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.     

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.     

Grapevine rupestris stem pitting virus: a new pathogen of grapevine in Serbia

Journal of Plant Diseases and Protection - Grapevine rupestris stem pitting-associated virus (GRSPaV), a member of the genus Foveavirus, is a commonly detected grapevine virus around the world....     

Viral sanitary status of declining grapevine Syrah clones and genetic diversity of Grapevine Rupestris stem pitting-associated virus

In order to determine the etiology of Syrah decline, virus detection was performed on 22 Syrah clones, chosen for their various levels of sensitivity to Syrah decline. All clones, including the sensitive ones, were free of 20 main grapevine viruses. In contrast, Grapevine Syrah virus-1 and Grapevine Rupestris stem pitting-associated virus (GRSPaV), were detected in 56 % and 100 % of the analysed Syrah clones respectively. This is the first report of GSyV-1 in a French vineyard. The genetic diversity of a 380 nt region within the GRSPaV coat protein gene was studied extensively in vines differing in their sensitivity to Syrah decline. Most GRSPaV variants were scattered between the four phylogenetic groups previously identified; 65 % of the sequences analysed were found to belong to the GRSPaV—group 1, 22 % to—group 2b, 10 % to—group 2a and 2 % to—group 3. Seventy percent of the 31 plants analysed harboured mixtures of genomic variants. Statistical analyses revealed no significant correlation between sensitivity and GRSPaV sequence variation. This suggests that GRSPaV is not the direct etiological agent of the Syrah decline.     

Distribution and partial molecular characterization of Grapevine leafroll-associated virus 2 (GLRaV-2) found in Croatian autochthonous grapevine (Vitis vinifera L.) germplasm

Journal of Plant Diseases and Protection - Study of Grapevine leafroll-associated virus 2 (GLRaV-2) distribution was conducted on 13 Croatian autochthonous grapevine cultivars or cultivars which...     

Detection of Apricot latent virus and Plum bark necrosis stem pitting-associated virus by RT-PCR in Eastern Anatolia (Turkey)

Field surveys were carried out to determine presence and incidence of Apricot latent virus (ApLV) and Plum bark necrosis stem pitting-associated virus (PBNSPaV) in the main stone fruit growing areas of Eastern Anatolia. RT-PCR and nested-RT-PCR techniques were used to detect ApLV and PBNSPaV, respectively. Three apricot samples out of 224 tested positive for ApLV, although infected trees showed no apparent disease symptoms. Of 45 sweet cherry and plum trees tested for PBNSPaV, 35 tested positive. Stem pitting symptoms were observed on the trunks of PBNSPaV-infected sweet cherries. Trunk bark was spongy and thick; pits and grooves were observed on the woody cylinder. The overall incidence of ApLV was 1.3% and of PBNSPaV was 77%. This is the first report of ApLV and PBNSPaV in Eastern Anatolia, Turkey.     

Assessment of Pierce's disease susceptibility in Vitis vinifera cultivars with different pedigrees

Pierce's disease (PD) of grapevine is caused by the bacterium Xylella fastidiosa. In this study, an integrated approach was applied to assess PD susceptibility among different Vitis vinifera cultivars that incorporated disease severity, bacterial pathogen abundance and loss of stem xylem hydraulic conductivity. It was hypothesized that levels of PD susceptibility in V. vinifera can be attributed in part to the host anatomical features that are shaped by its pedigree background. Two popular wine grape cultivars were initially selected from the occidentalis group, Merlot and Cabernet Sauvignon, and one from the orientalis group, Thompson Seedless. The more recently bred table grape cultivar Scarlet Royal, that has mixed pedigree parentage, was also included. PD susceptibility was compared to the known PD resistant b43-17 V. arizonica/candicans wild grape species from North America. The data indicated that Thompson Seedless was ranked as the most susceptible to PD because it significantly exhibited the most severe disease symptoms at 12 weeks post-inoculation and hosted the highest X. fastidiosa titre of the cultivars, and lost over 90% of its stem hydraulic conductivity. In contrast, the other three cultivars displayed less susceptibility to PD. The way in which the xylem anatomy could impact PD susceptibility in V. vinifera cultivars is discussed, together with how grape pedigrees and their cognate centre of domestication may have influenced xylem anatomical features. This work provides a reference framework to further test the hypothesis that V. vinifera cultivars with wide xylem vessels may be more prone to PD decline.     

Seasonal dynamics and virus translocation of Grapevine leafroll‐associated virus 3 in grapevine cultivars

Grapevine leafroll‐associated virus 3 (GLRaV‐3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV‐3; however, there is no PCR‐based assay available to quantify virus populations within plant tissues. A real‐time RT‐PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV‐3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA‐dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV‐3 in field‐grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV‐3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV‐3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV‐3 diagnosis, disease monitoring and population ecology studies.     

Characterization of Grapevine Algerian latent virus isolated from nipplefruit (Solanum mammosum) in Japan

Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato bushy stunt virus nipplefruit strain (TBSV-Nf), and an unknown spherical virus were isolated from nipplefruit (Solanum mammosum) cultivated in Chiba Prefecture, Japan. The spherical virus was identified as Grapevine Algerian latent virus nipplefruit strain (GALV-Nf) from the genus Tombusvirus, based on its physical properties, serological relationships, and analysis of genomic RNA. The genomic RNA of GALV-Nf is 4731 nucleotides long and encodes five open reading frames as well as those of other tombusviruses. Nipplefruit infected with GALV-Nf had severe stunting, leaf deformation, and clear mosaic symptoms. This is the first report of an isolation of GALV in Japan. An erratum to this article is available at .     

Elimination of phytoplasma by stem culture from mulberry plants (Morus alba) with dwarf disease

Stem culture in vitro was attempted for the elimination of mulberry dwarf phytoplasma. Stem segments, from mulberry plants ( Morus alba ) seriously infected with the phytoplasma, were cultured on Murashige and Skoog (MS) solid nutrient medium without phytohormones for 2–3 months. The tissue cultures, and regenerated plants grown under greenhouse conditions for 3 years, were tested by polymerase chain reaction (PCR) and 4,6-diamidino-2-phenylindole (DAPI) staining for the presence of the phytoplasma, and also observed for mulberry dwarf symptoms. From 70% to 90% of regenerated plants were apparently freed from the phytoplasma since they remained PCR-negative, DAPI-negative and symptom-free for as long as tested (3 years).     

Prognosis of phenological stages of Vitis vinifera (cv. Riesling) for optimizing pest management*

The prognosis of phenological stages of grapevine, linked with the assessment of leaf area development, is a valuable tool in support of timely plant protection treatments as well as optimum canopy management. Warm weather conditions from May to July combined with copious water supply may lead to an enormous growth in leaf area, so that volatile active substances have to be applied at shorter intervals than normally required. In addition, certain phenological stages are of special significance for the planning of appropriate plant protection measures. The expected phenological stages can be computed 10–14 days before their occurrence based on weather data, a 7‐day forecast and long‐term climatological data. In order to test the reliability of procedures aided by model approaches illustrated in the literature, a 30‐year observation series was used as a basis for this study. For the prognosis, mainly maximum and minimum temperatures, computed global radiation and daily precipitation totals were used. The computations were supplemented by long‐term daily mean values of these parameters.     

Incidence,distribution and limited genetic variability among Turkish isolates of Grapevine Pinot gris virus from different grapevine cultivars

Journal of Plant Diseases and Protection - Grapevine Pinot gris virus (GPGV) was firstly identified in northern Italy by deep sequencing from grapevine cv. Pinot gris, exhibiting mottling and...     

Effects of Uncinula necator on the yield and quality of grapes (Vitis vinifera) and wine

The effects of powdery mildew ( Uncinula necator ) on grape yield, juice and wine quality were quantified for cultivar Cabernet Sauvignon (CSa) in 1997 and 1999, and for Sauvignon blanc (Sa) in 1999. Analyses were carried out on batches of healthy berries to which known percentages (0–50%) of diseased berries were added, and on natural clusters that were classified into four visual classes from low (C₁) to high (C₄) disease severity. CSa diseased berries showed an average weight reduction of 12% (1997) and 20% (1999). The direct consequence of a higher percentage of smaller, diseased berries was a reduction in yield. The accompanying loss of weight in must from C₁ to C₄ clusters increased from 10 to 45%. Sugar content in diseased berries was not significantly different from disease-free berries in 1997, but was 20–21% (CSa) and 14% (Sa) higher in 1999. Severely infected batches also showed a higher total acidity than healthy ones. The total anthocyanin content of CSa was decreased by 0·91% (1997) and 0·66% (1999) per percentage mildewed berries added by weight. In Sa wines the concentration of 3-mercaptohexanol, a component of varietal aroma, was decreased by powdery mildew. Multidimensional analyses, based on all the variables studied, successfully grouped batches of CSa according to disease severity. Using directional triangular tests wine experts were able to recognize CSa wines produced from berries with ≈25% of powdery mildew; the threshold for nonexperts was 50%. CSa wines obtained from samples with more than 30% of diseased berries by weight were significantly classified as the worst according to preference order criteria, but below this value the preference was not significant. Sa wines with <50% mildewed berries could not be differentiated significantly by organoleptic tests performed by nonprofessionals.     

Potato virus X (PVX) resistance in tomato cultivars

Understanding how birds detect and react to deterrent methods which are employed to protect structures is important both for the effective control of property damage, and to ensure that human health and safety are not compromised. One such device is a shock strip that causes slight pain to birds when they use a perch. Our aims were to determine: (1) the efficacy of a shock strip to flocks of Brown-headed Cowbirds (Molothrus ater), and Rock Pigeons (Columba livia); (2) the length of time to extinction of effect; (3) whether members of a flock show a behavioural response to treatment; and (4) whether the birds habituate to the treatment. When activated, shock strips were effective in displacing birds from treated areas. Birds had to experience the treatment; there was no discernible indication of flock members reacting to affected birds. When strips were deactivated after an initial period of activation, birds required more than 2 hours to return to treated areas. No habituation was observed. Because birds had to experience the shock, reduced coverage of a structure or use of sham devices to lower costs is considered inadvisable.