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。     

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.     

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。     

Grapevine crown gall caused by <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> (Ti) in Japan

In 2005, characteristic symptoms of crown gall on grapevines (Vitis vinifera L. cv. Muscat of Alexandria, and cv. Seto Giants) were observed in a commercial greenhouse-orchard in Okayama Prefecture, Japan. Isolations from diseased tissues consistently yielded bacterial colonies that were white, glistening, and produced abundant polysaccharide on potato semi-synthetic agar (PSA) medium. Ten representative isolates were chosen for further characterization. A multiplex polymerase chain reaction (PCR) assay showed these strains were not Rhizobium vitis but did possess a Ti plasmid. The bacteriological characteristics of the isolates corresponded well with R. radiobacter. The almost complete 16S ribosomal DNA sequences of isolates AT06-1 and AT06-2, selected from 10 grapevine isolates, were determined and corresponded to sequences of R. radiobacter. The pathogenicity of the isolates was tested on young grapevine and tomato (Lycopersicon esculentum Mill.) plants. Gall symptoms developed on both plant species after inoculation, and bacteria with the same colony morphology as those inoculated were reisolated. Based on these results, the isolates were identified as R. radiobacter (Ti). This report is the first of the occurrence of R. radiobacter (Ti) on grapevine in Japan. Phylogenetic analyses using the partial nucleotide sequences of virC operon located on a Ti plasmid showed that the isolate of R. radiobacter (Ti) isolated from grapevine and some strains of R. vitis (Ti) belonged to the same monophyletic group, which differed from the groups of R. radiobacter (Ti) isolated from plants other than grapevine and of the majority of R. vitis (Ti) strains isolated from grapevine. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accessions AB306890, AB306891, and AB465432–AB465459.     

Crude garlic extract significantly inhibits replication of grapevine viruses

Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.     

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.     

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.     

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.     

Virulence affected by assay parameters during grapevine pathogenicity studies with Botryosphaeriaceae nursery isolates

This study assessed the symptoms that developed when 114 Botryosphaeriaceae isolates from grapevine nursery plant materials were used to inoculate excised green shoots and 1‐year‐old rooted canes of Sauvignon blanc. The experiments showed that all isolates and species were able to produce lesions. Overall, the Neofusicoccum species were shown to be highly pathogenic in both tissue types while the Diplodia species were highly pathogenic on canes but not on green shoots. Isolates of the most prevalent species, N. luteum and N. parvum, showed varying lesion lengths on excised green shoots and canes. An evaluation of the factors associated with lengths of lesions showed that they were significantly affected by experimental batch which reflected inherent host and environmental factors over time. Reisolation from inoculated canes also indicated that most isolates of all species except D. seriata were able to spread internally beyond the lesions. Genetic variability analysis using UP‐PCR showed that N. luteum isolates were genetically diverse but no association was observed between the phylogenetic group and degree of pathogenicity caused by the isolates. This study demonstrated that all Botryosphaeriaceae species from grapevine nurseries were pathogenic to grapevines and that the lesion lengths varied between species, among isolates within a species and among nursery sources, and was affected by the test method.     

A molecular epidemiology study reveals the presence of identical genotypes on grapevines and ground cover weeds and the existence of separate genetic groups in a Botrytis cinerea population

Botrytis cinerea, the grey mould agent, is one of the most important pathogens of grapevine, due to the great yield losses caused and the economic costs related to disease control. Ground cover plants are assumed to have a role in the complex epidemiology of the pathogen, even if no information on the genetic variability of the strains is available. In this study, a molecular epidemiology approach, based on the comparative analysis of the nucleotide sequences of multiple genes (ITS1-ITS2, G3PDH, NEP1, NEP2, BC-hch, and sdhB), was used to evaluate whether B. cinerea isolated from herbaceous species contributes to grey mould diffusion on grapevines. From 330 samples collected in two vineyards in Lombardy, Italy, 63 B. cinerea strains were isolated from tissues of grapevine with symptoms (50 strains) and spontaneous ground cover plants (13 strains). Capsella bursa-pastoris, Cardamine impatiens, Lamium purpureum, and Crepis tectorum were identified as novel B. cinerea hosts. Sequence analysis and phylogeny showed that the same genotypes were present on both grapevines and herbaceous plants, with no fitness (estimated from growth and sporulation on potato dextrose agar) or pathogenicity (on grapevine leaves and berries, and tomato leaves) penalties. This confirms that ground cover plants can be a source of inoculum for B. cinerea on grapevine. Moreover, phylogenetic analysis of the BC-hch gene allowed the identification of two genetically distinct clusters, characterized by vegetative incompatibility and different distributions of the mating types, fitness, and pathogenicity. Therefore, B. cinerea seems to be composed of two diverging subpopulations that do not differ for host specialization.     

TransgenicNicotiana Benthamiana and grapevine plants transformed with grapevine virus a (GVA) sequences

As part of a project to construct virus-resistant transgenic grapevines, grapevine virus A (GVA) coat protein (CP) sequences were introduced into grapevine and the model plantNicotiana benthamiana. The engineered constructs were tested for their ability to engender resistance inN. benthamiana. A number of GVA-CP-transformedN. benthamiana lines have become resistant to GVA. Preliminary biological and molecular analyses strongly suggest that in most cases the obtained resistance is RNA-mediated. Several lines of transgenic grapevine were obtained.     

Phylogeny,distribution and pathogenicity of Lasiodiplodia species associated with dieback of table grape in the main Brazilian exporting region

Botryosphaeria dieback is an important disease of table grape in the São Francisco Valley, the main Brazilian exporting region. The objectives of this study were to identify species of Lasiodiplodia associated with botryosphaeria dieback of table grapes in the São Francisco Valley, investigate the prevalence and distribution of the species in the region, and evaluate their pathogenicity and virulence in green shoots of table grape. A total of 112 Lasiodiplodia isolates were obtained from 14 vineyards, located in Casa Nova, Juazeiro and Petrolina. Fungal identifications were made using phylogenetic analysis based on partial sequences of translation elongation factor 1‐α (EF1‐α) and internal transcribed spacer (ITS) sequences, in combination with morphometric characteristics of conidia. Eight species of Lasiodiplodia were identified: L. brasiliense, L. crassispora, L. egyptiacae, L. euphorbicola, L. hormozganensis, L. jatrophicola, L. pseudotheobromae and L. theobromae. Except for L. crassispora, L. pseudotheobromae and L. theobromae, all the other species are reported for the first time on grapevine worldwide. The distribution of Lasiodiplodia species differed between the three table grape populations of São Francisco Valley. All Lasiodiplodia species isolated in this study were present in the population of Casa Nova and Lasiodiplodia theobromae was the most prevalent. All species of Lasiodiplodia were pathogenic on detached green shoots of grapevine, with L. brasiliense being the most virulent.     

我国部分地区常见农作物上黄瓜花叶病毒分离物核酸多样性分析

为明确我国黄瓜花叶病毒株系分化及系统进化基本情况,从湖南、新疆、青海和海南4省区采集1 367个样品对其进行酶联免疫和RT-PCR检测,并对分离获得的15个黄瓜花叶病毒(Cucumber mosaic virus,CMV)纯化分离物CP、MP、2b核苷酸序列进行相似性和进化树分析及生物学性状比较。结果表明,辣椒、龙葵和黄瓜的CMV阳性检出率较高,分别为54.13%、29.19%和18.46%。进化树分析显示CMV-Q5与CMV亚组II的亲缘性较高;CMV-N7为新发现的重组株系,其CP、2b基因属于CMV亚组IB,MP基因却属于CMV亚组II;其余13个分离物均属于CMV亚组IB。CMV-N7和CMV-Q5在系统寄主心叶烟和枯斑寄主苋色藜上引发的症状相似,但比对照株系CMV-P3613(IB)的发病时间要晚1~2 d,系统花叶较温和,枯斑较小。表明在以上4省区常见农作物上广泛流行的CMV存在分子变异。     

Genetic variability and pathological properties of <Emphasis Type="Italic">Grapevine Leafroll-associated Virus 2</Emphasis> isolates

The genetic variability among Grapevine leafroll-associated virus 2 (GLRaV-2) isolates was investigated in several grapevine accessions from various geographic origins in three genomic fragments, which encode the 70 kDa heat-shock protein homologue, the coat protein and the ∼60 kDa protein. The majority of the isolates were identical or only slightly different from one another and formed a monophyletic group. Several other variants were found which differed greatly from the main cluster and were mostly present in autochthonous or specific vine cultivars. Phylogenetic analyses on the CP gene sequences, including the isolates analyzed in this work and some others from related literature, allowed five clades to be identified (PN, H4, RG, BD and PV20). Biological trials for graft incompatibility and leafroll symptoms revealed that GLRaV-2 divergent variants had different pathological properties, mainly according to their phylogenetic grouping. The BD isolate seemed to be a mild variant of the virus, because it was unable to induce graft incompatibility and it rarely caused leafroll symptoms. The RG isolate appeared to be a more virulent variant, given the strong decrease in rooted grafted grapevines obtained in nursery, in particular with the use of certain rootstocks; however, it does not induce leafroll symptoms. The variants belonging to the PN group, which were the most widespread, caused both graft incompatibility and leafroll symptoms, according to the viral variant. Graft incompatibility also proved to depend strongly on the rootstock used.