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.     

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.     

Indexing of Citrus Viroids by Imprint Hybridisation

A method based on the hybridisation of tissue imprints was developed for routine indexing of citrus viroids. For maximum sensitivity and reliability, the inoculation of Citrus medica (Etrog citron) as a viroid amplification host is required. Hybridisation against Digoxigenin-labelled RNA- or DNA-probes followed by detection of viroid-probe hybrids using anti-DIG-alkaline phosphate conjugate and the chemiluminescence substrate CSPD was suitable for the detection of all citrus viroids with the same sensitivity as other available methods. The overall process is extremely simple and allows quick analysis of large numbers of samples by easily trained personnel and minimum equipment.     

Goat horns: Platforms for viroid transmission to fruit trees?

Mechanical inoculations with contaminating tools and propagation of infected budwood were considered the main causes for the omnipresence of multiple viroid species among citrus and other Middle Eastern and Mediterranean fruit trees and grapevines. However, neither means could explain viroid infections of wild trees — scattered on terrains inaccessible to humans — nor the finding of similar viroids among graft-incompatible plants. Northern hybridization of RNA extracts made of scrapings from the surfaces of goat (Capra hircus) horns that were rubbed against etrog (Citrus medica) stems infected with a citrus viroids complex, revealed accumulation of considerable amounts ofCitrus exocortis viroids (CEVd) andHop stunt viroids (HSVd). Experimental transmission of both CEVd and HSVd was obtained by rubbing healthy citrus plants with goat horns that had been rubbed 24 h earlier on infected etrog stems. These results implicate goats as possible vectors of viroids. Transmissionvia goats could have facilitated the long-range spread of viroids among cultivated and wild plants andvice versa and also among graft-incompatible plants.     

应用多重PCR技术同步检测柑橘4种重要嫁接传播病原

 柑橘衰退病毒(Citrus tristeza virus,CTV),柑橘碎叶病毒(Citrus tatter\|leaf virus,CTLV),柑橘裂皮病类病毒(Citrus exocortis viroid,CEVd)和柑橘黄龙病(Huanglongbing, HLB)亚洲种病原(Candidatus liberobacter asiaticus)是重要的柑橘嫁接传播病原。本文建立了同时检测HLB病菌、CTV、CEVd 和CTLV 4种柑橘嫁接病原的一步法、双温多重PCR检测技术体系,同时在体系中设置内参基因。应用该体系快速评价了4种嫁接传播病原在田间侵染情况,结果表明28个田间样品CTV、CEVd、CTLV和HLB感染率分别为89.3 %、17.9 %、10.7 %和28.6 %,接近半数样品为混合感染。并且将该方法应用于快速评价茎尖嫁接苗病毒的脱除情况。     

Interactions between citrus viroids affect symptom expression and field performance of clementine trees grafted on trifoliate orange

ABSTRACT Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), a noncachexia variant of Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) were co-inoculated as two-, three-, four-, and five-viroid mixtures to Clementine trees grafted on trifoliate orange to evaluate their effect on symptom expression, tree growth, and fruit yield. Most trees infected with CEVd-containing viroid mixtures developed exocortis scaling symptoms, as did CEVd alone, whereas most trees infected with HSVd- or CVd-IV-containing mixtures developed bark-cracking symptoms. Trees infected with mixtures containing both CEVd and CVd-IV revealed the existence of antagonism between these two viroids in terms of the expected bark-scaling and cracking symptoms. Synergistic interactions also were identified in trees infected with certain viroid combinations that, in spite of lacking CEVd, expressed exocortis-like scaling symptoms. Viroid interactions also affected the expected response of trees in terms of vegetative growth and fruit yield. Trees infected with viroid combinations containing CEVd or CVd-III were smaller and produced less fruit than trees infected with mixtures not containing these viroids. Viroid interactions on scion circumference and cumulative fruit yield, in terms of additivity of their effects, were statistically confirmed using a factorial analysis of variance model with two mean estimation approaches. In single-viroid infections, CEVd, CVd-III, and, to a lesser extent, CBLVd consistently and significantly reduced tree size and fruit yield. Conversely, HSVd and CVd-IV slightly increased fruit yield and reduced scion circumference. Rare and not consistent significant interactions were detected with the five-, four-, and three-viroid combinations. Antagonistic interactions between CEVd and CVd-III or CBLVd and CVd-III were revealed over the years with consistent significance. The antagonistic interaction between CEVd and CVd-IV was highly significant over the years when additional viroids were present; however, this antagonism appeared much later in the case of an exclusive interaction. HSVd and CVd-IV showed a consistent and significant synergistic interaction on yield only when both viroids were exclusively present. These results demonstrate antagonistic or synergistic relationships between citrus viroids depending on the viroid mixtures present in the host.     

Rapid in vitro microindexing of viroids in citrus

A new laboratory technique combining shoot-tip grafting in vitro and biological indexing on indicator plants was explored for the detection of citrus exocortis and related viroids. Τhree in vitro laboratory methods were used and compared with the classical biological method. With the classical in vivo method, diagnosis is based on the expression of symptoms on indicators 11–14 weeks after inoculation. In contrast, with the first in vitro method, microindexing in vitro of citron seedlings by graft inoculation, diagnosis was possible 12 days after inoculation; with the second method, microindexing in vitro of citron cuttings by graft inoculation, 20 days after inoculation; and with the third method, microindexing in vitro of citron cuttings by injection inoculation, 40 days after inoculation. Inoculated Etrog citron plantlets grown in vitro and tested by RT-PCR showed the same viroid content as the source plants. Of the three in vitro viroid indexing methods, microindexing on cuttings by grafting was easier and more reliable than microindexing either on seedlings or on cuttings by injection.     

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).     

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.     

<Emphasis Type="Italic">Citrus exocortis viroid</Emphasis> transmission through commercially-distributed seeds of <Emphasis Type="Italic">Impatiens</Emphasis> and <Emphasis Type="Italic">Verbena</Emphasis> plants

Surveys of Impatiens and Verbena species in local nurseries in Fredericton, Canada and Verbena species in New Delhi, India showed widespread infection of Citrus exocortis viroid (CEVd) in vegetatively-propagated and seed-grown plants. To determine viroid seed transmission, samples of eight varieties of Impatiens and 11 varieties of Verbena were obtained from four commercial sources. All 19 samples collected contained viroid infection irrespective of variety. The presence of viroid in non-germinated seed was 21%, while the transmission rate in seedlings was 66% in Impatiens walleriana in 2006. Following 2 years of seed storage, the respective figures were 6% and 26%. Similarly, in Verbena x hybrida the presence of viroid in seed was 13% in 2006 with a seed-transmission rate in seedlings of 28%, while the respective figures after 2 years of storage were 5% and 45%.     

柑桔裂皮病类病毒分子生物学研究进展

本文就柑桔裂皮病类病毒(citrus exocortis viroid,CEVd)的分类地位,分子结构及其生物学鉴定、聚丙烯酰胺凝胶电泳分析和分子杂交、分子生物学检测等方法进行了综述。     

A combined indexing and budwood propagation method for citrus

Several budwood-transmitted citrus diseases, including citrus tristeza virus, citrus psorosis, citrus impietratura and a range of citrus viroids, were tested both visually and biochemically on a combined indicator (CInd) plant consisting of an Alemow (Citrus macrophylla) rootstock grafted with Etrog citron (C.medica) and Sour orange (C.aurantium) or Sweet orange (C.sinensis) buds. Indexing on CInd plants is economical for limited testing space; an additional advantage is that, by collecting budwood directly from the CInd plants, the risk of diagnostic failure due to uneven pathogen distribution in the budwood source tree is considerably reduced.     

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.     

Separation of citrus viroids by shoot-tip grafting in vitro

Arizona 861-S1 citron ( Citrus media L.) infected with a severe exocortis isolate containing four citrus viroids was used as source of tissue for shoot-tip grafting in vitro. Out of 51 attempts, 25 successful grafts were obtained. Only 16 plants survived transplanting and of these 12 were viroid-free. The viroid profile of the other four plants showed fewer viroids than the original field source. The significance of these results, as compared with previous reports on the recovery of viroid-free plants, is discussed. The results show the usefulness of shoot-tip grafting in vitro as a tool to recover single viroid isolates from complex field sources.     

Host range and properties of <Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis>

We characterised the host range and physical properties of Tomato chlorotic dwarf viroid. Among the 46 plant species inoculated with the viroid, two in the family Compositae and 23 in the family Solanaceae were found to be systemic hosts. The viroids in the crude sap from diseased tomato plants were thermally inactivated by heating to 100°C for at least 40 min. These viroids also lost their infectivity when diluted in phosphate buffer to at least 10⁻⁶, or after 3 days of incubation at room temperature. However, the infectivity of the viroids in dried crude sap from the plants persisted throughout the 50-day test period.