Variability among Spanish citrus tristeza virus isolates revealed by double-stranded RNA analysis

A survey for citrus tristeza virus (CTV) strains, based on double-stranded RNA (dsRNA) analysis, was carried out in five locations on the eastern citrus-growing area of Spain. CTV was recovered from 137 trees of different ages, citrus species and varieties, sampled in 53 orchards. The best months for dsRNA recovery were April, May, September, October, and November, and the highest dsRNA yield was obtained from sweet orange cultivars. Sixteen dsRNA profiles differing by the number and/or position of subgenomic bands were detected. One of these profiles was detected in more than half the trees analysed. Maximum diversity of dsRNA patterns was found in the location with the oldest citrus orchards and the highest CTV incidence (Alzira-Carcaixent). In many instances, several dsRNA profiles were detected in neighbouring trees of the same orchard, notably in Alzira-Carcaixent, where 70% of the plots sampled contained more than one profile. The possible causes for the diversity of CTV isolates found in this specific area are discussed.     

Investigation of biological properties, double-stranded RNA patterns and antigen concentration in citrus species infected with citrus tristeza virus

Biological properties and dsRNA patterns of one Cyprus and three Turkish isolates of citrus tristeza virus (CTV) were investigated. In addition, CTV antigen concentration and effect of tissue sampling time from naturally infected Shamouti sweet orange trees grown in the field of Icel Province, Turkey, were also determined. The Cyprus isolate showed vein clearing symptoms on grapefruit, ‘Madam Vinous’ and Mexican lime and stem pitting symptoms on Mexican lime. The three Turkish isolates showed only vein clearing symptoms on Mexican lime. All four isolates showed a full-length major double-stranded RNA (dsRNA) band of 13.3 × 10⁶ Da mol. wt in extracts from infected Madam Vinous sweet orange trees, and major or minor dsRNA bands with 2.0. 0.8 and 0.5 × 10⁶ mol.wt. All seven different citrus varieties inoculated with the Igdir (D) strain contained full-length dsRNA. The additional two dsRNA of 0.8 and 0.5 × 10⁶ mol.wt were also detected as clearly as full-length dsRNA in these hosts, but were weaker inCitrus exelsa and ‘Interdonat’ lemon. Madam Vinous, rough lemon and Mexican lime were the best hosts for dsRNA analysis. ELISA values were highest in April (OD_405nm =0.476), decreased steadily until August, and then increased gradually through December. ELISA values were lowest in July and August (OD_405nm =0.157 and 0.141, respectively). dsRNA recovery from a field tree infected with isolate Igdir D was good in March, April and May and poor in January and February. No dsRNA band was detected in August or September. http://www.phytoparasitica.org posting July 9, 2002.     

广西柑橘衰退病和黄脉病调查及其病原病毒的遗传多样性分析

为明确柑橘衰退病毒(citrus tristeza virus, CTV)和柑橘黄脉病毒(citrus yellow vein clearing virus, CYVCV)在广西柑橘上的发生?分布及其遗传变异情况, 于2020年至2021年对百色?北海?崇左?贵港?桂林?河池?贺州?来宾?柳州?南宁?梧州和玉林等12个柑橘产区进行了病毒病调查?采用RT-PCR对采集样品进行了病毒检测, 并基于病毒分离物外壳蛋白(coat protein, CP)基因的核苷酸序列进行比对分析, 构建系统发育树?结果表明:采集的737份柑橘样品中, CTV的检出率为20.62%, CYVCV检出率为18.32%, CTV的检出率略高于CYVCV?病毒复合侵染的现象在采集的柑橘样品中普遍存在, CTV和CYVCV复合侵染率高达34.50%?对RT-PCR产物测序共获得12个CTV分离物和6个CYVCV分离物的CP基因序列?遗传多样性分析发现, CTV和CYVCV的CP基因序列都较保守, CTV分离物的遗传进化与地理来源?寄主来源均没有明显相关性, 但CYVCV分离物的遗传进化与地理位置具有相关性, 而与寄主来源无明显相关性?上述研究结果可为深入了解CTV和CYVCV在广西的流行情况以及柑橘病毒病的检疫和防控提供参考?     

Detection of plum pox virus by isolation of double-stranded ribonucleic acid (dsRNA)

Double-stranded RNA (dsRNA) associated with plum pox virus (PPV) in Nicotiana clevelandii and Prunus domestica has been isolated. While dsRNA was detected in N. clevelandii in considerable amounts by electrophoresis, only small amounts were found in P. domestica. This may be due to viscous substances in the leaves of this woody host. Different PPV strains (NAT - not aphid-transmissible; AT - aphid-transmissible) showed specific patterns in electrophoresis gels. When PPV was assayed in N. clevelandii by dsRNA detection or by standard ELISA or ISEM, all three methods were found to be efficient, with none being superior. ELISA, as a simple and fast routine method, is still the method of choice. DsRNA detection will be suitable for plant disease agents undetectable by ELISA and ISEM.     

Biological diversity of citrus tristeza virus (CTV) isolates in Spain

A survey of citrus tristeza virus (CTV) isolates was carried out in most citrus-growing areas in Spain. Twenty-two isolates were selected by geographical origin, cultivar of source tree, and symptoms observed on the host or in preliminary tests, and were biologically characterized.
A wide range of variation in transmissibility by aphids and symptom intensity on nine different indicator species or scion-rootstock combinations was observed among CTV isolates. Mexican lime. Citrus macrophylla , and to a lesser extent citron were the most useful hosts for characterizing these isolates, and leaf symptoms and stem pitting were the most discriminating traits. Positive correlation was observed between symptoms induced on Mexican lime and C. macrophylla , but not between the symptoms induced on these indicators under greenhouse conditions and the homologous symptoms on plants grown in the screenhouse. Some of the traits studied enabled us to establish relatively well-defined groups of isolates, but in most cases a continuous range of variation was obtained and no clear group could be defined.     

Molecular variability of the 5'- and 3'-terminal regions of citrus tristeza virus RNA

ABSTRACT Isolates of citrus tristeza virus (CTV) differ widely in their biological properties. These properties may depend on the structure of viral RNA populations comprising the different isolates. As a first approach to study the molecular basis of the biological variability, we have compared the sequences of multiple cDNA clones of the two terminal regions of the RNA from different CTV isolates. The polymorphism of the 5' untranslated region (UTR) allowed the classification of the sequences into three groups, with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. The variability of an open reading frame (ORF) 1a segment adjacent to the 5' UTR supports the same grouping. Some CTV isolates contained sequences of more than one group. Most sequences from Spanish isolates belonged to group III, whereas a Japanese isolate was composed mostly of sequences of groups I and II. The mildest isolates contained only sequences of group III, whereas the most severe isolates also contained sequences of groups I, II, or both. The most stable secondary structure predicted for the 5' UTR was composed of two stem-loops and remained essentially unchanged as a result of compensatory mutations in the stems and accommodation of most of the variability in the loops. In contrast to the 5'-terminal region, the variability of the 3'-terminal region of CTV RNA was very much restricted, with nucleotide identity values higher than 90%. The presence of a conserved putative "zinc-finger" domain adjacent to a basic region in p23, the predicted product of ORF 11, suggests that this protein might act as a regulatory factor during virus replication.     

First detection of a virulent strain of Citrus tristeza virus (Closteroviridae) in a citrus orchard of Chlef Valley (Algeria)

A large‐scale survey of Citrus tristeza virus (CTV) was carried out from 2016 to 2018 in the Chlef Valley, one of the main citrus growing areas in Algeria. In this study a total of 1680 citrus trees from 93 commercial orchards were sampled. The collected samples were tested by direct tissue blot immunoassay analysis and by the double antibody sandwich enzyme‐linked immunosorbent assay technique, and 54 trees were identified as being infected with CTV. This result confirmed that 54 trees were infected by the virus, corresponding to an infection rate of 3.21% throughout the studied area. Five of these local CTV sources were chosen for further molecular investigations to determine the genotype associated with the CTV isolates now spreading in the Chlef area. Characterization with multiple molecular markers showed the presence of the T30 and VT genotypes. This result allowed confirmation of the presence of a virulent strain belonging to the VT genotype. The other CTV isolates were similar to those from the Mitidja region, which showed 99% nucleotide identity with the Spanish mild CTV isolate. This early finding of a strain belonging to the VT genotype is an issue for Algerian citrus producers and needs rapid actions to be taken by the National phytosanitary services, extending the surveillance to other citrus production regions and uprooting the infected trees.     

Biological and molecular characterization of a distinct Citrus tristeza virus isolate originating from a lemon tree in Greece

A Citrus tristeza virus (CTV) isolate (L192GR) naturally occurring in lemon trees of more than 100 years old in Greece was fully characterized. Virus‐derived small interfering RNAs, induced by Dicer processing of dsRNAs formed during RNA virus replication, were isolated and used as targets for sequencing. Next‐generation high‐throughput sequencing using the Ion Torrent platform was performed. A total of 432 632 sequences, 94·05% of which corresponded to L192GR, were determined. Subsequent bioinformatics analysis enabled the determination of the full‐length 19 251 nt genome of the L192GR isolate (GenBank no. KC262793 ). Comparative analysis of complete genomes revealed molecular homology with CTV‐VT isolate FS2‐2 from Florida (GenBank no. EU937519 ) with 98·2% nucleotide sequence identity. Recombination events were detected in L192GR and they probably contribute to its unique characteristics. Specifically, although most isolates of the CTV‐VT group induce the seedling yellows syndrome and react positively with the monoclonal antibody MCA13, which is typically associated with severe CTV isolates, the MCA13‐positive L192GR gave very mild or even no symptoms in the seedling yellows indicator plants. Furthermore, experimental aphid transmissibility studies revealed a poor transmission efficiency of 20%. This is the first report of a CTV isolate originating from a lemon tree being fully characterized at biological, serological and molecular levels. The present study further confirms that, when the goal is the risk assessment associated with a new pathogen or isolate in a particular area, molecular data have to be combined with the biological properties of the pathogen.     

Contribution of uneven distribution of genomic RNA variants of citrus tristeza virus (CTV) within the plant to changes in the viral population following aphid transmission

The population of genomic RNA sequence variants of citrus tristeza virus (CTV) isolates was characterized by single-strand conformation polymorphism (SSCP) analysis of complementary DNA (cDNA) of the genes p18 and p20. Comparison of field and aphid-transmitted isolates showed that aphid transmission frequently altered the single-strand conformation polymorphism (SSCP) pattern of both genes, indicating changes in the population of genomic RNA variants. SSCP analysis of the cDNA of RNA extracted from small pieces of tissue sampled at different sites of the same plant sometimes yielded different patterns, indicating uneven distribution of the genomic RNA variants within the infected plant. Different SSCP patterns were also obtained when the RNA extracted from individual aphids probing in the same infected leaf was used as a template. Uneven distribution of the genomic RNA variants within the infected plant and sorting of some of these variants by individual aphids probably contribute to changes observed in the CTV population following aphid transmission.     

Molecular identification of a Cucumber mosaic virus subgroup II isolate from carrot (Daucus carota) based on RNA3 genome sequence analyses

Journal of Plant Diseases and Protection - Natural occurrence of severe chlorotic mottle disease of carrot (Daucus carota) with a significant incidence was observed in northern Uttar Pradesh,...     

柑橘衰退病、裂皮病和碎叶病的多重RT-PCR检测方法研究

 本研究以广东省农业科学院果树研究所隔离网室内通过嫁接分别感染CTV、CEVd和CTLV的柑橘树皮为实验材料,建立了以oligo(dT)为反转录引物的RT-PCR检测体系,成功检测到在病毒RNA 3'末端带有ploy_(A)加尾的CTV和CTLV。实验过程中,发现不具有ploy_(A)加尾的环状类病毒CEVd,同样可以用oligo(dT)反转录的RT-PCR检测出来,通过测序分析,其同源性在96%以上,并发现在CEVd序列中有一个富含A的区段。在此基础上,进一步研究了同时检测CTV、CEVd和CTLV的多重RT-PCR检测体系,该方法能为这3种病害的检测简化步骤、节省时间、降低成本。     

Infectivity analysis of a soybean isolate of Mungbean yellow mosaic India virus by agroinoculation

Yellow mosaic disease (YMD) of legumes endemic to South Asia are caused by begomoviruses transmitted by whiteflies. Based on molecular characterization, two distinct viruses – Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) – were found previously to be the etiological agents of YMD in legumes. Here, host range studies with a soybean isolate of MYMIV (MYMIV-[Sb]) were carried out by both whitefly transmission and agroinoculation. MYMIV-[Sb] was similar to a cowpea isolate of MYMIV (MYMIV-[Cp]) in its ability to infect cowpea, thus differing from blackgram (MYMIV) and mungbean (MYMIV-[Mg]) isolates, which do not infect cowpea. Genomic analysis of DNA A and DNA B components of these MYMIV isolates show characteristic differences in complete DNA B nucleotide sequence correlating with host range differences.     

番木瓜环斑病毒山东分离物的全基因组序列分析

Papaya ringspot virus (PRSV) is a major limiting factor to cucurbit production worldwide. One zucchini sample showing symptoms of leaf mosaic and fruit ringspot was collected from Ji’nan city of Shandong province. Primary experiments showed that the sample was infected with PRSV, which was designated as PRSV-SD accordingly. The complete genomic fragment of PRSV-SD was obtained with RT-PCR. The results of sequencing showed that the full genomic sequence of PRSV-SD was 1 0337 nucleotides (nt) excluding the 3′- terminal poly (A) tail. The 5′- and 3′-untranslated regions (UTR) were 90 and 206 nt, respectively. The putative polyprotein was 3 346 amino acids in length. Comparison of the PRSV-SD isolate with 18 other PRSV isolates revealed that they shared nucleotide identities of 82.1%~89.3% at the complete genomic levels and amino acid identities of 90.6%~94.7% for the polyprotein. No recombination was detected throughout the genome of PRSV-SD. Phylogenetic analysis with complete genomic sequences indicated that the PRSV isolates were clustered into two major groups: Asia and America and PRSV-SD was clustered to the Asia group. Selection pressure analysis revealed that all of the 11 proteins of PRSV-SD were under negative selection, but positive selection sites were detected in P1, P3, 6K1, NIa-pro and NIb. Our research results provide a theoretical foundation for the detection and prevention of PRSV. Key words:Papaya ringspot virus; complete genomic sequence; phylogenetic analysis; selection pressure; recombination 中图分类号:S436.429; Q939.46 文献标识码:A 文章编号:0412-0914(2018)02-0285-04 番木瓜环斑病毒(Papaya ringspot virus,PRSV)属于马铃薯Y病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus)^[1]。根据寄主范围可划分为P和W 2个株系,其中P株系侵染番木瓜(Carica papaya L.)和葫芦科(Cucurbitaceae)作物,而W株系只侵染葫芦科作物,两者血清学反应密切相关。PRSV可引起植株叶片褪绿、花叶、卷曲等症状,在果实表面形成环斑。 PRSV于20世纪40年代末在美国首次报道,目前该病毒在巴西、印度、波兰等国均有发生^[2]。2000~2001年,PRSV使我国海南番木瓜损失严重。2005年以来,我国广东、四川先后检测到PRSV侵染罗汉果、苦瓜等葫芦科作物^[3]。2016年从山东西葫芦上检测到PRSV,该分离物属于W株系^[4]。我国关于PRSV进化的研究大多集中于P株系,有关W株系全基因组序列分析的报道相对较少。为了进一步研究我国PRSV-W株系的基因组特性,为PRSV的监测预警提供依据,我们测定了PRSV山东分离物的全基因组序列,并进行了核苷酸和氨基酸序列一致率、重组、系统发育和基因选择压力分析。 2期黄显德,等:番木瓜环斑病毒山东分离物的全基因组序列分析 植物病理学报48卷 1 材料与方法 1.1 材料 发病西葫芦样品采自山东省济阳县。大肠杆菌DH5α由本实验室保存。植物总RNA提取试剂盒TRIzol、DNA凝胶回收试剂盒等均购自北京全式金公司;Taq DNA聚合酶、 pMD18-T克隆载体等购自TaKaRa公司;SuperScript^TM Ⅳ逆转录酶购自Invitrogen公司。其他生化试剂及普通化学试剂均为进口或国产分析纯。 1.2 实验方法 1.2.1 扩增策略及引物合成 根据GenBank中PRSV基因组序列,设计引物分4段扩增基因组序列。根据所得序列设计5′-RACE引物扩增5′-端片段。测序后用SeqMan拼接得到全基因组序列。所用引物详见表1。 1.2.2 植物总RNA提取及RT-PCR扩增 按照RNA提取试剂盒说明书进行植物总RNA提取。以总RNA为模板,用随机引物反转录合成cDNA。通过4次PCR和5′-RACE扩增PRSV-SD基因组片段。 1.2.3 克隆及序列测定 电泳分离PCR产物,切胶回收后连接到pMD18-T载体上,转化E. coli DH5α感受态细胞,挑选阳性克隆进行核苷酸测序。     

The 3' terminal sequence of RNA1 of wheat spindle streak mosaic virus canadian isolate (WSSMV-C)

The sequence of the 3 terminal 1722 nucleotides (nts) of RNA1 of the type (Canadian) isolate of wheat spindle streak mosaic bymovirus (WSSMV-C) was determined. The sequence started within a single open reading frame (ORF), which was expected to encode the carboxyl terminus of the nuclear inclusion b protein (NIb) and the capsid protein (CP) of 294 amino acids, followed by a 3 untranslated region (UTR) of 237 nucleotides. The NIb and CP of WSSMV-C share 99 and 100% amino acid sequence identity with the corresponding proteins of WSSMV-French isolate (WSSMV-F), but only 89 and 77% with wheat yellow mosaic virus (WYMV-J), respectively. The 3UTR of RNA1 of WSSMV-C shares 94% nucleotide sequence identity with that of WSSMV-F but only 73% with WYMV-J and WYMV-Chinese isolate (WYMV-Chi). The results support the classification of WSSMV-C and WSSMV-F as strains of the same virus species which is distinct from WYMV.     

侵染大豆的花生斑驳病毒公主岭分离物基因组测序及分析

本研究利用小RNA深度测序法在大豆叶片上检测到1株花生斑驳病毒Peanut mottle virus (PeMoV),根据小RNA深度测序结果和GenBank公布的PeMoV基因组序列设计引物克隆了PeMoV公主岭分离物(PeMoV-Gongzhuling)的基因组序列.测序结果经拼接后获得了PeMoV-Gongzhu...     

不同来源的柑橘衰退病毒分离物基因组5'端A、F变异区序列克隆及分析

 柑橘衰退病毒(Citrus tristeza virus,CTV)组群自然条件下存在株系分化现象。本研究利用RT-PCR技术扩增、克隆了来自我国不同地区的21个柑橘衰退病毒分离物的5'端A、F变异区。通过分析发现,不同来源的各分离物在5'端A、F区存在较大的变异。21个分离物A区序列相似性最低为85.8%,最高可达99.8%,平均为95.9%;与GenBank中9个代表性株系的平均相似性为84.2%。F区序列相似性较A区高,为98.0%;相似性最低为94.3%,最高达99.1%。结果显示不同来源的CTV分离物5'端序列A、F区变异较大。