Association between genetic variability and titre of Grapevine Pinot gris virus with disease symptoms

The present work was carried out in order to verify the possible association between a new grapevine disease, characterized by leaf mottling and deformation, and the genetic variability and concentration of Grapevine Pinot gris virus (GPGV), a recently identified virus tentatively associated with the pathology. After vineyard surveys and the establishment of real‐time qPCR assays, characterization of GPGV isolates and evaluation of GPGV titre were assessed in more than 100 samples of grapevine Glera, collected from plants regardless of whether or not they showed the symptomatology. Results showed that there was an important association between the GPGV variants and manifestation of the symptoms, and that grapevines with symptoms harboured significantly higher GPGV titre than symptomless vines. Moreover, an interesting relationship among the phylogenetic clustering of the isolates originating from plants with symptoms and some epidemiological characteristics of the disease was found. The current study confirmed the role of GPGV in the emergent disease characterized by grapevine leaf mottling and deformation.     

葡萄灰皮诺病毒微滴数字RT-PCR检测方法的建立

正葡萄灰皮诺病毒(Grapevine Pinot gris virus,GPGV)最早于2012年由意大利研究人员利用高通量测序技术发现(Giampetruzzi et al.,2012),该病毒可引起葡萄叶片褪绿、斑驳、皱缩、变形等症状,导致葡萄植株发育不良、成熟期延迟、产量降低、果实腐烂,还可与其它病毒共同作用产生复合负面效应。目前,葡萄灰皮诺病毒病尚缺乏有效防治药剂,为防控该病毒病的发生和传播,本研究拟设计特异性引物     

Occurrence of Grapevine Pinot gris virus in Poland and description of asymptomatic exhibitions in grapevines

Journal of Plant Diseases and Protection - Sixty five grapevines in productive areas from southern regions in Poland were tested for the presence of Grapevine Pinot gris virus (GPGV) by RT-PCR....     

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.     

我国灰比诺葡萄病毒分离物检测及基因序列分析

 灰比诺葡萄病毒(Grapevine Pinot gris virus, GPGV)是国内新近报道的葡萄病毒,能引起葡萄叶片褪绿斑驳、皱缩及变形等症状,严重影响葡萄长势。为明确我国GPGV的发生及基因序列变异情况,本研究采用RT-PCR方法对采自我国15个省(市)自治区的195个葡萄样品进行检测,其中55个葡萄样品检测出GPGV。对49个GPGV阳性分离物的外壳蛋白(coat protein, cp)和运动蛋白(movement protein, mp)基因进行克隆、测序, 序列分析结果显示:来源于国内外的GPGV分离物cp基因和mp基因的核苷酸和氨基酸序列同源性分别为85.30%~100%和95.00%~100%、85.73%~99.87%和95.58%~100%。基于cp和mp基因序列构建的进化树,将GPGV分离物划分为3个明显的组群。其中,大部分GPGV分离物被划分在组群1内,其它中国GPGV分离物形成2个新的组群2和3。本研究是关于中国GPGV分离物基因序列变异的初次报道,可为进一步开展GPGV分子检测的技术研发及不同变种的致病性研究提供依据。     

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.     

Pathogenic and genetic variability among Colletotrichum lindemuthianum isolates of different geographic origins

The pathogenic and genetic diversity of Colletotrichum lindemuthianum isolates collected from a total of 10 Central and South American, European and African countries were characterized using common bean differential cultivar pathogenicity tests and amplified fragment length polymorphism (AFLP) analysis. On the basis of pathogenicity tests, 74 isolates were attributed to 30 different pathogenic races using the CIAT-defined binary race-classification system. Twenty-one races were restricted geographically, being exclusive to different countries. Race 9 was the most widespread, being detected in four different countries. Cluster analysis of consensus AFLP data generated using three selective AFLP primer combinations grouped 86 isolates (including the 74 subjected to pathogenicity tests) into three clusters (with one isolate as an outlier). This analysis showed that the majority of South and Central American isolates were divided among two clusters, and that the limited number of European and African isolates used in this study were genetically most similar to Central American isolates. Overall, the results of this research showed some associations between the genetic diversity of the isolates and their country of origin, but not between genetic diversity and race classification of isolates.     

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.     

Occurrence of Grapevine Pinot gris virus in Friuli Venezia Giulia (Italy): field monitoring and virus quantification by real‐time RT‐PCR

Since 2003 the presence of a new syndrome characterized by symptoms of stunting, chlorotic mottling, leaf deformation, reduced yields and quality has been reported in some white berry varieties of Vitis vinifera in Trentino‐Alto Adige and Friuli Venezia Giulia vineyards. The identification of a new virus, provisionally called Grapevine Pinot gris virus (GPGV), in a cv. Pinot gris vine suggested an association between this new syndrome and the virus presence (Giampetruzzi et al., 2012), however the contemporary presence of GPGV in both symptomatic and asymptomatic plants has still to be explained. In this work, a large‐scale monitoring over a 3‐year period (2012–14) of Friuli Venezia Giulia vineyards and nurseries has shown a widespread presence of GPGV in symptomatic plants and also in asymptomatic vines, even if at a slightly lower percentage. Quantitative analyses of the virus titer revealed a great variability in the viral content of both symptomatic and asymptomatic plants but the mean GPGV quantity in symptomatic vines was significantly higher than in asymptomatic plants.     

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.     

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.     

Serological variability among European isolates of radish mosaic virus

Serological variability of radish mosaic virus (RaMV) isolates from white mustard, winter turnip rape, Camelina sativa and Chinese cabbage, collected in the Czech Republic and Russia, was studied using antisera against the Czech isolate RaMV1. In contrast to previous studies, reasonable serological differences were found between isolates from different locations and hosts, and even between neighbouring mustard plants in the field. Serological variability of European isolates was confirmed in extended experiments involving an Italian isolate, the American Type Strain (ATS) and its homologous antiserum. The results indicate that, in Europe, RaMV isolates may occur which differ both serologically and in host plant response, but no typical strains can be defined with the methods and isolates employed so far. Camelina sativa is reported as a new host of the virus.     

Genetic variability and virulence among Verticillium albo-atrum isolates from hop

Verticillium wilt, caused by Verticillium albo-atrum or V. dahliae, is an important disease of many worldwide crop species. In Europe, V. albo-atrum isolates infecting hop express different levels of virulence, inducing mild or lethal disease syndromes, and it is therefore an attractive model for studying the virulence of this pathogen. In this work, eleven amplified fragment length polymorphism (AFLP) primer combinations were used to analyze genetic variability among 55 V. albo-atrum hop isolates from four European hop growing regions, as well as isolates from other hosts and V. dahliae isolates. Cluster analysis divided V. albo-atrum and V. dahliae isolates into two well-separated groups. Within the V. dahliae cluster, isolates were separated without host specific grouping, although no host adapted isolates were included. In V. albo-atrum, the alfalfa isolates were distinct from isolates of other hosts, where a high association with virulence was observed in hop and tomato isolates. All lethal hop isolates were genetically different from mild hop isolates. The lethal hop isolates from England and Slovenia expressed the same virulence phenotype, although they showed a different AFLP pattern. The mild hop isolates formed two subgroups, to which isolates clustered irrespective of geographical location. These data suggest multiple origins of V. albo-atrum hop isolates, and the possible appearance of new virulent isolates in the future in other hop growing regions.     

Genetic variability among isolates of Fusarium oxysporum from sugar beet

Fusarium yellows, caused by the soil‐borne fungus Fusarium oxysporum f. sp. betae (Fob), can lead to significant yield losses in sugar beet. This fungus is variable in pathogenicity, morphology, host range and symptom production, and is not a well characterized pathogen on sugar beet. From 1998 to 2003, 86 isolates of F. oxysporum and 20 other Fusarium species from sugar beet, along with four F. oxysporum isolates from dry bean and five from spinach, were obtained from diseased plants and characterized for pathogenicity to sugar beet. A group of sugar beet Fusarium isolates from different geographic areas (including nonpathogenic and pathogenic F. oxysporum, F. solani, F. proliferatum and F. avenaceum), F. oxysporum from dry bean and spinach, and Fusarium DNA from Europe were chosen for phylogenetic analysis. Sequence data from β‐ tubulin, EF1α and ITS DNA were used to examine whether Fusarium diversity is related to geographic origin and pathogenicity. Parsimony and Bayesian MCMC analyses of individual and combined datasets revealed no clades based on geographic origin and a single clade consisting exclusively of pathogens. The presence of FOB and nonpathogenic isolates in clades predominately made up of Fusarium species from sugar beet and other hosts indicates that F. oxysporum f. sp. betae is not monophyletic.     

Genetic diversity and pathogenic variability among isolates of colletotrichum species from strawberry

ABSTRACT Ninety-five isolates of Colletotrichum including 81 isolates of C. acutatum (62 from strawberry) and 14 isolates of C. gloeosporioides (13 from strawberry) were characterized by various molecular methods and pathogenicity tests. Results based on random amplified polymorphic DNA (RAPD) polymorphism and internal transcribed spacer (ITS) 2 sequence data provided clear genetic evidence of two subgroups in C. acutatum. The first subgroup, characterized as CA-clonal, included only isolates from strawberry and exhibited identical RAPD patterns and nearly identical ITS2 sequence analysis. A larger genetic group, CA-variable, included isolates from various hosts and exhibited variable RAPD patterns and divergent ITS2 sequence analysis. Within the C. acutatum population isolated from strawberry, the CA-clonal group is prevalent in Europe (54 isolates of 62). A subset of European C. acutatum isolates isolated from strawberry and representing the CA-clonal and CA-variable groups was assigned to two pathogenicity groups. No correlation could be drawn between genetic and pathogenicity groups. On the basis of molecular data, it is proposed that the CA-clonal subgroup contains closely related, highly virulent C. acutatum isolates that may have developed host specialization to strawberry. C. gloeosporioides isolates from Europe, which were rarely observed were either slightly or nonpathogenic on strawberry. The absence of correlation between genetic polymorphism and geographical origin in Colletotrichum spp. suggests a worldwide dissemination of isolates, probably through international plant exchanges.     

Occurrence and molecular characterization of Turkish isolates of Turnip mosaic virus

A total of 142 samples of plants showing symptoms of Turnip mosaic virus (TuMV) were collected from fields planted to Brassicaceae and non‐Brassicaceae crops in the southwest Marmora region of Turkey, during the 2004?06 growing seasons. Using enzyme‐linked immunosorbent assay (ELISA) TuMV was detected in the main brassica‐crop fields of Turkey, with an overall incidence of 13·4%. TuMV was detected in samples from Brussels sprouts, cabbage, wild mustard, radish and wild radish, but not cauliflower or broccoli. The full‐length sequences of the genomic RNAs of two biologically distinct isolates, TUR1 and TUR9, were determined. Recombination analyses showed that TUR1 was an intralineage recombinant, whereas TUR9 was a non‐recombinant. Phylogenetic analyses of the Turkish isolates with those from the rest of the world showed that the TUR1 and TUR9 isolates belonged to world‐Brassica and Asian‐Brassica/Raphanus groups, respectively. This study showed that TuMV is widely distributed in the Asia Minor region of Turkey.