我国部分地区沙地葡萄茎痘相关病毒分离物外壳蛋白序列变异分析

为明确我国葡萄中沙地葡萄茎痘相关病毒（GRSPaV）的感染情况及病毒外壳蛋白（coat protein,CP）基因的变异特点,从而为其致病性、病害的防治以及抗病毒基因工程等研究提供依据,本研究对采自我国16个省市自治区的65个葡萄品种305株葡萄样品中的GRSPaV进行RT-PCR检测,根据地区与品种差异选取了24个阳性样品进行cp基因克隆与测序分析,并对不同RNA提取方法进行了比较。结果显示,114株样品被GRSPaV侵染,平均带毒株率为37.4%;分离物间及同一分离物不同克隆间的序列差异较大,从24个分离物克隆获得的37条cp基因序列与来源于不同国家的12个GRSPaV分离物的核苷酸序列同源性为80.5%~99.7%,氨基酸序列同源性为88.8%~100%;各个分离物的遗传距离无明显地域差异;SiO₂吸附法比SDS法和CTAB法更适宜葡萄样品RNA的提取。     

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.     

新疆三种葡萄病毒RT-PCR检测及序列分析

为研究新疆葡萄中沙地葡萄茎痘伴随病毒(Grapevine rupestris stem pitting associated virus,GRSPa V)、葡萄斑点病毒(Grapevine fleck virus,GFk V)及葡萄病毒A(Grapevine virus A,GVA)的发生情况和新疆分离株系统进化关系,分别克隆3种病毒新疆分离株部分基因区域,应用RT-PCR对新疆64份葡萄样品中上述3种病毒进行检测,并进行系统进化分析。结果显示,GRSPa V、GFk V和GVA的检出率分别为31.3%、62.5%和25.0%。新疆GRSPa V分离株(KJ801847)与美国GRSPa V分离株(AY368590)同源性达96.59%;新疆GFk V分离株(KJ801846)与日本GFk V分离株(AB222861)及中国辽宁GFk V分离株(JF927942)的同源性分别为91.70%和91.03%;新疆GVA分离株(KJ801845)与波兰GVA分离株(JN860997)同源性为93.88%,与中国四川GVA分离株(HQ671655)及辽宁GVA分离株(FJ445220)的同源性分别为92.92%和89.53%。表明3种葡萄病毒在新疆发生比较普遍,且新疆分离株与国内其它地方的分离株存在较大差异。     

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.     

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.     

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。     

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.     

Pathogenicity and Fungicide Sensitivity of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">R. cerealis</Emphasis> Isolates

The Rhizoctonia solani species consists of multinucleate isolates that belong to anastomosis groups AG1–AG3 and differ in virulence and host affinity. R. cerealis is a binucleate species of anastomosis group AG-D which causes sharp eyespot, a common plant disease in Poland. Rhizoctonia spp. is a ubiquitous soil pathogen that poses a significant threat for global crop production due to the absence of effective crop protection products. The aim of this study was to determine the virulence of R. solani and R. cerealis isolates towards Beta vulgaris, Zea mays, Triticum spelta and T. aestivum seedlings, to confirm the presence of endopolygalacturonase genes pg1 and pg5 in the genomes of the tested isolates and to evaluate the tested isolates’ sensitivity to triazole, strobilurin, imidazole and carboxamide fungicides. All tested isolates infected B. vulgaris seedlings. but none of them were virulent against Z. mays plants. R. solani isolates AG4 PL and AG2-2IIIB PL were characterized by the highest virulence (average infestation score of 2.37 and 2.53 points on a scale of 0–3 points) against sugar beet seedlings. The prevalence of infections caused by most of the analysed isolates (in particular R. solani AG4 J—11.8, and R. cerealis RC2—0.78) was higher in spelt than in bread wheat. The virulence of the analysed isolates was not correlated with the presence of pg1 and pg5 genes. The efficacy of the tested fungicides in controlling Rhizoctonia spp. infections was estimated at 100% (propiconazole + cyproconazole), 98.8% (penthiopyrad), 95.4% (tebuconazole) and 78.3% (azoxystrobin).     

Characterisation of a novel ilarvirus causing grapevine angular mosaic disease

Unusual symptoms were observed on ‘Baresana’ x ‘Baresana’ Vitis vinifera hybrid vines in the Grapevine Variety Collection of the Grapevine Institute, Athens. The affected vines showed sharp angular mosaic on leaves, along the veins and in vein angles, malformations, abortive flowers or very few berries with smaller, wrinkled and non-germinating seeds, as well as gradual decline, severe stunting and death of the vine. Serological tests on diseased vines for the presence of 13 known grapevine viruses gave negative results. An infectious agent was transmitted mechanically to several herbaceous indicator plants. Koch’s Postulates were fulfilled, and the agent, proven to be a virus, was named Grapevine angular mosaic virus (GAMV). Serological tests have been developed for the virus. The most conserved polymerase region showed significant similarity of GAMV with members of subgroup 1 of the Ilarvirus genus; however ML phylogenetic analysis could not support its clustering within this subgroup. GAMV differs serologically and in particle morphology from Grapevine line pattern virus (GLPV) a putative member of the Ilarvirus genus that infects grapevine. It is proposed that GAMV is a novel member of the Ilarvirus genus.     

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.     

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.     

Characterisation of a 16SrII phytoplasma strain associated with bushy stunt of hawkweed oxtongue (Picris hieracioides) in south-eastern Serbia and the role of the leafhopper Neoaliturus fenestratus (Deltocephalinae) as a natural vector

A 2-year study of host association, molecular characterisation and vector transmission of a phytoplasma related to the 16SrII group in a vineyard of south-eastern Serbia was conducted. Grapevine, eight common weeds and 31 Auchenorrhyncha species were collected and analysed for phytoplasma presence. PCR-RFLP analyses of the 16S rRNA gene identified the presence of a new strain of phytoplasma related to the 16SrII group in P. hieracioides with symptoms of stunting or bushy stunting. Grapevine samples, all without symptoms, were negative for phytoplasma presence. Plants of Erigeron annuus, Cynodon dactylon, Daucus carota and P. hieracioides, either exhibiting symptoms of yellowing or without symptoms, were positive for the presence of stolbur phytoplasma. Among the tested cicada species, seven were infected with phytoplasmas from the aster yellows group, two with stolbur phytoplasma, two with 16SrII phytoplasma, and one with the 16SrV-C phytoplasma subgroup. The phytoplasma strain of the 16SrII group was recorded in approximately 50?% of the collected leafhopper species Neoaliturus fenestratus and in a few specimens of the planthopper Dictyophara europaea. The vector status of N. fenestratus was tested using the second generation of the planthopper in two separate transmission trials with P. hieracioides and periwinkle seedlings. In both tests, the leafhopper successfully transmitted 16SrII phytoplasma to exposed plants, proving its role as a natural vector of this phytoplasma in Europe. A finer molecular characterisation and phylogenetic relatedness of the 16SrII phytoplasma strain by sequence analyses of the 16S rRNA and ribosomal protein genes rpl22-rps3 indicated that it was most closely related to the 16SrII-E subgroup.     

Multi-gene sequence analysis and phenotypic diversity of Phaeoacremonium species isolated from grapevines in Spain

Esca and Petri disease, caused by Phaeoa-cremonium, and other grapevine decline diseases, such as eutypa dieback, are responsible for serious economic losses to the wine industry worldwide. In the present work, 175 isolates of Phaeoacremonium spp. were collected (mostly in the region of Castilla y León, Spain) from vines either asymptomatic or symptomatic for grapevine decline diseases. The sequences of DNA fragments of 30 isolates were analysed. Amplicons of ITS, and partial gene of the β-tubulin, actin, calmodulin and translation elongation factor 1-α [EF1-α], were obtained by PCR. This is the first time that any exon of the EF1-α gene or exons 6–7 of the β-tubulin gene of seven Phaeoacremonium spp. have been amplified. Amplicons of the calmodulin and EF1-α exons were the most informative in terms of species differentiation: only 52.6 % and 56.6 % similarity across six and seven species was respectively seen. Phylogenetic analysis of combined DNA sequences revealed five Phaeoacremonium species isolated from the sampled plants; P. aleophilum was the most common. Four other species were found, however only one isolate each. P. mortoniae and P. iranianum are here reported for the first time on grapevines in the region of Castilla y León and the neighbouring region of La Rioja respectively. In total, 29 nucleotide differences among the studied gene fragments were seen across 23 isolates of P. aleophilum. Network analysis showed these Spanish P. aleophilum isolates to fall into two main groups of genotypes. P. aleophilum genotypes belonging to group 1 were mostly isolated from young grapevines affected by Petri disease but genotypes included in group 2 were isolated from adult plants showing esca or eutypa dieback symptoms.     

Survey on viroids infecting grapevine in Italy: identification and characterization of Australian grapevine viroid and Grapevine yellow speckle viroid 2

Five viroid species have been reported from grapevine. Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1) are distributed worldwide, whereas Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd) and Citrus exocortis viroid (CEVd) are found only sporadically. However, the presence of AGVd and GYSVd-2 in several countries, including China, Turkey and Tunisia, suggests a wider dissemination, possibly also in Europe, where AGVd has never been found and GYSVd-2 has been occasionally identified in Italy. Taking advantage of a multiplex RT-PCR assay recently developed for detecting simultaneously these five viroids, vines growing in Italy in commercial vineyards and germplasm collections were surveyed. Besides confirming the widespread presence of HSVd and GYSVd-1 in the field, GYSVd-2 and/or AGVd were identified in two grapevine table cultivars (Sultanina Bianca and Red Globe) from germplasm collections. Tests extended to vines cultivated in southern Italy confirmed the presence of both viroids, which were further characterized. No major sequence divergences between the AGVd and GYSVd-2 variants from Italy and those previously described from other countries were observed. Phylogenetic analysis supported the close relationships among AGVd variants from Italy, Tunisia and Australia. To our knowledge this is the first report of AGVd in Europe and the first molecular characterization of GYSVd-2 isolates from a European country.