or GLRaV‐3 and that the transmission of both GLRaVs could take place in the absence of GVA. This comparative study involving different viruses and vector species improves the current knowledge of the semi‐persistent transmission of these three viruses and contributes to the understanding of grapevine virus epidemiology.     

2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype

2-Methoxy-3-isobutylpyrazine (MIBP) contributes a bell pepper aroma to many grape cultivars and has a reported aroma threshold of ～2 ng L(-1) in water. The purpose of this study was twofold: (1) develop a procedure using headspace solid phase micro-extraction combined with GC-MS in the selected ion monitoring mode (HS-SPME-GC-MS-SIM) for analysis of MIBP in grape berries, and (2) determine the location of MIBP biosynthesis in grapevines by approach grafting clusters of Vitis vinifera L. cvs Cabernet Sauvignon and Muscat blanc onto each other. The soluble solids and pH of the grape juice/homogenate matrix from different grape berry developmental stages influenced the method precision; therefore, quantification via the method of standard addition was used. Using our developed method, the limit of detection (LOD) and limit of quantitation (LOQ) of MIBP were 0.1 ng L(-1) and 2 ng L(-1), respectively, measured in a model juice and non-MIBP containing Chardonnay juice. Spiked recoveries averaged between 91% and 112% in Cabernet Sauvignon and Pinot noir homogenates and the overall relative standard deviation was less than 10%. The method was used to analyze MIBP in 29 grape cultivars and in fruit from clusters grafted to Cabernet Sauvignon or Muscat vines. Quantifiable levels were found only in Cabernet franc, Cabernet Sauvignon, Merlot, Sauvignon blanc and Semillon, providing information on the genetic connection for the occurrence of MIBP in grapes. No MIBP was detected in the berries of Muscat blanc clusters grafted onto Cabernet Sauvignon vines when sampled at fruit maturity. MIBP was detected in all berries of Cabernet Sauvignon regardless the graft configuration. The data indicate that MIBP or its precursors originate in the berry and its formation depends upon grape genotype.     

Rootstock control of scion transpiration and its acclimation to water deficit are controlled by different genes

The stomatal control of transpiration is one of the major strategies by which plants cope with water stress. Here, we investigated the genetic architecture of the rootstock control of scion transpiration-related traits over a period of 3 yr. The rootstocks studied were full sibs from a controlled interspecific cross (Vitis vinifera cv. Cabernet Sauvignon × Vitis riparia cv. Gloire de Montpellier), onto which we grafted a single scion genotype. After 10 d without stress, the water supply was progressively limited over a period of 10 d, and a stable water deficit was then applied for 15 d. Transpiration rate was estimated daily and a mathematical curve was fitted to its response to water deficit intensity. We also determined δ(13) C values in leaves, transpiration efficiency and water extraction capacity. These traits were then analysed in a multienvironment (year and water status) quantitative trait locus (QTL) analysis. Quantitative trait loci, independent of year and water status, were detected for each trait. One genomic region was specifically implicated in the acclimation of scion transpiration induced by the rootstock. The QTLs identified colocalized with genes involved in water deficit responses, such as those relating to ABA and hydraulic regulation. Scion transpiration rate and its acclimation to water deficit are thus controlled genetically by the rootstock, through different genetic architectures.     

Early detection of grapevine leafroll virus in <Emphasis Type="Italic">Vitis vinifera</Emphasis> using <Emphasis Type="Italic">in vitro</Emphasis> micrografting

Micrografting of grapevine was investigated for its use as a tool in virus indexing of grapevine stock. Cabernet franc and Cabernet sauvignon scions infected with grapevine leafroll-associated closterovirus III (GLRaVIII) were grafted on to virus-free indicator rootstocks of LN 33 and Cabernet sauvignon growing in tissue culture. The two rootstocks and two scions were grafted in all four possible combinations along with two control grafts (virus-free scion on virus-free rootstock). A modified MS Murashige and Skoog (1962) tissue culture medium supplemented with 0.5 mg l^–1 6-benzylaminopurine was sufficient to induce multiple shoots. Shoots and micrografts readily produced roots in the basal medium. Micrografting gave an overall success rate of 77.8%, with no significant difference between LN 33 rootstock and Cabernet sauvignon. When leafroll infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5% in LN 33 and 63.9% in Cabernet sauvignon) or it showed leafrolling (28.5%, no significant difference between LN 33 and Cabernet sauvignon) within 2–3 weeks. After 12 weeks in culture, the extent of viral symptoms in the micrografted material was high (81.3%), with no significant difference between LN 33 and Cabernet sauvignon; however, the expression of symptoms was more severe on Cabernet sauvignon than on LN 33 rootstock. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) was used to validate the visual symptoms and the presence of virus was confirmed in 80% of the rootstock with visual symptoms of infection. Results indicate that micrografting is an effective method for viral indexing of grapevines. The method can be used in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.     

Long‐distance ABA transport can mediate distal tissue responses by affecting local ABA concentrations

Environmental stresses that perturb plant water relations influence abscisic acid (ABA) concentrations, but it is unclear whether long‐distance ABA transport contributes to changes in local ABA levels. To determine the physiological relevance of ABA transport, we made reciprocal‐ and self‐grafts of ABA‐deficient flacca mutant and wild‐type (WT) tomato plants, in which low phosphorus (P) conditions decreased ABA concentrations while salinity increased ABA concentrations. Whereas foliar ABA concentrations in the WT scions were rootstock independent under conditions, salinity resulted in long‐distance transport of ABA: flacca scions had approximately twice as much ABA when grafted on WT rootstocks compared to flacca rootstocks. Root ABA concentrations were scion dependent: both WT and flacca rootstocks had less ABA with the flacca mutant scion than with the WT scion under conditions. In WT scions, whereas rootstock genotype had limited effects on stomatal conductance under conditions, a flacca rootstock decreased leaf area of stressed plants, presumably due to attenuated root‐to‐shoot ABA transport. In flacca scions, a WT rootstock decreased stomatal conductance but increased leaf area of stressed plants, likely due to enhanced root‐to‐shoot ABA transport. Thus, long‐distance ABA transport can affect responses in distal tissues by changing local ABA concentrations.     

Effects of Water-Deficit Irrigation on Hormonal Content and Nitrogen Compounds in Developing Berries of Vitis vinifera L. cv. Tempranillo

Water-deficit irrigation to grapevines reduces plant growth, yield, and berry growth, altering the ripening process, all of which may influence fruit composition and wine quality. Therefore, the goals of this study were (1) to investigate the influence of the main endogenous berry hormones, abscisic acid (ABA), indole-3-acetic acid (IAA), salicylic acid (SA), and jasmonic acid (JA), on berry growth and ripening under water-deficit conditions and (2) to analyze changes in fruit composition, specifically N compounds, under water deprivation. The study was carried out using container-grown Tempranillo grapevines grown under controlled conditions in a greenhouse. Two irrigation treatments were imposed: control (well-watered) and sustained deficit irrigation (SDI). Water deficit decreased leaf area and the source-to-sink ratio, reduced yield and berry size, and decreased concentrations of the main phenolic compounds. SDI also modified berry hormonal status. At the pea-size stage, SDI berries had lower IAA and higher JA and SA than nonstressed berries. At veraison (onset of ripening), accumulation of ABA was less accentuated in SDI than in control berries. At harvest, the content of amino acids and free ammonium was low in both treatments but SDI-treated berries showed a significant accumulation of amines. Results suggest that water restrictions to grapevines might be playing a physiological role in reducing berry growth through affecting hormone dynamics, phenolic synthesis, and the berry amino acid content and composition, which could compromise fruit quality. Possible roles of endogenous IAA controlling berry size and endogenous ABA and SA controlling levels of anthocyanins and flavonols at harvest are discussed.     

Detection of Grapevine Viruses in Poland

During a 3‐year study, grapevines from 23 vineyards in Poland were surveyed for virus diseases and tested to determine the prevalence of the most economically important viruses by RT‐PCR. The rate of positive samples was 2.2% for grapevine leafroll‐associated virus 1 (GLRaV‐1), 1.9% for grapevine leafroll‐associated virus 2 (GLRaV‐2), 1.5% grapevine leafroll‐associated virus 3 (GLRaV‐3), 1.9% for grapevine virus A (GVA), 0.2% for grapevine virus B (GVB), 0.2% for grapevine virus E (GVE), 0.65% for grapevine fanleaf virus (GFLV), 20.4% for grapevine fleck virus (GFkV) and 71.9% for grapevine rupestris stem pitting‐associated virus (GRSPaV). These viruses were found to occur as single or mixed infections of different combinations in individual grapevines. The overall viral infection rate in the surveyed grapevines was 82.6%. GRSPaV is the most widely distributed virus of all the viruses currently detected in the region. DNA sequencing confirmed the identification of the viruses in selected samples, and analysis indicated that the Polish isolates shared a close molecular identity with the corresponding isolates in GenBank. To our knowledge, this is the first detection of GLRaV‐1, ‐2, ‐3, GVA, GVB, GVE, GFLV, GFkV and GRSPaV in Poland.     

Scion and Rootstock Effects on ABA-mediated Plant Growth Regulation and Salt Tolerance of Acclimated and Unacclimated Potato Genotypes

Tolerance of salt stress in potato (Solanum tuberosum L.) increased when the plants were pre-exposed to low concentrations of salt (salt acclimation). This acclimation was accompanied by increased levels of abscisic acid (ABA) in the shoot. To further study the role of roots and shoots in this acclimation process, reciprocal grafts were made between a salt-tolerant (9506) and salt-sensitive ABA(−) mutant and its ABA(+) normal sibling potato genotype. The grafted plants were acclimated with 75 or 100 mM NaCl for 3 weeks and then exposed to 150–180 mM NaCl, depending on the salt tolerance of the rootstock. After 2 weeks of exposure to the salt stress, the acclimated and unacclimated plants were compared for physiologic and morphologic parameters. The response to the salt stress was strongly influenced by the rootstock. The salt-tolerant 9506 rootstock increased the salt tolerance of scions of both the ABA-deficient mutant and its ABA(+) sibling. This salt tolerance induced by the rootstock was primarily modulated by salt acclimation and manifested in the scion via increased plant water content, stem diameter, dry matter accumulation, stomatal conductivity, and osmotic potential, and is associated with a reduction in leaf necrosis. There was also a pronounced scion effect on the rootstock. Using 9506 as a scion significantly increased root fresh and dry weights, stem diameter, and root water content of ABA(−) mutant rootstocks. Specific evidence was found of the role of exogenous ABA in the enhancement of water status in grafted plants under salt stress beyond that of grafting alone. This was verified by more positive stomatal conductivity and upward water flow in ABA-treated grafted and nongrafted plants and the absence of upward water flow in nontreated grafted plants through NMR imaging. Grafting using either salt-tolerant scions or rootstocks with inherently high ABA levels may positively modify subsequent responses of the plant under salt stress.     

Untersuchungen über endophytische Pilze der Rebe unter besonderer Berücksichtigung des Gefäßsystems der Unterlage

Investigations on endophytic fungi of grapevine with special emphasis on the vascular system of rootstocks In three vegetation periods endophytic fungi from rootstocks of grafted grapevines were isolated, and their influence on the host-plant was determined. Before grafting (pre-nursery) about 40 to 50% of grapevines were colonized by endophytic fungi; in grafted material after growing in a nursery this rate increased up to 50 to 90% (post-nursery). Most of these fungi are Deuteromycetes, especially Moniliales and Coelomycetes. Only a few Ascomycetes were found. The spectrumof fungi differs, mainly depending on the season (pre- and post-nursery), but less on the variety and year of sampling. Contradictory results from investigations of the relationship between the isolated fungi and the vitality of grafted grapevines were found, however the endophytes clearly showed no pathogenic character. On the contrary in some cases a higher vitality of the grapevines correlated with presence of endophytes.     

Effects of Grapevine leafroll‐associated virus 3 on the physiology in asymptomatic plants of Vitis vinifera

Grapevine leafroll disease is one of the most important viral diseases of grapevine (Vitis vinifera) worldwide. Grapevine leafroll‐associated virus 3 (GLRaV‐3) is the most predominant virus species causing this disease. Therefore, it is important to identify GLRaV‐3 effects, especially in plants which do not systematically show visual symptoms. In this study, effects of GLRaV‐3 on grapevine physiology were evaluated in asymptomatic plants of Malvasía de Banyalbufar and Cabernet Sauvignon cvs. Absolute virus quantification was performed in order to determine the level of infection of the treatment. The net carbon dioxide (CO₂) assimilation (A_N) and electron transport rate (J_flux) were the main parameters affected by the virus. The A_N reduction in infected plants was attributed to restrictions in CO₂ diffusion caused by anatomical leaf changes and a reduction of Rubisco activity. Those effects were more evident in Malvasia de Banyalbufar plants. The reduction of A_N leads to a decrease in the total oxygen uptake rate by the activity of the cytochrome oxidase pathway, producing slight differences in plant growth. Therefore, even though no symptoms were expressed in the plants, the effects of the virus compromised the plant vital processes, showing the importance of early detection of the virus in order to fight against the infection.     

ABA and GA3 increase carbon allocation in different organs of grapevine plants by inducing accumulation of non‐structural carbohydrates in leaves,enhancement of phloem area and expression of sugar transporters

Grape quality for winemaking depends on sugar accumulation and metabolism in berries. Abscisic acid (ABA) and gibberellins (GAs) have been reported to control sugar allocation in economically important crops, although the mechanisms involved are still unknown. The present study tested if ABA and gibberellin A₃ (GA₃) enhance carbon allocation in fruits of grapevines by modifying phloem loading, phloem area and expression of sugar transporters in leaves and berries. Pot‐grown Vitis vinifera cv. Malbec plants were sprayed with ABA and GA₃ solutions. The amount of soluble sugars in leaves and berries related to photosynthesis were examined at three points of berry growth: pre‐veraison, full veraison and post‐veraison. Starch levels and amylase activity in leaves, gene expression of sugar transporters in leaves and berries and phloem anatomy were examined at full veraison. Accumulation of glucose and fructose in berries was hastened in ABA‐treated plants at the stage of full veraison, which was correlated with enhancement of Vitis vinifera HEXOSE TRANSPORTER 2 (VvHT2) and Vitis vinifera HEXOSE TRANSPORTER 6 (VvHT6) gene expression, increases of phloem area and sucrose content in leaves. On the other hand, GA₃ increased the quantity of photoassimilates delivered to the stem thus increasing xylem growth. In conclusion, stimulation of sugar transport by ABA and GA₃ to berries and stems, respectively, was due to build‐up of non‐structural carbohydrates in leaves, modifications in phloem tissue and modulation in gene expression of sugar transporters.     

Occurrence of grapevine leafroll-associated virus complex in Napa Valley

Grapevine leafroll disease (GLD) is caused by a complex of several virus species (grapevine leafroll-associated viruses, GLRaV) in the family Closteroviridae. Because of its increasing importance, it is critical to determine which species of GLRaV is predominant in each region where this disease is occurring. A structured sampling design, utilizing a combination of RT-PCR based testing and sequencing methods, was used to survey GLRaVs in Napa Valley (California, USA) vineyards (n = 36). Of the 216 samples tested for GLRaV-1, -2, -3, -4, -5, and -9, 62% (n = 134) were GLRaV positive. Of the positives, 81% (n = 109) were single infections with GLRaV-3, followed by GLRaV-2 (4%, n = 5), while the remaining samples (15%, n = 20) were mixed infections of GLRaV-3 with GLRaV-1, 2, 4, or 9. Additionally, 468 samples were tested for genetic variants of GLRaV-3, and of the 65% (n = 306) of samples positive for GLRaV-3, 22% were infected with multiple GLRaV-3 variants. Phylogenetic analysis utilizing sequence data from the single infection GLRaV-3 samples produced seven well-supported GLRaV-3 variants, of which three represented 71% of all GLRaV-3 positive samples in Napa Valley. Furthermore, two novel variants, which grouped with a divergent isolate from New Zealand (NZ-1), were identified, and these variants comprised 6% of all positive GLRaV-3 samples. Spatial analyses showed that GLRaV-3a, 3b, and 3c were not homogeneously distributed across Napa Valley. Overall, 86% of all blocks (n = 31) were positive for GLRaVs and 90% of positive blocks (n = 28) had two or more GLRaV-3 variants, suggesting complex disease dynamics that might include multiple insect-mediated introduction events.