Detection and characterization of two strains of Grapevine fanleaf nepovirus in Tunisia

The nematode-borne Grapevine fanleaf nepovirus (GFLV) causes severe degeneration of grapevines in vineyards worldwide. In a recent survey of the sanitary status of grapevine plants in north Tunisian vineyards, we were interested to study the polymorphism of GFLV. Purified virus, from mechanically inoculated Chenopodium quinoa , was used to produce anti-GFLV antiserum, which specifically recognized GFLV in different Tunisian grapevine samples using the DAS-ELISA technique. Positive samples were subjected to oligoprobe-RT-PCR to amplify a 606 bp region of the viral coat protein sequence. PCR products used for RFLP analysis after digestion with endonuclease Alu I produced 3 restriction profiles. RFLP data allowed clear distinction of two GFLV strains in Tunisia. The nucleotide sequence of the PCR-generated amplicons from each strain was determined showing 93.4% identity at the nucleic acid level and 97.5% similarity at the aminoacid sequence level compared to the previously characterized GFLV-F13 French isolate. This paper is the first report on molecular variability of GFLV in Tunisia.     

Agroecological characterization of Xiphinema index in Spain1

The agroecological characteristics of Xiphinema index were determined by studying its spatial and temporal distribution in different environmental conditions in Spain. The host range, life cycle in the Mediterranean continental climate and its relationships with grapevine fanleaf nepovirus are also reported. X. index is widespread in grape-growing areas in a wide range of soil textures and pH, even at high percentage of carbonates. However, it seems to prefer sandy-loam and sandy-clay loam soils, and populations decrease when soil carbonates increase. Its spatial distribution is random but it has a tendency to be found in the deepest clay horizons, where soil moisture is retained. Availability of moisture seems to be the limiting factor for nematode development. Grapevine and fig are the main host plants but X. index is also associated with fruit trees, ornamental crops, vegetable crops, woodlands and other uncultivated soils. Its life cycle in central Spain, where temperature ranges from <0°C until March to above 40°C from May—June until September, takes 6–8 weeks to complete. X. index was found in 14% of all vineyards sampled and in 50% of the fanleaf virus-infected vineyards. Transmission tests using bait plants were successful and the detection of the virus by means of ELISA test in a mimimun of five nematodes was achieved. Fanleaf-like symptoms often occurred in the absence of the virus, as demonstrated by ELISA.     

Control of Xiphinema index populations by fallow plants under greenhouse and field conditions

The dagger nematode Xiphinema index has a high economic impact in vineyards by direct pathogenicity and above all by transmitting the Grapevine fanleaf virus (GFLV). Agrochemicals have been largely employed to restrict the spread of GFLV by reducing X. index populations but are now banned. As an alternative to nematicides, the use of fallow plants between two successive vine crops was assessed. We selected plant species adapted to vineyard soils and exhibiting negative impact on nematodes and we evaluated their antagonistic effect on X. index in greenhouse using artificially infested soil, and in naturally infested vineyard conditions. The screening was conducted with plants belonging to the families Asteraceae (sunflower, marigold, zinnia, and nyjer), Poaceae (sorghum and rye), Fabaceae (white lupin, white melilot, hairy vetch, and alfalfa), Brassicaceae (rapeseed and camelina), and Boraginaceae (phacelia). In the greenhouse controlled assay, white lupin, nyjer, and marigold significantly reduced X. index populations compared with that of bare soil. The vineyard assay, designed to take into account the aggregative pattern of X. index distribution, revealed that marigold and hairy vetch are good candidates as cover crops to reduce X. index populations in vineyard. Moreover, this original experimental design could be applied to manage other soilborne pathogens.     

Frequency of occurrence and genetic variability of Grapevine fanleaf virus satellite RNA

Little is known about the natural occurrence and genetic variability of nepovirus large satellite RNA (satRNA). This study screened 71 Grapevine fanleaf virus (GFLV) isolates mainly from Slovenia, but also from other countries in Europe and the USA, for the presence of satRNA, using a newly developed RT‐PCR assay. GFLV satRNA (satGFLV) was detected in 72% of naturally GFLV‐infected grapevines analysed, which is the highest frequency of occurrence of satGFLV reported to date. From 39 naturally GFLV‐infected grapevines, 122 satGFLV clones were sequenced and compared to publicly available sequences of satGFLVs and the closely related satRNAs from Arabis mosaic virus (satArMVs). Phylogenetic analyses of these satRNAs revealed that their evolution was driven by substitutions, insertions, deletions, recombinations and reassortments between closely related helper viruses. Phylogenetic relationships of the satGFLVs and satArMVs show their separate and subsequent common evolution. Furthermore, the satGFLVs varied in size and showed higher variability at the amino acid level than at the nucleotide level, just as the 2AHP gene of their helper virus. This study shows that satGFLVs are also similar to their helper virus with respect to their quasispecies nature and their transmission route through anthropogenic exchange of propagation material.     

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%.     

Spatial distribution of the dagger nematode Xiphinema index and its associated Grapevine fanleaf virus in French vineyard

The nematode Xiphinema index is, economically, the major virus vector in viticulture, transmitting specifically the Grapevine fanleaf virus (GFLV), the most severe grapevine virus disease worldwide. Increased knowledge of the spatial distribution of this nematode, both horizontally and vertically, and of correlative GFLV plant infections, is essential to efficiently control the disease. In two infested blocks of the Bordeaux vineyard, vertical distribution data showed that the highest numbers of individuals occurred at 40 to 110 cm depth, corresponding to the two layers where the highest densities of fine roots were observed. Horizontal distribution based on a 10 x 15 m grid sampling procedure revealed a significant aggregative pattern but no significant neighborhood structure of nematode densities. At a finer scale ( approximately 2 x 2 m), nematode sampling performed in a third block confirmed a significant aggregative pattern, with patches of 6 to 8 m diameter, together with a significant neighborhood structure of nematode densities, thus identifying the relevant sampling scale to describe the nematode distribution. Nematode patches correlate significantly with those of GFLV-infected grapevine plants. Finally, nematode and virus spread were shown to extend preferentially parallel to vine rows, probably due to tillage during mechanical weeding.     

Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes

ABSTRACT The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.     

Host suitability of Vitis rootstocks to root‐knot nematodes (Meloidogyne spp.) and the dagger nematode Xiphinema index,and plant damage caused by infections

The host suitability of commercial Vitis rootstocks commonly used in Spain (161‐49C, 41B, 1103P, 110R, 140Ru and SO4) to root‐knot nematodes (Meloidogyne arenaria, M. incognita, M. javanica) and Xiphinema index, and damage caused by nematode infection were determined under controlled conditions. The three root‐knot nematodes reproduced with a rate higher than one in all rootstocks, indicating that they are suitable hosts for these nematodes. Growth of rootstocks infected with the root‐knot nematodes was less vigorous than that of nematode‐uninfected controls in the majority of the rootstocks studied. Root infection resulted in moderate to severe root galling in all rootstocks. The shoot and main stem diameters appeared to be the most sensitive variables of damage caused by infection by Meloidogyne spp., with reduction rates from 36% and 53% in 161‐49C to 57% and 66% in 140Ru, respectively. The shoot height was not significantly affected by the root‐knot nematodes and the root fresh weight generally increased as a consequence of intensive galling. The nematode X. index caused significant root damage with a reproduction factor higher than one in all rootstocks. However, reproduction factor was significantly influenced by the rootstock and significantly decreased by about 12‐fold (5·7 to 18·1‐fold) with the increase in inoculum density from 100 to 1000 nematodes per plant. The root dry weight was reduced by X. index infections, and was the plant growth variable most affected by the nematode infection in all rootstocks at both inoculum densities. Meloidogyne arenaria, M. incognita, M. javanica and X. index, prevalent in many world vineyards, are all shown to have a damaging effect on the six tested rootstocks.     

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.     

Distinct phylogenetic positions of <Emphasis Type="Italic">Grapevine fanleaf virus</Emphasis> isolates from Iran based on the movement protein gene

In DAS-ELISAs of 86 grapevine samples from northwestern Iran, Grapevine fanleaf virus (GFLV) was detected in 18 samples. RT-PCR with two primer pairs (M2/M4 or M0/M4) corresponding to GFLV movement protein (MP) amplified the expected 854- and/or 1,489-bp fragment(s), respectively, from all ELISA-positive samples. Four smaller and three larger PCR products were cloned and sequenced, which revealed that the MP region of the isolates was 1,044 nucleotides (nt) long, corresponding to the GFLV MP. There were 83–86% nucleotide and 93–94% amino acid identities deduced between the MPs of the sequenced isolates. Nucleotide sequence identities of 81–87 and 75–79% were found between the MP regions of these isolates and that of previously published GFLV and Arabis mosaic virus (ArMV) strains/isolates, respectively. On a consensus parsimony tree based on the nucleotide sequences, isolates La208 and X300 remained distinct from previously reported GFLVs. This is the first molecular characterization of GFLV MP isolates from Iran. The sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank databases and have been assigned accession numbers DQ286901 to DQ286916.     

Phytosanitary status of grapevine in Montenegro

During 2006 and 2007, a survey on the incidence and distribution of fourteen grapevine viruses was carried out in the Skadar Lake basin, one of the two main grapevine‐growing areas of Montenegro. In total 165 samples were collected from four red (‘Vranac’, ‘Krato?ija’, ‘Merlot’ and ‘Cardinal’), two white (‘Chardonnay’ and ‘Rkaciteli’) and a few unknown grapevine varieties in the vicinity of Podgorica and Bar. The phytosanitary status of the collected samples was analysed by DAS‐ELISA and the presence of Grapevine fanleaf virus (GFLV), Grapevine leafroll‐associated virus 1 (GLRaV‐1), Grapevine leafroll‐associated virus 2 (GLRaV‐2) and Grapevine leafroll‐associated virus 3 (GLRaV‐3) was confirmed in some of them. The most frequently found virus in assayed samples was GLRaV‐3 (54.5%), followed by GFLV (23%), GLRaV‐1 (20%) and GLRaV‐2 (0.6%). These serological analyses showed the absence of Grapevine leafroll‐associated virus 6 (GLRaV‐6), Grapevine leafroll‐associated virus 7 (GLRaV‐7), Raspberry bushy dwarf virus (RBDV), Strawberry latent ringspot virus (SLRSV), Tomato ringspot virus (ToRSV), Raspberry ringspot virus (RpRSV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV), Tomato black ring virus (TBRV) and Cherry leaf roll virus (CLRV) from all tested samples.     

Sanitary status of grapevines in south-eastern and central Anatolia (Turkey)

Surveys were carried out in commercial vineyards in the main grapevine-growing areas of south-eastern (Adiyaman, Diyarbakir, Merdin, Sanliurfa, Elazig) and central (Nevşehir) Anatolia (Turkey) to assess the presence and incidence of virus and virus-like diseases. Typical fanleaf symptoms were observed in most of the surveyed areas, but they were particularly frequent in Elazig in cvs Kirmizi, Agin, Sirfoni and Kohnu. Leafroll symptoms were present in most vineyards in Adiyaman, Sanliurfa and Elazig, primarily in the red-fruited cvs Antep Karasi, Humusi, Kohnu and Siyah Kabarcik, and in Nevşheir. Rugose wood symptoms were common in Adiyaman, where vineyards were established with grafted planting material, but not in any of the self-rooted Cappadocian cultivars. Phytoplasma-like symptoms were sometimes observed in Elazig and Nevşheir. Biological (sap inoculation to herbaceous plants and graft transmission to woody indicators) and serological (ELISA) assays were used for virus detection and identification. A total of 55.3% of ELISA-tested vines (296 out of 535) were infected by one (11.4%) or more (43.9%) viruses. GVA ¹ was the most widespread virus (42.4%), followed by GLRaV-1 (38.5%), GFLV (10.7%) and GFkV (7.1%). Surprisingly low (2.4%) was the infection rate by GLRaV-3, and even lower (<1%) that of the other viruses tested, i.e. GLRaV-2, GLRaV-6, GVB and ArMV. GLRaV-7 was not detected. The occurrence of vein mosaic and vein necrosis was ascertained by testing on woody indicators. A putative nepovirus was isolated from a single vine of cv. Kizlar Tahtasi, the identification and characterization of which is still under way.     

Detection of Grapevine A vitivirus in Tunisian grapevines

Grapevine A vitivirus (GVA), Grapevine B vitivirus (GVB), Grapevine leaf-roll associated closterovirus 3 (GLRaV3) and Grapevine fanleaf nepovirus (GFLV) were detected by DAS-ELISA in samples of grapevine showing symptoms of rugose wood from different viticultural areas of Tunisia. After optimization of experimental conditions, PCR and IC-RT–PCR were found to be more efficient and sensitive than DAS-ELISA for the detection of GVA.     

<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.     

Prevalence, polyphasic identification, and molecular phylogeny of dagger and needle nematodes infesting vineyards in southern Spain

The occurrence and geographic distribution of longidorid nematode species inhabiting the rhizosphere of grapevine plants in southern Spain were investigated. Nematode surveys were conducted on 77 vineyards during the spring seasons of 2006, 2007 and 2008 in the main Andalusian grapevine-growing areas, including the provinces of Cádiz, Córdoba, and Huelva. Morphological and morphometrical studies identified two Longidorus and nine Xiphinema species, viz.: Longidorus alvegus, L. magnus, Xiphinema adenohystherum, X. hispidum, X. index, X. italiae, X. lupini, X. nuragicum, X. pachtaicum, X. rivesi, and X. turcicum. Overall, frequencies of infestation were, in decreasing order: X. pachtaicum 90.8%, X. index 30.3%, X. italiae 13.2%, L. magnus 11.8%, X. hispidum 7.9%, X. lupini 3.9%, L. alvegus and X. rivesi 2.6%, and X. adenohystherum, X. nuragicum and X. turcicum 1.3%. Xiphinema hispidum, X. lupini, L. alvegus and L. magnus were compared with nematode type specimens and are reported for the first time in Spain. Furthermore, the male of L. alvegus is described for the first time in the literature. Molecular characterisation of these species using D2–D3 expansion regions of 28S rRNA, 18S rRNA and ITS1-rRNA was carried out and maximum likelihood and Bayesian inference analysis were used to reconstruct phylogenetic relationships among these species and with other longidorids. The monophily of the genera Xiphinema and Longidorus was accepted and the genera Paralongidorus and Xiphidorus were rejected by the Shimodaira-Hasegawa test based on tree topologies.     

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.