河南甜樱桃病毒病害调查及病原检测

在河南省郑州市、巩义市、荥阳市、新郑市选择具有代表性的甜樱桃生产园对病毒病发生情况进行调查,采集表现为疑似病毒病症状的样本65份,利用7种病毒引物进行RT-PCR检测。5种病毒检测结果呈阳性,分别是李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV)、李矮缩病毒(Prune dwarf virus,PDV)、樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)、樱桃坏死锈斑病毒(Cherry necrotic rusty mottle virus,CNRMV)及樱桃病毒A(Cherry virus A,CVA);序列分析结果表明,5种病毒扩增片段与GenBank中注册的相应病毒核苷酸序列均具有较高的一致性;样本病毒检出率为100%,其中13份样本为单独侵染,其余52份样本均为多病毒复合侵染,占比高达80%,复合侵染比例随着侵染病毒种类的增多逐渐降低;病毒侵染组合与叶片表型症状无明显对应关系。     

我国部分地区樱桃病毒病害初步调查和病原检测

对山东泰安、辽宁大连和北京的樱桃病毒病发生情况进行调查,发现8个果园/栽培区均有病毒病发生,主要症状为叶片皱缩、畸形、卷叶、花叶、植株矮缩等。采集20份样品,利用12种病毒的引物进行RT-PCR检测。结果表明,在样品中扩增出与樱桃病毒A(Cherry virus A,CVA)、李属坏死环斑病毒(Prunus necrotic ringspot virus,PNRSV),李矮缩病毒(Prune dwarf virus,PDV)、李树皮坏死与茎痘伴随病毒(Plum bark necrosis stem pitting-associated virus,PBNSPaV)、樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)、樱桃小果病毒-1(Little cherry virus-1,LChV-1)预期大小一致的目的片段;序列分析表明,与GenBank中注册所测的病毒核苷酸序列均具有较高的一致性。其中,大连、泰安和北京样品均检测到CVA;大连和北京样品中检测到PNRSV和PDV;北京样品中检测到PBNSPaV;大连苗木样品枝条中检测到CGRMV和LChV-1。这是在我国樱桃上首次检测到LChV-1。     

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.     

Partial characterization of a closterovirus associated with apple mealybug-transmitted little cherry disease in north america

ABSTRACT Little cherry disease (LChD) is a serious economic problem of sweet cherry production in western North America where apple mealybug is the principle vector. LChD is associated with a distinct species of double-stranded (ds) RNA. In this study, filamentous virus particles were purified from LChD-infected trees and shown to contain single-stranded RNA corresponding to the previously reported dsRNA isolated from infected trees. The virus particles were characterized and were similar to monopartite members of the genus Closterovirus. A portion of the genome was sequenced and found to be most closely related to the RNA-dependent RNA polymerase of Grapevine leafroll-associated virus-3, a mealybug-transmitted closterovirus. The characteristics of the mealybug-transmitted Little cherry virus in North America are very different from those of a closterovirus associated with a similar disease in Europe.     

樱桃小果病毒1号胶体金免疫层析试纸的研发与应用

近年来，病毒病在甜樱桃主产区的发生呈上升趋势，制约了甜樱桃产业升级和发展。能够侵染甜樱桃的病毒种类中，樱桃小果病毒1号(little cherry virus 1,LChV-1)导致樱桃果实缩小，彻底丧失经济价值，对甜樱桃产量影响较大。LChV-1通常具有潜伏侵染的特性，在樱桃苗期难以通过症状进行判断。同时，多数品种对LChV-1敏感，因此该病毒的早期检测对甜樱桃的生产尤为重要。传统的病毒检测手段需要专业仪器，不能满足田间快速检测的需求。本研究获取了LChV-1外壳蛋白的多克隆抗体，并研发了一种能够准确检测LChV-1病毒的胶体金免疫层析试纸，确定了胶体金的最佳抗体标记浓度为0.24 mg/mL,检测线最佳重组蛋白浓度为0.25 mg/mL,质检线最佳羊抗兔IgG抗体浓度为0.04 mg/mL。运用该试纸检测只需10～15 min,检测时间短、成本低、结果容易判断，可用于田间快速检测。本研究为樱桃小果病的综合防控提供了有效的监测和检测手段。     

The location of virus-like particles in the male gametophyte of birch, walnut and cherry naturally infected with cherry leaf roll virus and its relevance to vertical transmission of the virus

Virus-like particles (VLPs) in close-packed paracrystalline arrays were observed in cells forming the anther walls and in the sperm cell cytoplasm of immature pollen grains developing within cherry leaf roll virus (CLRV)-infected birch ( Betula pendula Roth.). VLPs within tubules, that were in some instances multiple and membrane bound, were also observed in anther cells and in pollen grains of CLRV-infected walnut ( Jugians regia L.). VLPs rarely coated the outer surfaces of developing grains. Washings from intact freshly collected pollen did not contain infective agents but pollen triturates were infectious after 12 months storage at-70°C. Purified CLRV (concentration 6·4 ng/ml) was readily detected by enzyme-linked immunosorbent assay (ELISA). CLRV-specific antigens (detected by ELISA) and VLPs (detected on grids coated with an antiserum prepared against CLRV) were readily removed by washing intact pollen grains from infected birch, walnut and cherry ( Prunus avium L. cv. F12/1). The antigen was less tenaciously held to the surfaces of anemophilous (birch and walnut) than entomophilous (cherry) pollen. Treatment of grains before ELISA testing with CLRV-specific γ globulin virtually eliminated the antigenicity of pollen washings whereas γ globulin from a pre-immune serum had no such effect. When anti-CLRV γ globulin-treated pollen grains were disrupted, CLRV-specific antigens were liberated. VLPs trapped on CLRV-antiserum coated grids to which pollen washings or extracts from disrupted grains had been applied were identified by decoration; a halo of antibody molecules enveloped VLPs treated with CLRV-antiserum but not those treated with antiserum prepared against poplar mosaic virus.     

Serological differences between red currant spoon leaf virus,virus isolates from Eckelrade-diseased cherry trees and the Scottish raspberry ringspot virus

Antisera were made to red currant spoon leaf virus (SLV), an isolate of the Scottish raspberry ringspot virus (RRV), and two virus isolates from Eckelrade-diseased cherry trees (EV). Different virus isolates, including one from Belgium, were tested against these antisera. The results indicate that we are dealing with a group of virus isolates with different antigenic properties. SLV is very closely related to RRV, being undoubtedly a strain of this virus. Dutch EV isolates differ from SLV and RRV and from each other, the Belgian isolate being closely related to one of the Dutch EV isolates. The serological differences found do not correspond with the geographical distances between the localities where the viruses were collected.Samenvatting Antisera werden bereid tegen het lepelbladvirus van rode bes (SLV), een isolatie van het Schotse raspberry ringspot-virus (RRV) en twee virusisolaties uit kersebomen met Eckelraderziekte (EV). Met behulp van deze antisera werden verschillende virusisolaties getoetst, waaronder één uit België van kers met Eckelraderziekte. De resultaten zijn vermeld in de tabellen 1 en 2. Ze duiden erop, dat we te maken hebben met een groep van virusisolaties met verschillende antigene eigenschappen. SLV is zeer nauw verwant aan RRV en is ongetwijfeld een stam van dit virus. Nederlandse EV-isolaties verschillen zowel van SLV en RRV, als van elkaar. De Belgische isolatie is nauw verwant aan één van de Nederlandse EV-isolaties. De grootte van de gevonden verschillen correspondeert niet met de geografische afstand tussen de plaatsen waar de virusisolaties werden verzameld.     

Effects of cherry leaf spot on photosynthesis in tart cherry 'Montmorency' foliage

Results described here span a total of three field seasons and quantitatively depict the effects of an economically important fungal pathogen (Blumeriella jaapii) on tart cherry (Prunus cerasus 'Montmorency') leaf physiology. For the first time, leaf photosynthesis, stomatal conductance (g(s)), maximum ribulose-1,5-bisphosphate carboxylation rate (V(cmax)), and maximum electron transport (J(max)) were measured as functions of visible cherry leaf spot disease (CLS) severity. Defined as the proportion of chlorotic and necrotic tissue per leaf, CLS severity was estimated from leaves of mature 'Montmorency' trees in 2007, 2008, and 2009. Briefly, as visible disease severity increased, all of the leaf-level physiological parameters decreased significantly (P < 0.01) and disproportionately. Thus, the effects of visible symptoms on leaf photosynthetic metabolic function encroached upon asymptomatic tissue as well. Impairment of photosynthetic metabolism in 'Montmorency' tart cherry leaves due to CLS appears to be mediated through disproportionately large perturbations in g(s), V(cmax), and J(max). These findings offer a new perspective on the amount of damage that this serious disease can inflict.     

First detection and complete genome characterization of a Cherry (C) strain isolate of plum pox virus from sour cherry (Prunus cerasus) in Germany

Journal of Plant Diseases and Protection - An isolate of plum pox virus (PPV) was detected in sour cherry (Prunus cerasus) in the Eastern region of Germany. Symptoms observed on infected sour...     

环渤海湾地区甜樱桃小果病毒及樱桃病毒A的鉴定与调查

为了解环渤海湾地区甜樱桃[Cerasusavium (Linn) Moench]中樱桃病毒的发生情况,选取该地区代表性樱桃示范园中甜樱桃品种‘红灯’为研究对象,以生长期功能叶片为材料,根据樱桃小果病毒-1(Little cherry virus-1,LChV-1)、樱桃小果病毒-2(Little cherry virus-2,IChv-2)和樱桃病毒A(Cherry virus A,CVA)基因组序列设计特异性检测引物进行RT-PCR扩增,结果扩增出与LChV-2、CVA预期片段337 bp和652 bp大小相符的目的片段,测序结果与基因组(基因登录号NC-005065、NC-003689)对应片段一致性分别为89.0％、98.2％,未检测到LChV-1.本研究完成了对环渤海湾地区甜樱桃‘红灯’样品LChV-2、CVA的检测调查,LChV-2检出率43.1％,CVA检出率60.8％;在该地区首次发现LChV-2、CVA复合侵染植株,复合检出率37.3％0.结果表明该地区CVA发生较为普遍,可与LChv-2等其他病毒共同加重对甜樱桃的危害.     

Inhibition by bacterial lipopolysaccharide of wilt induced by phytopathogenic bacteria in cut cherry shoots, and of cherry canker disease

Virulent phytopathogenic bacteria and their less virulent mutants induced wilt in cut cherry shoots treated with cell suspensions. Both heterologous and homologous bacteria induced the will reaction. The wilt-inducing isolates also caused the hypersensitive response in tobacco. An avirulent capsulaled mutant of Erwinia amylovora did not elicit either response. Non-phytopathogens did not cause wilting to occur. All the tested isolates aggregated after entry into plant tissues whether wilting occurred or not, but did not aggregate in suspensions in contact with cut shoots. Bacterial lipopolysaccharide or sidechain carbohydrate derived from lipopolysaccharide of Pseudomonas syringae pv. morsprunorum protected shoots against wilt when administered with the same bacterial isolate. These substances also inhibited cherry canker disease in susceptible mature trees when applied before inoculation to leaf-scar inoculation sites. Inhibition of bacterial adhesion to mature plant tissues treated with lipopolysaccharide or sidechain is suggested as a cause of disease inhibition. The wilt reaction may provide a rapid test of phytopathogenicity.     

甜樱桃流胶病原菌的分子鉴定和致病性检测

 为明确甜樱桃流胶病的病原菌,采用普通细菌学方法从15个病样中分离获得10个代表性菌株,对病原菌的形态学特征、生理生化特性以及致病性进行了研究。利用PCR对菌株的16S-23S rDNA转录间隔区(ITS)序列进行扩增,扩增产物克隆测序,结果显示该菌株属于丁香假单胞菌(Pseudomonas syringae)。用MegAlign构建系统发育树,结果显示该菌株与丁香假单胞菌丁香致病变种(P. syringae pv. syringae)亲缘关系最近,两者最先聚在一起。分离菌株中均可检测到syrB基因。研究结果表明P. syringae pv. syringae为甜樱桃流胶病的病原菌。     

Partial Nucleotide Sequence and Genome Organization of a Canadian Isolate of Little cherry virus and Development of an Enzyme-Linked Immunosorbent Assay-Based Diagnostic Test

ABSTRACT Approximately 12.4 kb of the genome of a mealybug-transmissible, North American isolate of Little cherry virus (LChV-3, previously designated LChV-LC5) has been cloned and sequenced. The sequenced portion of the genome contains 10 open reading frames (ORFs) and, based on sequence comparisons, encodes a putative RNA helicase (HEL), RNA-dependent RNA polymerase (POL), two coat proteins (CPs), a homologue of HSP70, a 53K protein (p53) that is similar to an equivalent-size protein in other closteroviruses, and a 22K (p22) protein of unknown function. The genome also potentially encodes two small proteins (p5 and p6), one of which is similar to the small hydrophobic proteins of other closteroviruses. Phylogenetic analyses utilizing sequences of the HEL, POL, and HSP70 homologue suggest that LChV-3 is most similar to other mealybug-transmitted closteroviruses. Further comparisons between LChV-3 and a 4.7-kb region of the recently described Little cherry virus-2 (LChV-2) reveals 77% nucleotide sequence identity. Based on this low sequence identity, we propose that LChV-3 be considered a separate species, designated LChV-3. Unexpectedly, the LChV-3 CP duplicate ORF was found to lie upstream of the HSP70 ORF; therefore, the genome organization of LChV-3 is distinct from that of other closteroviruses. Polyclonal antiserum raised to bacterially expressed LChV-3 CP was useful for detection of LChV-diseased trees in the cherry-growing districts of British Columbia, Canada.     

Plum pox potyvirus on sour cherry in Moldova

Plum pox potyvirus (PPV) was identified in six cultivars of sour cherry in the collection orchard of the Moldavian Horticultural Research Institute. Study of biological properties of the sour cherry isolate in herbaceous indicators showed its similarity or identity with the PPV isolate of Van Oosten and a significant difference from isolates widespread in Moldova. A purified viral preparation was used to develop antiserum with a working titre of 1:1024. Comparative serological examination of the sour cherry and conventional plum PPV isolates using ELISA, ISEM and SDS-PAGE of the protein capsid could not differentiate these isolates. The sour cherry isolate was transferred to plum resulting in weak but distinctive PPV symptoms in susceptible cv. Sopernitsa.