Correction to: Droplet-vitrification cryotherapy and thermotherapy as efficient tools for the eradication of apple chlorotic leaf spot virus and apple stem grooving virus from virus-infected quince in vitro cultures

European Journal of Plant Pathology -     

Chenopodium quinoa,a herbaceous test plant for chlorotic leaf spot virus in apple

The chlorotic leaf spot virus which occurs latent in apples, is widespread in Dutch orchards. In the present paper sap inoculation onChenopodium quinoa is recommended for rapid indexing of trees. Young leaves gave satisfactory results when tested during the first 4–6 weeks after expansion of the buds.The best results were obtained when homogenated petals were used as inoculum.Samenvatting Het chlorotische-bladvlekkenvirus komt in Nederland zeer algemeen latent voor in alle appelrassen. Het virus kan door middel van sapinoculatie opChenopodium quinoa worden aangetoond. Een week na de inoculatie reageren de toetsplanten met lokale vlekken op de bladeren. Deze methode is veel sneller dan de indicatie met behulp van houtige toetsplanten. Hoewel gedurende het gehele seizoen virusoverdracht mogelijk is, kan het virus goed worden aangetoond met jong blad in de eerste 4 tot 6 weken na het uitlopen van de bladknoppen. Nog betere resultaten worden verkregen met bloemblaadjes.     

Exogenous application of melatonin improves eradication of apple stem grooving virus from the infected in vitro shoots by shoot tip culture

Production and maintenance of virus-free planting materials is pivotal for the control of viral diseases. The present study attempted to test exogenous application of melatonin for eradication of apple stem grooving virus (ASGV) from virus-infected in vitro shoots of apple cultivar Gala. Exogenous application of 15 μm melatonin to the shoot proliferation medium significantly increased the number of shoots and shoot length. The level of endogenous indole-3-acetic acid (IAA) was the highest in the shoots proliferating on the shoot proliferation medium containing 15 μm melatonin. Shoot regrowth levels were significantly higher in shoot tips of the virus-infected shoots cultured for 4 weeks on this medium than the control. In addition, culture of shoot tips of the virus-infected in vitro shoots proliferated for 4 weeks on this medium resulted in 95% of shoots being virus-free, while no virus-free shoots were obtained in shoot tips of the virus-infected shoots cultured without melatonin. Analyses by microtissue direct RT-PCR and RT-qPCR showed that ASGV concentration decreased in shoot tips of the virus-infected shoots proliferating on the medium containing 15 μm melatonin for 4 weeks. Virus localization showed that exogenous application of melatonin enlarged the virus-free area in the virus-infected shoot tips. These data provide explanations as to why exogenous application of melatonin can efficiently eradicate ASGV. Exogenous application of melatonin provides an alternative means for plant virus eradication and has the potential to produce virus-free plants.     

来源于桃和苹果的苹果褪绿叶斑病毒的部分分子生物学特性和cp基因的原核表达

 从桃和苹果上分离得到苹果褪绿叶斑病毒ACLSV-HBP和ACLSV-C2个分离物,采用RT-PCR法进行扩增,所获扩增片段经序列测定,其全长分别为1768nt(ACLSV-HBP)和1751nt(ACLSV-C)。这2个分离物扩增片段全长的同源性为83%,mp基因片段核苷酸和推导编码氨基酸序列同源性分别为82.6%和87.1%;cp基因均由582nt组成,其核苷酸和推导编码氨基酸序列同源性分别为87.8%和95.9%。将2个分离物的cp基因与已报道ACLSV分离物进行序列同源性比较,结果显示ACLSV-HBP与SX/2的cp基因核苷酸序列及推导编码氨基酸序列同源性最高,分别为94.0%和96.4%。将ACLSV-HBP分离物的cp基因克隆到原核表达载体pGEX-KG,在大肠杆菌BL21(DE3)中诱导表达,SDS-PAGE分析表明,融合蛋白大小约为46kDa。Western-blot分析表明,该基因在大肠杆菌内得到高效表达,融合蛋白具有抗原性。     

来源于新疆的3 个苹果褪绿叶斑病毒分离物的分子变异研究

Apple chlorotic leaf spot virus (ACLSV) isolates from Korla pear (KI-2), New pear no. 7 (XI-1) and Red Fuji apple (API-4) were collected from XinJiang and characterized by analyzing sequences of their near genomic 3忆-terminal. The RT-PCR products were cloned, and analyzed by single-strand conforma-tion polymorphism (SSCP). Eight out of 39 collected positive clones showing different SSCP patterns were sequenced. The results showed that the amplified products had sizes ranging 676 - 703 bp, including partial coat protein (CP) gene (506 bp, accounts for 87% of the complete cp gene) and 3忆-terminal non-coding re-gion (3忆NCR) sequences. The cp gene sequences from isolate KI-2 showed a high intra-isolate divergence,with 84. 8% - 85. 4% identities at the nucleotide (nt) level, and the intra-isolate identities were 99. 8 % and 92.5% - 99. 8 % for isolate XI-1 and API-4, respectively. Phylogenetic analysis on the nt sequences of cpgene showed that the analyzed ACLSV variants from three isolates fell into two different clusters. A variant KI-2-6 from KI-2 was clustered into a group with an apple isolate aclsv-c from China and a plum isolated from France, and all other variants fell into a large cluster. The 3忆NCR sequences of these variants were identical ranging 80. 6% - 100 % .     

Pollen grains infected with apple stem grooving virus serve as a vector for horizontal transmission of the virus

Journal of General Plant Pathology - Here, Nicotiana benthamiana plants became infected with apple stem grooving virus (ASGV) after pollination with pollen grains produced by ASGV-infected apple...     

苹果褪绿叶斑病毒双引物对PCR检测体系的建立及应用

<正>病毒病在我国苹果树上普遍发生,其中苹果褪绿叶斑病毒(apple chlorotic leaf spot virus,ACLSV)、苹果茎沟病毒(apple stem grooving virus,ASGV)和苹果茎痘病毒(apple stem pitting virus,ASPV)最常见,常混合侵染,引起树体衰弱,苹果产量和品质下降,经济损失严重(CieniewiczFuchs,2016)。因此,建立快速、准确的检测技术对病毒病防控具有重要意义。Hu et al.(2015)认为在病毒检测过程中,需     

利用Real-time RT-PCR方法检测苹果中苹果茎沟病毒

以含有苹果茎沟病毒(Apple stem grooving virus,ASGV)的苹果枝条为试材,设计扩增产物片段为176 bp的特异性引物,优化反应条件,构建标准曲线,建立了苹果茎沟病毒的SYBR Green Ⅰ实时荧光定量RT-PCR检测方法.对该方法进行了灵敏度和重复性试验,并且利用建立的方法对受ASGV感染的苹果枝条和树叶进行定量检测.最后,比较了实时荧光定量RT-PCR与常规RT-PCR检测的灵敏度.结果表明:样品的荧光定量PCR有良好的扩增曲线和溶解曲线,该方法的灵敏度可达6.8× 102拷贝/μL,检测其灵敏度是传统RT-PCR的100倍,所建立的荧光定量技术可用于田间样品中此病毒的检测,丰富了ASGV的检测手段,提高了检测灵敏度,具有较好的应用前景.     

Molecular variability of Apple chlorotic leaf spot virus in Shaanxi,China

Apple chlorotic leaf spot virus (ACLSV) is one of the latent viruses that occur in apple orchards worldwide but usually without visible symptoms. In 2010–2012, a total of 550 apple leaf samples from 12 different major apple-producing areas in Shaanxi, China, were tested by serological assay for ACLSV; the results revealed an infection level of 51.5 %. Because of the known variability in the putative amino acid sequences of the coat protein (CP), and thus the potential for non-detection by serological assay, the molecular variability of isolates of ACLSV collected in Shaanxi was analyzed using PCR and compared with isolates from the rest of the world. Sequences of 504 nt corresponding to 87 % of the CP gene of 12 isolates were acquired by RT-PCR and deposited in GenBank with the accession numbers KF134387–KF134298. Comparisons of the partial CP gene sequences of these 12 isolates as well as isolates previously reported in the world revealed the pairwise identities ranging from 68.9–99.8 % and 73.8–100 % at the nucleotide and amino acid level, respectively. Phylogenetic analysis based on these nucleotide sequences showed that the 72 isolates deposited in GenBank fell into three groups (P205, B6 and Ta Tao 5 Group). Our 12 ACLSV isolates were separated into the P205 and B6 groups, respectively. Multiple alignment analysis of the amino acid sequences of CP revealed that there was a combination of six amino acids at positions 40, 59, 75, 86, 130 and 184 in isolates from each group that could be used to distinguish among the three groups. Two recombination events were identified from all isolates by recombination analysis, and three ACLSV isolates collected in this study participated in these two events. Our results show that molecular variation was present in isolates of ACLSV collected in Shaanxi province and this may reflect introductions of the virus associated with different sources of germplasm.     

Prevalence and molecular variability of Apple chlorotic leaf spot virus capsid protein genes in Lithuania

Journal of Plant Diseases and Protection - Apple chlorotic leaf spot virus (ACLSV) is a most common pathogen of apples in the world. The occurrence, genetic diversity, recombination patterns and...     

苹果茎沟病毒梨分离物外壳蛋白基因的克隆和序列分析

 选取生物学和血清学特性存在较大差异的苹果茎沟病毒(Apple stem grooving virus,ASGV)梨分离物P-6-1-17、P-4-1-69、P-L2和P-D-21,采用RT-PCR法对3'端进行扩增,所获扩增片段经序列测定,全长分别为1089nt(P-6-1-17、P-L2)和1069nt(P-4-1-69、P-D-21)。4个分离物的CP基因均由714nt组成,其核苷酸和推导编码蛋白氨基酸序列同源性分别为89.8%~96.4%和94.9%~97.9%。将我国4个分离物的CP基因与已报道ASGV分离物及分子变种进行序列同源性比较和系统进化树分析,结果表明我国ASGV分离物至少可分为2个组群,其中生物学表现明显不同于其它分离物的P-L2,其CP基因核苷酸序列显示较大的变异。     

来源于砂梨和桃的苹果褪绿叶斑病毒分离物部分CP基因和3′端非翻译区的序列分析

 苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus, ACLSV）是引起果树病害的一种重要病毒。ACLSV寄主范围广、发生较普遍,可侵染苹果、梨等仁果类果树和桃、扁桃、李、樱桃、杏等核果类果树,据报道我国梨产区感染ACLSV达80%以上。ACLSV引起植物症状的类型与寄主种类、病毒株系有关。ACLSV为线形病毒科（Betaflexiviridae）、纤毛病毒属（Trichovirus）的代表成员^［1］。ACLSV的CP相对比较保守,研究表明不同的ACLSV分离物的CP基因具有序列多样性,存在分子变异^［2～5］,CP基因分子特性的研究可为ACLSV株系划分提供依据。来源于欧洲、亚洲和北美的桃、李等核果类果树,以及苹果寄主上的ACLSV分离物的分子变异报道较多^［2,3,5］,来源于梨寄主上的ACLSV分子变异研究较少^［4,5］。     

Characterization of a strain of Apple stem grooving virus in Actinidia chinensis from China

A new strain of Apple stem grooving virus (ASGV) has been identified in Actinidia chinensis imported from China. The leaves of these plants exhibited a variety of symptoms including interveinal mottling, chlorotic mosaics and ringspots. Capillovirus-like particles were observed under the electron microscope, and the virus could be mechanically transmitted to a range of herbaceous indicators. The virus was detected using ELISA with antisera raised against ASGV. Sequencing of the virus revealed that it had more than 95% amino acid identity with ASGV in the putative coat and movement proteins. From the morphological, transmission, serological and molecular evidence, it was concluded that the virus is a strain of ASGV. It is not known how this strain of ASGV is transmitted, other than by grafting, nor is it known what effect the virus has on the growth of infected vines. The Actinidia -infecting strain of ASGV does not occur in New Zealand, and infected plants will not be released from quarantine. The detection methods used during the research will assist quarantine and the safe movement of breeding material.     

Serological Comparison and Molecular Characterization for Verification of Calla lily chlorotic spot virus as a New Tospovirus Species Belonging to Watermelon silver mottle virus Serogroup

ABSTRACT Calla lily chlorotic spot virus (CCSV) isolated from central Taiwan was recently identified as a tospovirus serologically but distantly related to Watermelon silver mottle virus (WSMoV). To clarify the serological relationship between the two viruses, rabbit polyclonal antibody (PAb) to CCSV and mouse monoclonal antibodies (MAbs) to WSMoV NP or CCSV NP were produced in this investigation, using purified nucleocapsid protein (NP) as immunogens. The PAb to CCSV NP reacted stronger with the homologous antigen than with the heterologous antigen, with much lower A(405) readings in indirect enzyme-linked immunosorbent assay (ELISA) and low-intensity banding in immunoblotting. MAbs produced to CCSV NP or WSMoV NP reacted specifically with the homologous antigens but not with the heterologous antigens in both ELISA and immunoblot analyses. The CCSV S RNA was determined to be 3,172 nucleotides in length, with an inverted repeat at the 5' and 3' ends and two open reading frames encoding the NP and a nonstructural (NSs) protein in an ambisense arrangement. A typical 3'-terminal sequence (5'-AUUGCUCU-3') that is shared by all members of the genus Tospovirus also is present in the CCSV S RNA. The CCSV NP and NSs protein share low amino acid identities of 20.1 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. Phylogenetic dendrogram analysis indicates that CCSV is a distinct member in the genus Tospovirus. The results provide evidence that CCSV is a new species in the genus Tospovirus and belongs to WSMoV serogroup.     

The promotion of thiophanate-methyl and tebuconazole for the continuous control of Glomerella leaf spot in apple leaves by adding pellouxite as a synergistic reagent

Journal of Plant Diseases and Protection - Some site-specific fungicides were usually mixed with synergistic agrochemicals to prolong the active life and lower the risk of the resistance. As an...     

西瓜叶斑病病原菌的鉴定及室内药剂筛选

2022年在上海市浦东新区西瓜上发生一种深褐色、不规则至近圆形斑的叶部病害。采用组织分离法对病害样本进行分离, 分离物SHCCC01经柯赫氏法则验证具有较强致病性。根据分离物的菌落特征、分生孢子形态、生物学特征以及基于核糖体内部转录间隔区(internal transcribed spacer, ITS)、组蛋白基因(histone, His)、真核翻译延伸因子1α(translation elongation factor 1α, TEF-1α)和肌动蛋白 (actin, Act)基因联合系统发育分析等, 将病原菌鉴定为瓜类尾孢Cercospora citrullina。10种杀菌剂对该病原菌的毒力测定试验结果表明:氟啶胺、己唑醇、氟环唑、戊唑醇等4种药剂对病原菌的抑菌活性较高, 其EC50分别为0.375 2、0.575 4、1.472 3、1.890 2 μg/mL。这些结果为西瓜尾孢叶斑病的防控和杀菌剂登记提供了重要依据。     

茼蒿叶斑病病原菌的鉴定及室内药剂筛选

茼蒿是上海地区设施叶菜主要种类之一?近年来, 上海市部分地区的茼蒿上出现了一种叶部病害, 叶片出现外缘灰褐色?中央灰白色的圆形病斑, 严重影响茼蒿产量与商品质量?本文采用组织分离法进行病原菌的分离纯化, 通过柯赫氏法则验证?形态学?生物学特性以及基于核糖体转录间隔区(internal transcribed spacer, ITS)?真核翻译延长因子1α (translation elongation factor, TEF)?钙调蛋白 (calmodulin, CAL) 基因和肌动蛋白 (actin, ACT) 基因联合系统发育分析等方法进行了病原菌鉴定, 结果表明, 茼蒿叶斑病的病原菌为芹菜尾孢Cercospora apii, 菌丝生长最适温度为26℃, 复合氮源利于菌丝的生长?杀菌剂的离体抑菌活性测定结果表明:己唑醇?苯醚甲环唑?戊唑醇?氟环唑?嘧菌酯的EC50分别为0.042 3?0.060 4?0.317 4?0.452 4?0.863 3 μg/mL, 对该病原菌菌丝生长表现出较强的抑制作用?这些结果为小宗作物病害的防控或杀菌剂登记提供重要依据?     

Multiple Alternaria species groups are associated with leaf blotch and fruit spot diseases of apple in Australia

Leaf blotch and fruit spot of apple caused by Alternaria species occur in apple orchards in Australia. However, there is no information on the identity of the pathogens and whether one or more Alternaria species cause both diseases in Australia. Using DNA sequencing and morphological and cultural characteristics of 51 isolates obtained from apple leaves and fruit with symptoms in Australia, Alternaria species groups associated with leaf blotch and fruit spot of apples were identified. Sequences of Alternaria allergen a1 and endopolygalacturonase gene regions revealed that multiple Alternaria species groups are associated with both diseases. Phylogenetic analysis of concatenated sequences of the two genes resulted in four clades representing A. arborescens and A. arborescens‐like isolates in clade 1, A. tenuissima/A. mali isolates in clade 2, A. alternata/A. tenuissima intermediate isolates in clade 3 and A. longipes and A. longipes‐like isolates in clade 4. The clades formed using sequence information were supported by colony characteristics and sporulation patterns. The source of the isolates in each clade included both the leaf blotch variant and the fruit spot variant of the disease. Alternaria arborescens‐like isolates were the most prevalent (47%) and occurred in all six states of Australia, while A. alternata/A. tenuissima intermediate isolates (14%) and A. tenuissima/A. mali isolates (6%) occurred mostly in Queensland and New South Wales, respectively. Implications of multiple Alternaria species groups on apples in Australia are discussed.