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.     

The role of the mealybug Phenacoccus aceris in the spread of Grapevine leafroll-associated virus −1 (GLRaV-1) in two French vineyards

Spread patterns of a Grapevine leafroll-associated virus 1 (GLRaV-1) epidemic and a mealybug infestation survey over 10 year were recorded in two Burgundy French vineyards to investigate the relation between them. The temporal evolution of leafroll spread at both study sites was compared on disease incidence data with logistic regression models. We first tested if the spatial distribution of the disease and the mealybug were aggregated using permutation methods, then we tested the independence between the two spatial patterns by randomly shifting one pattern. In Bonzon, an increase from 5 % to 86 % of leafroll prevalence was observed over an 8-year time span, whereas leafroll prevalence remained stable around 5 % in Marsannay-la-Côte during the same period. In Bonzon, the disease spread rapidly from older neighbouring vineyards in four main patches while no spread of the disease was recorded from infected vines in Marsannay-la-Côte. The mealybug Phenacoccus aceris was recorded on 74 % of vines in Bonzon throughout the study and only 6 % of vines in Marsannay-la-Côte. In the latter location, the disease was not associated with the presence of the mealybug, so that it may have arisen from infected plant material escaping the sanitary inspection. In Bonzon, the significant statistical correlation between the mealybug distribution and diseased plants suggests that P. aceris was responsible for the rapid spread of GLRaV-1 in the vineyard. This is the first report of GLRaV-1 natural spread in Europe.     

Survey of mealybug (Hemiptera: Pseudococcidae) vectors of <Emphasis Type="Italic">Ampelovirus</Emphasis> and <Emphasis Type="Italic">Vitivirus</Emphasis> in vineyards of northwestern Italy

Mealybugs (Hemiptera: Pseudococcidae) are sap-sucking insects which infest a broad range of crops worldwide. They represent an important threat to viticulture as they are vectors of viruses associated with leafroll and rugose wood complex diseases. In this study, we surveyed the presence of mealybugs and their associated viruses in vineyards of the Piemonte and Liguria regions, northwestern Italy. In order to determine the collected specimens correctly, we added a species-specific marker for Heliococcus bohemicus to an existing molecular identification key. The only species collected in Piemonte was H. bohemicus, whereas in Liguria, H. bohemicus, Planococcus ficus and Pseudococcus longispinus were found; Ps. longispinus has never before been reported in Italian vineyards. Several specimens of all three species were infected by the ampeloviruses GLRaV-1 and GLRaV-3 and the vitivirus GVA. Both nymphs and adult females tested positive for the viruses and mixed infections were commonly found within the same insect. Population levels and virus incidence were higher in Liguria than in Piemonte, suggesting a greater risk of disease spread. We conclude that the mild, Mediterranean climate of Liguria favors the development of a diverse mealybug fauna while only H. bohemicus, known to be tolerant to the severe continental winter temperatures, colonize grapevines in colder viticultural areas.     

Spatio-temporal association of GLRaV-3-infected grapevines,and effect of insecticidal treatments on mealybug populations in Virginia vineyards

Grapevine leafroll disease (GLD) is caused by a number of viruses in the grapevine leafroll-associated virus complex (GLRaVs). GLRaV-3 is the most commonly found GLRaV and is transmitted by mealybugs (Hemiptera: Pseudococcidae) and soft scale insects (Hemiptera: Coccidae). A series of investigations were conducted to understand spatio-temporal patterns of GLRaV-3 and mealybugs, and to examine management strategies of mealybugs under the environmental conditions of Virginia, USA. During the study, a rapid increase (185 % per year) of GLRaV-3 positive vines was observed at an experimental vineyard. Spatial Analysis by Distance IndicEs (SADIE) indicated a mix of aggregated, random, and uniform distributions of GLRaV-3 within six surveyed vineyards, and a temporal association of GLRaV-3-positive vines over three years at the experimental vineyard. Results from a field experiment in 2009–2011 showed the effect of a delayed dormant application of acetamiprid (Assail, 0.182 L/ha) to be not significantly differing in mealybug abundance from the unsprayed check in 2010 and 2011. Moreover, an application of the contact insecticide beta-cyfluthrin (Baythroid XL, 0.219 L/ha) resulted in significantly higher numbers of mealybugs counted in 2011. This study also demonstrated that GLRaV-3 was spread from mature vines to newly planted sentinel vines during the first growing season, with no apparent effect of prevailing wind direction. Results from another insecticide trial conducted in 2010–2011 demonstrated that both dinotefuran (Scorpion, 0.292 L/ha) and spirotetramat (Movento, 0.439 L/ha) treatments significantly reduced mealybug counts when mealybug populations were high, but only spirotetramat significantly reduced mealybug counts when mealybug populations were low.     

No evidence of transmission of grapevine leafroll-associated viruses by phylloxera (<Emphasis Type="Italic">Daktulosphaira vitifoliae</Emphasis>)

Grapevine leafroll disease is associated with several species of phloem-limited grapevine leafroll-associated viruses (GLRaV), some of which are transmitted by mealybugs and scale insects. The grape phylloxera, Daktulosphaira vitifoliae (Fitch) Biotype A (Hemiptera: Phylloxeridae), is a common vineyard pest that feeds on the phloem of vine roots. There is concern that these insects may transmit one or more GLRaV species, particularly GLRaV-2, a species in the genus Closterovirus. A field survey was performed in vineyards with a high incidence of grapevine leafroll disease and D. vitifoliae was assessed for acquisition of GLRaV. In greenhouse experiments, the ability of D. vitifoliae to transmit GLRaV from infected root sections or vines to co-planted virus-free recipient vines was tested. There were no GLRaV-positive D. vitifoliae in the field survey, nor did D. vitifoliae transmit GLRaV-1, ?2, ?3, or -4LV in greenhouse transmission experiments. Some insects tested positive for GLRaV after feeding on infected source vines in the greenhouse, however there was no evidence of virus transmission to healthy plants. These findings, in combination with the sedentary behaviour of the soil biotype of D. vitifoliae, make it unlikely that D. vitifoliae is a vector of any GLRaV.     

Viruses and virus diseases of grapevine in Egypt

Surveys for virus and virus-like diseases were carried out in commercial vineyards of the main grapevine-growing areas of Egypt along the river Nile and in recently reclaimed desert lands. The only symptoms observed and identified with reasonable confidence in the field were those of leafroll disease in red-berried cultivars. No virus was transmitted to herbaceous hosts by mechanical inoculation from glasshouse-forced cuttings of about 300 vines (40% of total samples). By contrast, ELISA tests showed that 78% of the assayed European vines (521 out of 664) were infected by one (29%) or more (49%) viruses. Grapevine virus A (GVA) was the most widespread virus (67.9% infection), followed by Grapevine leafroll-associated virus 3 (GLRaV-3) (55.9% infection). All the other viruses tested for were scarcely represented, i.e. Grapevine leafroll-associated virus 1 (GLRaV-1) 1.8% infection, Grapevine leafroll-associated virus 2 (GLRaV-2) 1.4% infection, Grapevine virus B (GVB) (0.6% infection) and Grapevine fleck virus (GFkV) (0.2% infection), or, like Grapevine fanleaf virus (GFLV), were totally absent. The infection rate of native cultivars (86%) was particularly heavy. 'Banaty Abiad' and 'Romy Ahmer', the two major Egyptian cultivars, had infection levels of 78% and 89%, respectively, and 'Fayoumy', the most important cultivar in the Fayoum area, had 96% infection. Totally infected were the tested samples of several minor native cultivars such as 'Farg El-Tair', 'Siwi Abiad', 'Ta'afi', 'Romy Abiad', 'Eswid El-Wady', 'Edkawy' and 'Bez El-Anza'. Slightly better was the sanitary situation of imported European grapevine cultivars (60% infection) and of American rootstocks (11.5% infection). In rootstocks, infection rate by GVA and GLRaV-3 was 5.5%, whereas GVB and GLRaV-1 were only sporadically detected.     

Planococcus ficus and the spread of grapevine leafroll disease in vineyards: a 30-year-long case study in north-West Spain

European Journal of Plant Pathology - The mealybug Planococcus ficus is one of the main vectors of Grapevine leafroll associated virus-3 (GLRaV-3), which was commonly detected in cv...     

Genetic diversity in the 3' terminal 4.7-kb region of grapevine leafroll-associated virus 3

Grapevine leafroll-associated virus 3 (GLRaV-3; Ampelovirus, Closteroviridae), associated with grapevine leafroll disease, is an important pathogen found across all major grape-growing regions of the world. The genetic diversity of GLRaV-3 in Napa Valley, CA, was studied by sequencing 4.7 kb in the 3' terminal region of 50 isolates obtained from Vitis vinifera 'Merlot'. GLRaV-3 isolates were subdivided into four distinct phylogenetic clades. No evidence of positive selection was observed in the data set, although neutral selection (ratio of nonsynonymous to synonymous substitution rates = 1.1) was observed in one open reading frame (ORF 11, p4). Additionally, the four clades had variable degrees of overall nucleotide diversity. Moreover, no geographical structure among isolates was observed, and isolates belonging to different phylogenetic clades were found in distinct vineyards, with one exception. Considered with the evidence of purifying selection (i.e., against deleterious mutations), these data indicate that the population of GLRaV-3 in Napa Valley is not expanding and its effective population size is not increasing. Furthermore, research on the biological characterization of GLRaV-3 strains might provide valuable insights on the biology of this species that may have epidemiological relevance.     

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%.     

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.     

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.     

Presence of Grapevine leafroll-associated virus 3 in primary salivary glands of the mealybug vector Planococcus citri suggests a circulative transmission mechanism

Grapevine leafroll-associated virus 3 (GLRaV-3) is a mealybug-transmissible ampelovirus. Though the transmission mechanism has been described as semipersistent on the basis of temporal parameters, definitive proof of this mechanism has never been provided. In the present study, we carried out preliminary assays to establish the location of the virus in its vector, Planococcus citri. After dissecting the insects, GLRaV-3 was detected by means of IC-RT-PCR in the salivary glands, intestine and Malpighian tubes, but not in the sucking apparatus. Immunogold labelling of the capsid protein revealed the presence of the virus in some cells of the primary salivary glands, but not in the alimentary channel of the stylet, or in the accessory salivary glands. The strong labelling of the electron-dense secretion vesicles in some cells of the primary salivary glands, together with the non-detection of the virus in the sucking apparatus suggests that the transmission mechanism may be different from that previously described. We propose a circulative transmission mechanism based on a specific transportation route for the viral particles from the midgut or hindgut to the salivary glands. As the transmission mechanism is generally a common feature of a viral genus, the existence of a circulative transmission mechanism for other mealybug-transmitted ampeloviruses is expected. Organ by organ analysis of GLRaV-1, another ampelovirus not transmissible by P. citri, showed the absence of the virus in the salivary glands, thus providing further, though indirect, evidence in favour of circulative transmission for this virus genus.     

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.     

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).     

Distribution of grapevine leafroll associated virus-3 variants in South African vineyards

Three genetic variants of Grapevine leafroll-associated virus 3 (GLRaV-3) were identified from vineyards of the Western Cape, South Africa. In a previous study, three full genome sequences of isolates representing each of the variant groups were determined. The three variant groups were represented by accessions 621, 623 and PL-20, of variant groups I, II and III respectively. A specific single strand conformation polymorphism (SSCP) profile was assigned to each variant which was used as a quick, reliable detection and differentiation method. In this study we analysed the occurrence of these three GLRaV-3 variants in mother blocks in different cultivars and from different vine growing regions using SSCP. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of three GLRaV-3 variants within a spatio-temporally recorded cluster of diseased plants was studied by means of SSCP profile analysis of ORF5 amplified PCR products. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows.     

<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.