Segregation of a viroid complex from a graft-transmissible dwarfing agent source for grapefruit trees

A marked variation in tree size occurred in grapefruit trees budded in trifoliate orange rootstock and inoculated with a graft-transmissible dwarfing agent (GTD) from grapefruit. Etrog citron (Citrus medico) inoculated with budwood collected from two GTD sub-types displaying very mild (VM) and mild (M) dwarfing effects showed only mild leaf epinasty, whereas the severe (S) subtype induced severe epinasty and stunting typical of infection by the original GTD source 225-T. The characteristic symptoms of the three sub-types were persistent following three serial transfers to 'Etrog' indicators.
Extracts from 'Etrog' citron containing either the 225-T or its derivative S displayed a profile of five viroid bands when analysed by the sequential PAGE system for detection of circular RNAs. The profile obtained included the well characterised 371 nucleotide citrus exocortis viroid (CEV) and four additional viroids of approximately 330, 300, 295 and 275 nucleotides. Sub-types M and VM lacked the CEV band and each contained a complement of only three viroids of 330, 295, 275 and 300, 295, 275 nucleotides, respectively. These results indicate that a segregation of the viroid complex in grapefruit budwood was a major factor in the variation seen among trees inoculated from the GTD source 225-T.     

Microarray analysis of Etrog citron (Citrus medica L.) reveals changes in chloroplast, cell wall, peroxidase and symporter activities in response to viroid infection

Viroids are small (246-401 nucleotides), single-stranded, circular RNA molecules that infect several crop plants and can cause diseases of economic importance. Citrus are the hosts in which the largest number of viroids have been identified. Citrus exocortis viroid (CEVd), the causal agent of citrus exocortis disease, induces considerable losses in citrus crops. Changes in the gene expression profile during the early (pre-symptomatic) and late (post-symptomatic) stages of Etrog citron infected with CEVd were investigated using a citrus cDNA microarray. MaSigPro analysis was performed and, on the basis of gene expression profiles as a function of the time after infection, the differentially expressed genes were classified into five clusters. FatiScan analysis revealed significant enrichment of functional categories for each cluster, indicating that viroid infection triggers important changes in chloroplast, cell wall, peroxidase and symporter activities.     

Variations in the "cross protection" effect between two strains of citrus exocortis viroid

In a survey conducted to evaluate the biological properties of several field sources that induced a severe exocortis reaction on citron (Citrus medica L.), a viroid isolate which induced mild symptoms on Gynura aurantiaca was detected. This isolate was characterised as a strain of the citrus exocortis viroid (CEV) by size and homology, and was designated as CEV-129. Cross protection assays using CEV-129 as a “protecting” strain against the severe type strain of CEV demonstrated that a mild strain of CEV could provide apparent ‘protection’ against challenge inoculations with the severe strain. The protection effect, however, displayed a variability which ranged from only a brief delay to almost total impairment of symptom expression. The level of protection was dependent upon the length of the interval between the inoculations with the mild and the severe strains. In all cases the effect was temporary since the symptoms and viroid concentration which ultimately prevailed reflected the predominance of the severe strain in the mixed infection. The interpretation of these results and their relationship with previous reports of ‘cross protection’ reactions are discussed along with the consideration of the efficacy and limitations of this term with viroid infection.     

Two nucleotide positions in the Citrus exocortis viroid RNA associated with symptom expression in Etrog citron but not in experimental herbaceous hosts

Citrus exocortis viroid (CEVd) is the causal agent of exocortis disease of citrus. CEVd has a wide host range that includes woody and herbaceous species. A new CEVd strain (CEVd(COL)), phylogenetically clustering with CEVd variants of Class A inducing severe symptoms in tomato, was identified in Colombia and shown to induce only extremely mild symptoms in Etrog citron indicator plants. Using site-directed mutagenesis, two nucleotide substitutions (314A → G and 315U → A) in the lower strand of the P domain of the predicted CEVd(COL) secondary structure resulted in a severe artificial CEVd(MCOL) variant. Conversely, two nucleotide exchanges (314G → A and 315A → U) in the same region of the severe variant CEVd(E-117) resulted in a symptomless artificial CEVd(ME-117) variant. Infectivity assays conducted with the natural and mutated variants showed that all induced severe symptoms in Gynura aurantiaca, tomato and chrysanthemum. This is the first report of the identification of pathogenic determinants of CEVd in citrus, and shows that these pathogenicity determinants are host dependent.     

The movement and distribution of citrus tristeza virus and citrus exocortis viroid in citrus seedlings1

The systemic movement of citrus tristeza virus (CTV) in sour orange (Citrus aurantium) seedlings and of citrus exocortis viroid (CEVd) in Etrog citron (C. medica) seedlings was studied. The movement of the two pathogens was analysed by detection in sections of roots and stems at different time intervals. Both pathogens were detected initially in the basal parts and the roots and subsequently spread to the shoot. CTV and CEVd moved in young citrus seedlings at similar rates. The findings are consistent with long distance phloem transport of the virus and the viroid. The practical implications of the pattern of systemic movement for diagnosis of infected trees are discussed.     

Intra-population diversity between citrus viroid II variants described as agents of cachexia disease

The citrus viroid II (CVd‐II, Hop stunt viroid) variant, cachexia 909 (Ca‐909) has been designated as a “cachexia” disease isolate on the basis of inducing extremely mild symptoms on the cachexia indexing host Parson's Special mandarin (PSM). However, Ca‐909 lacks the six‐nucleotide cluster demonstrated to control the pathogenicity of cachexia inducing agents such as CVd‐IIc. Progeny populations of CVd‐IIc and Ca‐909 from the bioamplification host, Etrog citron, and the indexing host PSM were surveyed for clones with possible mutations in the locus of the “cachexia cluster”. The intra‐population diversity and the genealogical relationships among clones of CVd‐IIc and Ca‐909 populations were also analysed using principles of the coalescent theory. CVd‐IIc progeny was found not to mutate in the “cachexia cluster” and Ca‐909 did not acquire any mutations related to the nucleotide sites of the “cachexia cluster”. Specific mutations of the Ca‐909 progeny were found to be similar to the non‐cachexia variant, CVd‐IIa. Population profiles and genealogical patterns of CVd‐IIc and Ca‐909 in Etrog citron were not significantly different. However, although CVd‐IIc progeny were more conserved in PSM, Ca‐909 progeny displayed titre, population profiles, and genealogical patterns more uniform in selected tissues of Parson's Special mandarin than CVd‐IIc. These experimental approaches demonstrate the genetic stability of the cachexia‐agent CVd‐IIc and question the inclusion of Ca‐909 as a cachexia disease agent.     

Comparative oligonucleotide fingerprints of three plant viroids.

5' Phosphorylation in vitro with gamma-32P-ATP and T4 phage induced polynucleotide kinase was used to obtain RNAase A and RNAase T1 fingerprints of three plant viroids: Potato spindle tuber viroid from tomato (PSTV-tom), chrysanthemum stunt viroid from cineraria (ChSV-cin) and citrus exocortis viroid from Gynura aurantiaca (CEV-gyn). These three viroids differ significantly from each other as judged from their oligonucleotide patterns. This supports the concept of individual viroid species.     

Tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy

Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.     

The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid.

The primary structure of a grapevine viroid (GVs) isolated in Spain was determined. The sequence consisted of 369 nucleotide residues forming a circular molecule. GVs presented extensive homology with viroids of the potato spindle tuber viroid (PSTV) group, that was specially high in the case of citrus exocortis viroid (CEV) both with variants found in isolates inducing severe (92% with CEV-A) and mild (89% with CEV-DE26) symptoms on tomato. The secondary structure proposed for GVs showed that the changes in the sequence in relation to CEV-A generated modifications of the secondary structure particularly important in the left terminal (Tl), variable (V) and pathogenesis (P) viroid domains that have been postulated. Nevertheless it was noted in GVs a central core in the P domain that is conserved in the class A sequence variants characteristic of severe isolates, but not in the class B ones found in mild isolates of CEV. These observations indicate that GVs should be considered as a severe isolate of CEV from grapevine (CEV-g), a suggestion that correlates with the biological properties of CEV-g both in tomato and in Gynura aurantiaca. The presence of this central core in the P domain seems to characterize all the variants of CEV inducing severe symptoms in tomato.     

Australian grapevine viroid--evidence for extensive recombination between viroids.

Australian grapevine viroid (AGV, 369 residues) is a novel viroid with less than 50% sequence similarity with any known viroid. Nevertheless its entire sequence can be divided into regions, each with a high sequence similarity with segments from one of citrus exocortis, potato spindle tuber, apple scar skin, and grapevine yellow speckle viroids. AGV contains the entire central conserved region of the apple scar skin viroid group and is proposed as a member of this group. AGV appears to have originated from extensive RNA recombination involving other viroids. The vegetatively propagated grapevines which have been exposed to multiple viroid infections during their long history of cultivation may have allowed such recombination.     

An artificial chimeric derivative of Citrus viroid V involves the terminal left domain in pathogenicity

The recently described Citrus viroid V (CVd-V) induces, in Etrog citron, mild stunting and very small necrotic lesions and cracks, sometimes filled with gum. As Etrog citron plants co-infected with Citrus dwarfing viroid (CDVd) and CVd-V show synergistic interactions, these host–viroid combinations provide a convenient model to identify the pathogenicity determinant(s). The biological effects of replacing limited portions of the rod-like structure of CVd-V with the corresponding portions of CDVd are reported. Chimeric constructs were synthesized using a novel polymerase chain reaction-based approach, much more flexible than those based on restriction enzymes used in previous studies. Of the seven chimeras (Ch) tested, only one (Ch5) proved to be infectious. Plants infected with Ch5 showed no symptoms and, although this novel chimera was able to replicate to relatively high titres in singly infected plants, it was rapidly displaced by either CVd-V or CDVd in doubly infected plants. The results demonstrate that direct interaction(s) between structural elements in the viroid RNA (in this case, the terminal left domain) and as yet unidentified host factors play an important role in modulating viroid pathogenicity. This is the first pathogenic determinant mapped in species of the genus Apscaviroid .     

Fatal Yellowing of Oil Palms: Search for Viroids and Double-Stranded RNA

Fatal yellowing is a serious disease of still unknown origin affecting oil palms in several regions of Central and South America. In this study a search for viroids and viroid-like RNAs in oil palms was performed using two-dimensional gel electrophoresis and return gel electrophoresis of nucleic acid extracts. Although RNAs showing viroid-like gel-electrophoretic properties were detected, the presence of the known viroids was excluded by hybridization experiments using probes specific for potato spindle tuber viroid (PSTVd), coconut cadang-cadang viroid (CCCVd), or Coleus blumei viroid 1 (CbVd1). By using double-stranded RNA (dsRNA) specific monoclonal antibodies, which do not react with viroid RNA, we were able to show that oil palm RNAs, migrating like viroids are double-stranded RNA species. Since the same dsRNA pattern was found in extracts from diseased as well as from healthy oil palms, the dsRNAs can neither be part of the causative agent of fatal yellowing, nor are they associated with the disease. Their possible origin is discussed. In addition to the standard electrophoretic methods, which have been used for identification of viroids and viroid-like RNAs, we describe additional control experiments to differentiate unequivocally between circular single stranded and linear dsRNA.     

Some properties of the viroid inducing peach latent mosaic disease

Analysis by polyacrylamide gel electrophoresis of nucleic acid extracts from different peach samples, healthy or infected with the peach latent mosaic (PLM) disease, demonstrated the association of this disease with an RNA exhibiting the electrophoretic properties typical of circular viroid molecules. This RNA was called peach latent mosaic viroid (PLMV), since a purified preparation of it, when inoculated into GF 305 peach seedlings induced characteristic symptoms of PLM disease. PLMV was estimated to have a molecular size in the range of 330–340 bases, by comparison of its electrophoretic mobility under denaturing conditions with those of several viroid RNA. Dot-blot analysis showed that PLMV has a sequence clearly different from other viroids, including citrus exocortis viroid, apple scar skin viroid (ASSV), hop stunt viroid (HSV) and avocado sunblotch viroid. The possible significance of the limited sequence homology shared by PLMV with HSV, and especially with ASSV, is discussed.     

Molecular,Biological and Phylogenetic Analysis of Chinese Isolates of Hop stunt viroid Associated with Citrus Cachexia Disease

Citrus cachexia is an economically important disease of citrus hosts caused by specific variants of Hop stunt viroid (HSVd) that are usually referred to as Citrus cachexia viroid (CCaVd). Eight cachexia‐associated HSVd isolates were collected from six citrus growing areas of China, where citrus cachexia had not been reported previously. Forty‐seven independent cDNA clones were used for genetic diversity and phylogenetic analysis. There were no sequence variant‐cultivar correlation and no distinct regional specificity among or within the cachexia‐associated HSVd populations analysed. Three clusters consisting of three major HSVd variants were identified by phylogenetic analysis, suggesting that most Chinese isolates contain a mixture of cachexia and non‐cachexia variants. Biological properties of eight CCaVd isolates were determined by inoculating Parson’s Special mandarin (Citrus reticulata). Our results suggest that the interactions between CCaVd and non‐CCaVd variants might play an important role in suppressing cachexia symptom expression.     

Some properties of chrysanthemum stunt, a virus with the characteristics of an uncoated ribonucleic acid

Grafting to virus-free Mistletoe chrysanthemums was the most reliable method of detecting the chrysanthemum stunt agent, but specific light and temperature conditions were required for the diagnostic ‘measles’ symptoms to develop. Although stunt agent was highly infectious, leaf-rubbing inoculations with chrysanthemum sap gave erratic results. Colorimetric and electrophoretic tests were unreliable for indexing chrysanthemums. Stunt agent infected eight of twenty-nine species in the family Compositae, but none of 116 species in forty-seven other families. Stunt spread rapidly by foliage contact and by handling plants, but dipping the hands in 2% trisodium orthophosphate when handling plants increased the amount of spread. Stunt agent was not transmitted by four species of aphids, the glasshouse redspider mite, dodder (Cuscuta campestris) or through chrysanthemum seed. Stunt agent withstood 10 min at c. 98 °C and dilution to 10^-4, was not pelleted by ultracentrifugation, and was inactivated by RNase in weak, but not strong, buffer, suggestive of an uncoated RNA ‘viroid’. Partially purified preparations were made by homogenizing frozen chrysanthemum leaves in 0.5 m phosphate buffer with antioxidant at c. 2 °C, and clarification by n-but-anol and chloroform, followed by centrifugation. Highly infective RNA was precipitated from the supernant fluid by 2.5 vol. cold ethanol, and resuspended in a small volume of buffer. The u.v. absorption spectra of infective preparations and the u.v. absorbance profiles of density-gradients, were very similar to those of preparations from healthy chrysanthemum. Infective partially purified preparations of stunt agent withstood exposure to 2% formaldehyde or tri-sodium orthophosphate, u.v. irradiation, and sonication. Stunt preparations contained no virus particles recognizable by electron microscopy, gave no distinct peak on analytical ultracentrifugation, and did not consistently contain any specific antigen. Although similar to the ‘viroids’ potato spindle tuber and citrus exocortis, stunt agent did not infect Citrus limon, Gynura aurantiaca or tomato.     

利用RT—PCR扩增和分析柑桔裂皮病类病毒

参考国外CEVd-A株中央保守区段(C区)和左端区段(T区),设计并合成引物Cl(-)、C2( )、C3( )、T1(-)、T2( )。对感染 CEVd中国分离物的柑桔(Citrus L.)和香橼(Citrus medica L.)总核酸进行了cDNA第一链合成和PCR扩增,其中C1/C3、C1/C2分别能从感病香橼和柑桔总核酸中扩增出210bp和370bp左右的特异DNA,分别相当于CEVd的左半部和全长片段,T1/T2未能扩增出产物;健康香橼和柑桔总核酸中均未能扩增出产物。扩增结果用DIG标记的CEVd-cDNA探针进行了确证。扩增结果说明:CEVd中国分离物在左端T区与CEVd-A株存在差异。PAGE-银染法分析扩增产物表明:建立的RT-PCR方法可从约0.1ng柑桔总核酸中扩增出全长CEVd-cDNA。     

Water relations of citrus exocortis viroid-infected grapefruit trees in the field

Citrus viroids (CVds) were used to limit citrus tree size ('dwarfing'). It is hypothesized that changes also occur in the hydraulic properties of the conducting system, thereby affecting water relations. Grapefruit trees (var. Star Ruby on Troyer Citrange rootstock) in northern Israel were infected with citrus exocortis viroid (CEVd). The orchard was drip irrigated. The infected (I) and apparently healthy (H) control plots were subdivided into wet (W) and dry (D) irrigation treatments receiving 100% and 70% of the recommended irrigation quantity for the region. Soil water content, soil temperature, water uptake, leaf and stem water potential, and leaf conductance were measured in addition to climatic variables. Water uptake response to increasing spring-time soil temperatures was greater in healthy than in infected trees. Leaf conductance in the infected trees was lower than the non-infected trees only in the sixth year after inoculation. Stem water potential was significantly lower in the CEVd-infected trees than in the control trees. Water uptake was lower and hydraulic resistance higher in infected than in healthy trees. It was concluded that CEVd causes a reduction in the water uptake ability of the root and canopy system. Possible mechanisms for this are discussed.