Correlation of hop stunt viroid variants to cachexia and xyloporosis diseases of citrus

ABSTRACT Citrus viroid (CVd) group II is comprised of hop stunt viroid (HSVd)-related variants of 295 to 302 nucleotides. Included in this group are the cachexia-inducing agents citrus cachexia viroid (or CVd-IIb), CVd-IIc, Ca-903, and Ca-909 as well as the non-cachexia-inducing variant CVd-IIa. The cachexia indexing hosts 'Parson's Special' mandarin and 'Orlando' tangelo as well as Citrus macrophylla responded with symptoms of gumming, discoloration, and stem pitting when infected by CVd-IIb, CVd-IIc, or Ca-903. However, 'Palestine' sweet lime, the indicator host used to describe the xyloporosis disease, displayed a distinctly different fine-pitting reaction and no discoloration or gumming when infected by the same viroids. Cachexia-inducing variants contain a number of nucleotide changes more similar to hop-type HSVd sequences than to the citrus-type HSVd sequences, as typified by CVd-IIa. The nucleotide sequence of CVd-IIc was identical to CVd group II isolates common to trees expressing xyloporosis. Experimental evidence indicates that either CVd-IIb or CVd-IIc can cause citrus diseases known as cachexia and xyloporosis and that the two disease designations reflect the distinct responses of different indexing hosts to the same viroids.     

A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China

Citrus plants are natural hosts of five viroid species and large numbers of sequence variants. In this paper a simple and sensitive one step multiplex RT-PCR protocol with an internal control was utilised to simultaneously detect and differentiate five citrus viroids: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid-III (CVd-III) and Citrus viroid-IV (CVd-IV). In addition, a micro and rapid total nucleic acid extraction method was developed and the protocol applied to evaluate the occurrence and distribution of citrus viroids in China.     

Citrus viroid II variants associated with ‘Gummy Bark’ disease

A viroid etiology for citrus gummy bark (CGB) disease of sweet orange is supported by the similarity of symptom expression to cachexia disease of mandarins and tangelos caused by the hop stunt viroid (HSVd) related citrus viroid II (CVd-II), as well as the detection of CVd-II variants in CGB infected Washington navel and Dörtyol sweet orange, a Turkish cultivar. A survey was made of 67 clones of CVd-II related variants recovered from severe CGB symptomatic and non-symptomatic trees of the same cultivars growing in close proximity. Only CVd-IIa, a non-cachexia inducing variant, was found in non-symptomatic Washington navel trees and no CVd-II variants were recovered from the Dörtyol control. CGB infected sources contained a number of CVd-II related variants with the predominant species detected closely related to CVd-IIc, a known cachexia inducing viroid. Biological activity of representactive variants from CGB sources was determined by transmission to citron (Citrus medica) as well as by bioassay on the indexing host for cachexia, Parson's Special mandarin (Citrus reticulata).     

Identification and characterization of known and novel viroid variants in the Greek national citrus germplasm collection: threats to the industry

Seventy four percent of the budwood tree sources samples from the Greek national citrus germplasm foundation collection were positive for one or more viroids. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd), the two potentially damaging viroids for the Greek citriculture, especially after transitioning to Citrus tristeza virus resistant/tolerant rootstocks and scions, were detected along with Citrus bark cracking viroid, Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). All samples tested negative for Citrus viroid V (CVd-V), CVd-VI and CVd-I-LSS (CBLVd variant). An HSVd isolate related to the non-cachexia variant contained two critical cachexia-related nucleotide changes, while two more isolates were unique among the previously reported HSVds. Unusual CDVd isolates with altered RNA secondary structure were identified in trees additionally co-infected with CEVd and HSVd. Budwood sources that had previously undergone therapy tested negative for all targeted viroids, suggesting that budwood sources in Greece can be protected against graft-transmissible pathogens, even under severe inoculum pressure. Therapied and tested citrus propagative material requires a comprehensive program not available currently in Greece, involving regulators, scientists, and the private sector, for the establishment and successful operation of a national citrus germplasm collection.     

法国进境葡萄砧木中啤酒花矮化类病毒的检测与序列分析

为检测法国进境葡萄砧木中啤酒花矮化类病毒（Hop stunt viroid,HSVd）,对其进行了RT-PCR检测及序列测定,并构建系统进化树比较了不同地域来源HSVd间的分子差异性。结果表明基于HSVd基因序列设计合成的1对引物能够扩增出302 bp大小的目标片段,而健康植株无此扩增产物。法国葡萄砧木中检出的HSVd分离物,与国内外已报道毒株的核酸序列同源性达90%~97%,与已报道的HSVd全基因组序列间差异较小,表明HSVd的序列变异与地域、寄主等无明显相关性。     

Incidence and genetic diversity of Peach latent mosaic viroid and Hop stunt viroid in stone fruits in Serbia

Tissue-imprint hybridization (TIH) assay was validated for large-scale detection of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). All 72 collected leaves (100%) from 2 PLMVd- and 2 HSVd-infected trees were positive in TIH, regardless of the geographic orientation of the scaffold, level of the canopy and position of the leaf in the shoot. In a large-scale survey in Serbia, we tested by TIH 871 trees of stone fruits, representing 602 cultivars from fruit collections in Belgrade, Čačak and Novi Sad. PLMVd was detected in 185 (50%) peach trees or 95 (54%) cultivars and HSVd in 2 apricot trees and cultivars (2%). The occurrence of HSVd is a new report for Serbia. No viroid infection was found in European plums, sweet cherries, sour cherries and wild Prunus spp. PLMVd-infected peach cultivars originated from the world’s main breeding centres of this crop. Western European and Asian cultivars were the most infected (58%) followed by those originating from North America (50%). Nine PLMVd and two HSVd isolates were sequenced and analyzed. All showed PMLVd sequences clustered together in the previously reported phylogenetic group III. Both HSVd isolates were found to be derived from recombinant events, but that of the cv. Saturn represented a putative new phylogenetic group of HSVd.     

A preliminary account of the sanitary status of stone-fruit trees in Morocco

Field surveys were carried out in the main stone-fruit growing areas of Morocco to evaluate the sanitary status of commercial orchards, varietal collections and nurseries. The presence of virus and virus-like diseases was checked by ELISA, sap transmission to herbaceous hosts, testing on woody indicators and molecular hybridization (dot-blot and tissue-printing). 1211 samples (382 almond, 339 peach, 291 plum, 150 apricot and 49 cherry) were tested by ELISA for the presence of Prunus necrotic ring spot virus (PNRSV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV) and Plum pox virus (PPV). The overall average of virus infection rate was 16.4%, whereas that of single species was: 22.6% for almond, 17.8% for plum, 15% for peach, 10.2% for cherry, and 2.7% for apricot. The following viruses were detected: PNRSV, PDV, ACLSV and ApMV. 565 samples were tested by dot-blot and tissue-printing hybridization for the presence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). 48 samples were infected, 41 by PLMVd and 7 by HSVd. In addition, nested-PCR tests identified Plum bark necrosis and stem-pitting associated virus (PBNSPaV) in a few almond trees affected by stem pitting.     

Characterization of a new Apple dimple fruit viroid variant that causes yellow dimple fruit formation in ‘Fuji’ apple trees

A 303-nucleotide viroid was isolated from an apple tree (Malus × domestica, ‘Fuji’) cultivated in Japan. The viroid had 84.9% overall nucleotide sequence homology to Apple dimple fruit viroid (ADFVd), a member of Pospiviroidae, reported from Italy. This viroid differed from the Italian variant by 47 mutations (38 substitutions, six deletions and three insertions), and most of these mutations occurred on either side of the central conserved region. The leaves and branches of the infected trees did not have any disease symptoms, but the fruits were dimpled and yellow. The infected scions were top-grafted onto a healthy ‘Fuji’ apple tree, which tested positive for this viroid in a northern hybridization analysis, and yellow dimple fruits were produced in the second growing season. We propose that this viroid is a new variant of ADFVd and causes yellow dimple fruit formation in ‘Fuji’ apple trees.     

Survey on viroids infecting grapevine in Italy: identification and characterization of Australian grapevine viroid and Grapevine yellow speckle viroid 2

Five viroid species have been reported from grapevine. Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd-1) are distributed worldwide, whereas Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd) and Citrus exocortis viroid (CEVd) are found only sporadically. However, the presence of AGVd and GYSVd-2 in several countries, including China, Turkey and Tunisia, suggests a wider dissemination, possibly also in Europe, where AGVd has never been found and GYSVd-2 has been occasionally identified in Italy. Taking advantage of a multiplex RT-PCR assay recently developed for detecting simultaneously these five viroids, vines growing in Italy in commercial vineyards and germplasm collections were surveyed. Besides confirming the widespread presence of HSVd and GYSVd-1 in the field, GYSVd-2 and/or AGVd were identified in two grapevine table cultivars (Sultanina Bianca and Red Globe) from germplasm collections. Tests extended to vines cultivated in southern Italy confirmed the presence of both viroids, which were further characterized. No major sequence divergences between the AGVd and GYSVd-2 variants from Italy and those previously described from other countries were observed. Phylogenetic analysis supported the close relationships among AGVd variants from Italy, Tunisia and Australia. To our knowledge this is the first report of AGVd in Europe and the first molecular characterization of GYSVd-2 isolates from a European country.     

Molecular characterization of Chinese Hop stunt viroid isolates reveals a new phylogenetic group and possible cross transmission between grapevine and stone fruits

A survey for the incidence of Hop stunt viroid (HSVd) infection in China was conducted using dot-blot hybridization. Out of 553 tested samples, 127 samples of stone fruits (apricot, peach, plum and almond), grapevine and hop were positive for HSVd, giving a mean infection rate of 23?%. Phylogenetic analysis revealed that most of the HSVd variants isolated from stone fruits, grapevine and hop were clustered into known hop and plum groups. However, two grapevine variants, HSVd.g50 and HSVd.g57, could not be clustered into any known groups, indicating a previously unknown phylogenetic group of HSVd isolates. HSVd.g38 was the single grapevine variant that clustered with the plum group isolates, supporting cross transmission between grapevine and stone fruits and the heterogeneity of grapevine isolates.     

Simultaneous detection and genetic variability of stone fruit viroids in the Czech Republic

In this paper, we report a large-scale survey for the incidence of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) in stone fruit collections and commercial orchards in the Czech Republic. From the 645 samples analysed, PLMVd was detected in 80 (26.6%) of peaches and the HSVd in 3 (1.3%) of apricot and 1 (0.33%) of peach trees. Sixty-seven accession of peach (44.6%) from the Czech Clonal GeneBank were infected by PLMVd. In addition, we used naturally infected trees to standardise the simultaneous detection of PLMVd and HSVd plus host mRNA as the control by means of one-step multiplex RTC-PCR. Eleven PLMVd and two HSVd isolates were sequenced and analysed. All the PLMVd variants were highly homologous (97–100%) to previously reported PLMVd variants from Tunisian peach and almond trees, and clustered together in the previously reported phylogenetic group III. The HSVd variants obtained from apricot and peach trees were included in the previously proposed recombinant group PH/cit3.     

An important new apricot disease in Spain is associated with <Emphasis Type="Italic">Hop stunt viroid</Emphasis> infection

The province of Murcia (Murcia Region) is the one of the most important apricot growing regions in Europe. In recent years a fruit disorder named by growers as “degeneración” has been detected in apricot commercial orchards of this region, mainly in the variety Velázquez Fino. Affected fruits are characterized by changes in their external appearance involving rugosity and a loss of organoleptic characteristics, which leads to an unmarketable product. In order to identify the causal agent of this disorder, the presence of the most important viruses affecting stone fruit trees and Hop stunt viroid (HSVd) was tested. While negative results were obtained for all viruses analysed, the viroid was detected in all the symptomatic trees. Within a single tree, the viroid was restricted to branches bearing fruits with the characteristic symptoms but was usually absent from the rest of the tree. Sequence analysis of several isolates of HSVd obtained from these affected trees revealed the presence of two variants previously detected in other apricot cultivars. Taken together, these results suggest that degeneration is associated with HSVd.     

Studies on the diagnosis of hop stunt viroid in fruit trees: Identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents

A non-radioactive digoxigenin-labelled RNA probe specific for hop stunt viroid (HSVd) diagnosis has been developed. The high sensitivity and specificity of this RNA probe in dot blot hybridizations to nucleic acids from field samples, allowed the confirmation of the presence of HSVd in apricot (Prunus armeniaca L.) and its detection in two fruit tree species not previously described as hosts of this pathogen, almond (Prunus dulcis Miller) and pomegranate (Punica granatum L.). This result supports and extends the notion of the world wide distribution of HSVd, infecting cultivated fruit trees. HSVd was also found to accumulate to much higher levels in mature apricot fruits than in leaves. Additionally, a sample processing procedure which does not involve the use of organic solvents was demonstrated to render faithful results when used for viroid detection. The combined reliability and facility of use of both this extraction procedure and the non-radioactive probe will benefit agronomic investigations addressing the detection and eradication of HSVd. Other applications of the work described here, as the study of possible causal relations between specific disorders and HSVd infection, are also discussed.     

Molecular Characterization of an Almond Isolate of Hop Stunt Viroid (HSVd) and Conditions for Eliminating Spurious Hybridization in its Diagnosis in Almond Samples

Hop stunt viroid (HSVd) has a wide range of hosts including herbaceous and woody plants. Recently, HSVd was demonstrated to infect almond trees (Astruc et al., 1996). In this work, we present the molecular characterization of an almond HSVd isolate and report on the problems encountered in the diagnosis of HSVd in almond tissue through dot-blot non-radioactive hybridization procedures. False positives were eliminated through incubation of the membranes with RNase at high ionic strength after hybridization. Further experiments which included Northern hybridization, RT-PCR coupled to Southern hybridization, and cloning and sequencing suggested that spurious hybridization signals were due to host RNAs with sequence similarity to HSVd. Genetic characterization of the almond isolate demonstrated the existence of two new HSVd sequence variants named HSVd.alm1 and HSVd.alm2. The changes of HSVd.alm1 and HSVd.alm2 were located at residues variable among HSVd sequences, in loops on the left part of the rod-like molecule that includes the pathogenic (P) domain. Multiple alignments with all the available HSVd sequences and subsequent phylogenetic analyses revealed that the two new almond sequence variants were included in the previously described Prunus group of HSVd sequences.     

Molecular characterization and phylogenetic analysis of Citrus viroid I-LSS variants from citrus in Pakistan and China reveals their possible geographic origin

CVd-I-LSS (low sequence similarity), a variant of Citrus bent leaf viroid (CBLVd), was first discovered in Japan, and its distribution is currently limited to Japan and Iran. In the present study, seven CVd-I-LSS isolates were detected from different citrus hosts (Citrus sinensis, C. reticulata and C. limettioides) in Pakistan and China. Genetic diversity analysis of 49 cDNAs of CVd-I-LSS isolates showed that the Pakistan population was more diverse than that tested from Japan or China. Phylogenetic analysis clustered the predominant sequences examined into three main clades. Only sequences from the Pakistan isolates were found in all three clades, suggesting Pakistan may be the original source of CVd-I-LSS. Cultivar import records and the close phylogenetic relationship found between CVd-I-LSS from China and Japan suggested that the viroid isolated from China might have originated from Japan.