Somatic embryogenesis efficiently eliminates viroid infections from grapevines

Indirect somatic embryogenesis is effective at eliminating the most important viruses affecting grapevines. Accordingly, this technique was tested as a method for eradicating two widespread viroids, Grapevine yellow speckle viroid 1 (GYSVd-1) and Hop stunt viroid (HSVd), from four grapevine cultivars. Both viroids were detected by RT-PCR in grapevine floral explants used for initiating embryogenic cultures, as well as in undifferentiated cells of embryogenic and non-embryogenic calli from anthers and ovaries. In contrast, somatic embryos differentiated from these infected calli were viroid-free, and viroids were not detected in embryo-derived plantlets even 3 years after their transfer to greenhouse conditions. A wider spatial distribution of HSVd than GYSVd-1 within proliferating calli was revealed by in situ hybridization, whereas no hybridization signal was detected in the somatic embryos. In addition, GYSVd-1 and HSVd were localised in the nucleus of infected cells, conclusively showing the nuclear accumulation of representative members of Apscaviroid and Hostuviroid genera, which has been only an assumption so far. Somatic embryogenesis was compared to in vitro thermotherapy, a technique routinely used for virus eradication. After thermotherapy, HSVd and GYSVd-1 were detected in all in vitro plantlets of the cultivar Roussan, and in all lines analysed after 3 years of culture in greenhouse. The high efficiency with which somatic embryogenesis may eliminate viroids and viruses from several infected grapevine cultivars, should allow the availability of virus- and viroid-free material, which would be useful not only for sanitary selection but also for basic research on plant-virus and plant-viroid interactions in grapevine.     

<Emphasis Type="Italic">In situ</Emphasis> localization of <Emphasis Type="Italic">Grapevine fanleaf virus</Emphasis> and phloem-restricted viruses in embryogenic callus of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

In grapevine, somatic embryogenesis is particularly effective in eliminating several important virus diseases. However, the mechanism whereby regenerated somatic embryos are freed of the viruses is not clear. The distribution of Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus-3 (GLRaV-3) and Grapevine virus A (GVA) in embryogenic callus of grapevine was investigated by in situ hybridization using digoxygenin-labelled oligonucleotide probes. Four months after culture initiation, in callus originated by GFLV-infected explants we observed a mosaic of infected and uninfected cells, with high concentrations of viruses in some cell groups in peripheral zones of the callus. In addition some abnormal somatic embryos showed a high hybridization signal. In callus originated by GVA- and GLRaV-3-infected explants the viruses were concentrated in few cells surrounded by areas of virus-free cells. The two viruses were generally localized in different clusters of cells inside the callus and the levels of infection were lower than those observed in GFLV-infected callus. No virus was detected in callus nor in somatic embryos after 6 months of culture. The results highlight the difficulties of some viruses at stably invading callus tissues and the differential ability of GFLV to spread in the callus cells compared to the phloem-limited viruses.     

Crude garlic extract significantly inhibits replication of grapevine viruses

Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.     

Viruses of grapevine in Syria

Surveys for virus and virus-like diseases were carried out in commercial vineyards and nurseries in seven different Syrian provinces (Aleppo, Dara'a, As Suwayda, Al Qunaytirah, Homs, Hamah, Tartous). Samples were collected at random from 835 individual vines (735 Vitis vinifera and 100 rootstock accessions) for laboratory testing. Grapevine fanleaf virus (GFLV) , Arabis mosaic virus (ArMV), and Grapevine virus A (GVA) were the only viruses recovered by mechanical transmission to herbaceous hosts. Vein necrosis developed in c. 53% of graft-inoculated 110R indicators and vein mosaic in V. riparia inoculated with material from cv. Corna Alegra. A total of 71% of the ELISA-tested V. vinifera plants (522 out of 735) were infected by one (14.8%) or more (55.8%) viruses. GVA was the most widespread (54.7%), followed by Grapevine leafroll-associated virus 1 (GLRaV-1, 47.3%), Grapevine fleck virus (GFkV, 29.7%), and Grapevine leafroll-associated virus 3 (GLRaV-3, 23.9%). Other economically relevant viruses were scarcer, i.e. Grapevine leafroll-associated virus 2 (GLRaV-2, 9%), GFLV (0.8%) and ArMV (0.1%). The most important Syrian grapevine varieties, i.e. Hellwany, Salty, Balady, and Zeiny, had average infection rates that ranged between 44% and 91%. The highest incidence of infections was observed at Damascus (90%), whereas it ranged between 68% and 79% in the other provinces, except for Hama (36%). Rootstocks were in much better sanitary condition (25% infection). GFkV (22%) was the most common virus, whilst the presence of GLRaV-3 (3%), GLRaV-1, and GFLV (1%) was negligible. Grapevine rupestris stem pitting associated virus (GRSPaV) was detected in 72.3% of the samples by RT-PCR. A high percentage of the GRSPaV-positive vines (80%) induced vein necrosis reactions in 110R, thus confirming the recently established correlation between this virus and vein necrosis.     

Viruses and virus diseases of grapevine in Tunisia

Mature canes were collected from vines in the main grapevine-growing areas in Tunisia (Cape Bon, Bizerte, Ben Arous), from commercial vineyards and mother-plant plots, to assess the presence of virus and virus-like diseases. Biological (mechanical transmission onto herbaceous hosts and grafting onto indicator woody plants) and serological detection (ELISA) methods were applied. ELISA showed that 96.4% of 669 vines tested were infected, most of them (88.1%) by at least two viruses. Grapevine leafroll-associated 3 closterovirus (GLRaV-3) was the most widespread virus (87.9%), followed by grapevine A vitiviras (GVA, 69.4%), grapevine fleck virus (GFkV, 51.9%), grapevine leafroll-associated 1 closterovirus (GLRaV-1, 36.8%), grapevine leafroll-associated 2 closterovirus (GLRaV-2, 19.1%), grapevine fan leaf nepovirus (GFLV, 18.2%) and grapevine B vitiviras (GVB, 14.8%). ELISA tests yielded negative results for grapevine leafroll-associated 7 closterovirus (GLRaV-7) and potato X potexvirus (PVX). The highest infections were found in Bizerte and Cape Bon regions (100 and 99.2%), and in vineyards aged over 20 years (98.5%) as compared with the younger ones (81.1%). Rootstocks in mother-plant plots were practically free from all the viruses tested (1 plant infected out of 81), whereas severe infections were found in Vitis vinifera mother plants (67.4% of 341 samples), in particular table grapes (92.6%) compared with wine grapes (47.9%). In these mother-plant plots, the prevailing viruses were GLRaV-3 (41.3%), followed by GFkV (36.7%), GVA (27.9%), GLRaV-1 (17%) and GLRaV-2 (15.2%). GFLV and GVB were far more limited (1.5 and 0.6%, respectively). The presence of vein necrosis and vein mosaic was ascertained by transmission onto 110R and Vitis riparia indicators, whereas only GFLV was mechanically transmitted onto herbaceous hosts (from about 20% of the samples).     

Grapevine leafroll-associated viruses and grapevine virus A in selected Vitis vinifera cultivars in northern Italy

The occurrence of grapevine leafroll-associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine virus A (GVA) was demonstrated in a viticultural region of northern Italy (Emilia-Romagna) using immunoelectron microscopy. Virus incidence was subsequently assessed using ELISA. A total of 60.6% of the 150 clone selections tested, from 18 local Vitis vinifera cultivars, were found to be infected. ELISA did not reveal the presence of grapevine leafroll-associated virus 2 (GLRaV-2) or grapevine leafroll-associated virus 5 (GLRaV-5). GLRaV-1, GLRaV-3 and GVA were found individually and in various combinations. The most common findings were GLRaV-1 alone (25.3%) and associated with GVA (33%). Serological data confirmed that the majority (91%) of the clones known to be affected by grapevine leafroll (GLR), on its own or in association with rugose wood (RW), contained viruses. On the other hand, where the RW phenomenon was present on its own, only 40% of these clones were ELISA-positive. The implications for the biology of GLR and RW are discussed and the complex aetiology of these grapevine diseases is confirmed.     

4种葡萄卷叶伴随病毒多重RT-PCR检测

 葡萄受卷叶伴随病毒侵染后,树势减弱,抗逆性变差,果穗着色不良,成熟期推迟,含糖量降低。目前已报道11种葡萄卷叶伴随病毒(Grapevine leafroll-associated virus,GLRaV)。为提高检测效率,降低检测费用,本文在研究单个卷叶伴随病毒RT-PCR检测技术基础上,对4种葡萄卷叶伴随病毒的多重RT-PCR模板浓度、引物浓度和退火温度进行优化,建立了同时检测葡萄卷叶伴随病毒-1(GLRaV-1)、葡萄卷叶伴随病毒-3(GLRaV-3)、葡萄卷叶伴随病毒-4(GLRaV-4)和葡萄卷叶伴随病毒-5(GLRaV-5)的多重RT-PCR技术体系。模板浓度、引物浓度、Taq DNA聚合酶浓度、退火温度和循环次数对多重RT-PCR检测结果均有较大影响,而在一定范围内改变延伸时间和dNTP浓度对检测结果影响较小。对4种葡萄卷叶伴随病毒的PCR产物进行克隆和测序,扩增基因片段与GenBank中登录的基因序列同源性为95%~99%。所建立的多重RT-PCR技术检测田间样品效果良好。     

Elimination of Citrus psorosis virus by somatic embryogenesis from stigma and style cultures

Somatic embryogenesis was used to eliminate Citrus psorosis virus (CPsV) from three citrus species (common mandarin, sweet orange and Dweet tangor), all of which regenerated somatic embryos with different embryogenic potential from stigma and style explants. CPsV was detected by double antibody sandwich‐indirect‐enzyme‐linked immunosorbent assay (DASI-ELISA) in explants and embryogenic callus, but was not detected in any of the plants obtained from somatic embryos, even 24 months after regeneration. Loss of juvenile characters (disappearance of thorns) was observed in the first year of growth and was retained in plants propagated by grafting from thornless stems. Somatic embryogenesis appears to be a very promising technique for the production of healthy citrus stocks.     

Transmission of six ampeloviruses and two vitiviruses to grapevine by Phenacoccus aceris

Grapevine leafroll disease is caused by grapevine leafroll-associated viruses (GLRaVs). These viruses are common in vineyards worldwide and often associated with vitiviruses that are involved in the rugose wood complex of grapevine. Ten mealybug species are known as vectors of one or several of these grapevine viruses, including the apple mealybug Phenacoccus aceris which is widespread in Holarctic regions and able to transmit Grapevine leafroll-associated virus-1 and -3 (GLRaV-1 and -3). Our aim was to characterize the transmission features of leafroll viruses by Phenacoccus aceris in order to better understand the contribution of this mealybug to leafroll epidemics. Results showed that Phenacoccus aceris is able to transmit GLRaV-1, -3, -4, -5, -6, and -9 to grapevine but not GLRaV-7. This is the first report of GLRaV-6 transmission by a mealybug. Also, for the first time it was shown that Phenacoccus aceris could vector vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). First instar nymphs were the most efficient stage in transmitting GLRaV-1, -3, and GVA. This research sheds light on the transmission biology of grapevine viruses by Phenacoccus aceris and represents a step forward to leafroll disease management.     

Elimination of Grapevine rupestris stem pitting-associated virus (GRSPaV) from two Vitis vinifera cultivars by in vitro chemotherapy

The grapevine (Vitis vinifera L.) cultivars ‘Agiorgitiko’ and ‘Malagouzia’, naturally infected with Grapevine rupestris stem pitting-associated virus (GRSPaV), were subjected to in vitro chemotherapy using the antiviral inosine 5′-monophosphate dehydrogenase inhibitors tiazofurin (TR), ribavirin (RBV) and mycophenolic acid (MPA). The chemotherapy lasted 80 days and was carried out as two consecutive treatments. Severe phytotoxicity, estimated after 40 days of culture, was observed in drug-treated explants, especially when high doses of TR were used. Phytotoxicity exhibited a cultivar- and chemical compound-dependent profile. The virus eradication status of the survived plantlets was determined by nested RT-PCR using total RNA templates, after 80 days of drug treatment and one year later, after the passage of one dormancy period, in potted plants grown in a greenhouse. Data indicated that the highest GRSPaV elimination in ‘Agiorgitiko’ was obtained with 10 μg? ml^?1 TR, 30 μg ?ml^?1 RBV and 20 μg? ml^?1 MPA. The eradication rates were lower in the case of ‘Malagouzia’, where the highest ones were achieved after treatments with 15 μg ml^?1 TR and 80 μg ml^?1 MPA. This is the first report on GRSPaV elimination in grapevine following treatment with antiviral compounds, which could provide an alternative to the traditional methods of virus eradication through meristem culture and thermotherapy.     

Synergy between grapevine vitiviruses and grapevine leafroll viruses

An interactive relationship between vitiviruses and grapevine leafroll viruses was characterized in grapevine. Grapevine viruses A and B (GVA and GVB) were found more frequently in the presence of co-infecting Grapevine leafroll associated viruses (GLRaV-1, ?2 or ?3) than in their absence. The titers of the vitiviruses in co-infection with leafroll viruses were found to be higher than were their titers in the absence of leafroll virus infection. The occurrence of vitivirus-associated stem-pitting symptoms was correlated with leafroll virus co-infection. Specific pairing associations on the species level were found between different viti- and leafroll virus species: GVB was associated preferentially with GLRaV-2; GVA was associated preferentially with GLRaV-1 and GLRaV-3. In contrast to the increase in vitivirus titer seen with leafroll virus co-infection, the incidence and titer of grapevine leafroll virus appeared to be unaltered by vitivirus co-infection. The potential for a synergistic enhancement of grapevine disease in co-infected vines is discussed.     

High prevalence of viruses in table grape from Spain detected by real-time RT-PCR

Table grapes from one of the most important growing area in Spain (Vinalopó, Alicante) protected by the Designation of Origin “Vinalopó bagged table grape”, were surveyed and analysed to determine the prevalence of the five viruses included in the Spanish certification program: Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV), Grapevine leafroll associated virus-1 (GLRaV-1) and Grapevine leafroll associated virus-3 (GLRaV-3). Ninety five sampling points were selected and the position of grapevine plants georeferenced. Samples were collected in two different vegetative periods and analyses were performed by ELISA and real-time RT-PCR. Purified RNA and immobilized viral targets from plant extracts on nylon membranes were used in parallel assays as templates for PCR assays. In order to analyse these five viral species by real-time RT-PCR, new specific primers and TaqMan probes were designed for detection of ArMV and GFkV. Real time RT-PCR from purified RNA was more sensitive than spot version and ELISA tests. The most prevalent virus was GFLV (95.8%) followed by GLRaV-3 (94.7%), GLRaV-1 (66.3%) and GFkV (65.3%). ArMV was not detected in any sample. The high level of viral infections and the presence of mixed infections suggest that initial infected plant material and uncontrolled traffic of propagation material have played an important role in the spread of viruses.     

沙地葡萄茎痘相关病毒的RT-LAMP检测方法

 本研究建立了一种用于沙地葡萄茎痘相关病毒（Grapevine rupestris stem pitting-associated virus, GRSPaV）的RT-LAMP检测方法。以GRSPaV的RdRp基因序列（GenBank登录号：GQ478314）为靶序列,设计3组RT-LAMP引物,从中筛选出1组有效引物,并确定了适宜的反应温度和反应时间。对RT-LAMP产物进行Hha Ⅰ酶切,酶切片段与理论片段大小一致,证明了RT-LAMP产物的特异性。RT-LAMP方法能够检测出GRSPaV的RNA最大稀释倍数为10^-4,与RT-PCR方法相比更为灵敏。田间葡萄样品RT-LAMP检测结果与已知样品带毒情况相同,表明RT-LAMP检测GRSPaV具有较好的可靠性。在RT-LAMP反应产物中加入染料SYBR Green Ⅰ （×1000）可直接观察反应结果。建立的GRSPaV RT-LAMP检测方法具有简便、快速、灵敏、可视化等特点,尤其适合基层使用,具有良好的应用前景。     

Genetic diversity of Grapevine rupestris stem pitting-associated virus isolates from Tunisian grapevine germplasm

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most widespread grapevine viruses and is transmitted mainly by grafting. GRSPaV presence was tested in 487 samples representative of the Tunisian grapevine germplasm (including autochthonous, table, wine, wild grape, and rootstock varieties) from different Tunisian regions. GRSPaV infection was detected in 51.3% of samples from different Tunisian regions, among which the table grapevine cultivars were the most commonly infected (68.7%). Genetic variability of GRSPaV isolates from wild and cultivated grapevines was assessed by sequencing the partial capsid protein (CP) gene of 19 Tunisian isolates and 1 Italian GRSPaV isolate from Sicily, and the partial RNA-dependent RNA polymerase (RdRp) gene of 13 Tunisian GRSPaV isolates. According to phylogenetic analysis of CP nucleotide sequences obtained in this study and sequences retrieved from GenBank, Tunisian isolates fell into four phylogenetic groups already described (I, II, III, and IV) and two new phylogenetic groups (VI and VIII). Phylogenetic analysis of the partial RdRp gene revealed that Tunisian isolates of GRSPaV are distributed into four phylogroups. This study highlights the importance of regular monitoring of GRSPaV infections in Tunisia, with special regard to those grapevine accessions employed in conservation and selection programmes. In particular, the presence of new GRSPaV genetic variants and infection of wild grapevines must be taken into account in order to choose a correct control strategy.     

Viruses and virus diseases of grapevine in Lebanon

Grapevines were surveyed for the presence of virus and virus-like diseases in the main viticultural areas of Lebanon (Bekaa valley, Mount Lebanon, South and North Lebanon). Symptoms of rugose wood were observed in vines ofall cultivars and areas surveyed, whereas leafroll was observed only in some vineyards of the Bekaa valley and, to a lesser extent, in South Lebanon on cvs Tfaifihi, Cinsaut and Cardinal. Symptoms of fanleaf and of phytoplasma-induced yellows were also observed with low frequency in the Bekaa valley on wine-grape cultivars. ELISA tests showed that 53% of 1536 Vitis vinifera vines individually checked were infected by one or more viruses. Grapevine trichovirus A (GVA) was the prevailing virus (32.4%), followed by grapevine fleck virus (GFkV) (19.5%) and grapevine leafroll-associated closterovirus 3 (GLRaV-3) (12.4%). Grapevine leafroll-associated closterovirus 1 (GLRaV-l), grapevine trichovirus B (GVB) and grapevine fanleaf nepovirus (GFLV) were also detected to a lesser extent, their incidence ranging between 1.1 and 3.6%.