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.     

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.     

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 .     

Phospholipids, free sterols and adenosine triphosphatase of plasma membrane-enriched preparations from roots of citrus genotypes differing in chloride exclusion ability

Plasma membrane-enriched preparations from fibrous roots of three citrus genotypes differing in their abilities for chloride exclusion, and grown in the presence of 0,50 or 100 mM NaCl for 4 weeks, were analysed for phospholipid and free sterol content and vanadate-sensitive adenosine triphosphatase (ATPase) activity over a range of temperatures. The best chloride excluder, Rangpur lime (Citrus reticulaia Blanco var. austera hyb.?), had significantly higher maximal ATPase activity than both the moderate chloride excluder. Kharna khatta (Citrus kharna Raf.), and the worst chloride excluder, Etrog citron (Citrus medico L.), at all assay temperatures below 28°C. Salt treatment had no effect on maximal ATPase activity of either Rangpur lime or Etrog citron but resulted in increased activity of the enzyme in Kharna khatta at temperatures below 28°C. Arrhenius plots of ATPase activity from the three citrus genotypes showed that, in controls, the activation energy (E.,) of Rangpur lime ATPase was significantly lower than that of both Kharna khatta and Etrog citron. The thermotropic phase transition temperature (T_f) for Rangpur lime (27°C) was also lower than for the other citrus genotypes (31°C). Salt treatment resulted in increases in both Ea and T, for Rangpur lime, decreases in both parameters for Kharna khatta and no change of either parameter for Etrog citron. An inverse relationship between E_a and the phospholipid to free sterol ratio was evident for plasma membrane preparations from all three citrus genotypes in the presence and absence of salt treatment suggesting that changes in membrane fluidity, particularly those induced by free sterols, have the potential to influence active as well as passive ion transport processes and thus may play a significant role in the chloride exclusion mechanism.     

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.     

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.     

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.     

4-Desmethylsterol composition of citrus rootstocks of different salt exclusion capacity

Fibrous roots from seedlings of three citrus rootstocks (Rungpur lime, Kharna khatta and Etrog citron) grown hydroponically for 6 weeks in the presence or absence of 50 mM NaCl were analysed for their content of free, esterified, and glycosidic sterols. Leaf chloride analyses indicated that Rangpur lime was a good Cl- excluder and the other two rootstocks were Cl- accumulators.
On a dry weight basis and in the absence of NaCI only the free 4-desmethyIsterol levels showed significant rootstock differences. Kharna khatta had the highest, and Rangpur lime the lowest, free stcrol levels. Sitostcrol was the major component of all sterol fractions of Rangpur lime, the esterified fraction of the other rootstocks, and the glycosidic sterol fraction of Kharna khatta. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron in all cases and the ratio of apos;more-planar apos; to apos;less-planar apos; free sterols was highest in Etrog citron and lowest in Rangpur lime.
Treatment with 50 mM NaCI resulted in an increase in free sterol levels in Rangpur lime and a decrease in Kharna khatta. Steryl ester levels were unaffected in Rangpur lime but were significantly reduced in the other rootstocks. In all three sterol fractions the sitosterol/stigmasterol ratio was decreased. A decrease in the ratio of apos;more-planarapos; to apos;less-planarapos; free sterols was observed only in Kharna khatta and, more notably, Etrog citron. Salt-induced changes in the apos;more-planar apos; to apos;less-planar apos; free sterol ratio correlated well with salt exclusion capacity.     

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.     

NaCl effects on 4-desmethylsterol composition of plasma-membrane-enriched preparations from citrus roots

Abstract The free 4-desmethylsterol composition of plasma-membrane-enriched preparations from white fibrous roots of Rangpur lime (Citrus reticulata var. austera hybrid?), Kharna khatta (C. kharna Raf.) and Etrog citron (C. medica L.) seedlings grown in the presence of 0, 50, or 100 mol m^?3 NaCl for 28 d was quantitated by gas chromatography (GC) on analytical capillary (SE-54 fused silica) columns and the sterols were identified by combined gas chromatography-mass spectrometry (GC-MS). Only three 4-desmethylsterols were positively identified by GC-MS, viz. campesterol, stigmasterol and sitosterol. Cholesterol could not be positively identified in any of the membrane preparations. Campesterol levels were generally similar for all treatments and for all three genotypes, approximating 30% of the total free 4-desmethylsterol content of the plasma membranes. At all levels of salinity (0, 50 or 100 mol m^?3 NaCl) sitosterol levels decreased in the order Rangpur lime > Kharna khatta > Etrog citron and stigmasterol levels decreased in the reverse order. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron at each level of salinity and was reduced by salt treatment in all three genotypes. Salt-induced reductions in the ratio of ‘more planar’ to ‘less planar’ sterols correlated inversely with the accumulation of Cl^? in the leaves of the three genotypes suggesting a role for plasma membrane sterols in the Cl^? exclusion mechanism. A model relating sterol structure, membrane sterol composition and membrane permeability to Cl^? exclusion ability in citrus is presented.     

Influence of virus and virus-like agents on the development of citrus buds cultured in vitro

Tissue culture in vitro was used to determine the effect of six major citrus virus and virus-like agents. Nodal stem segments from inoculated Pineapple sweet orange (Citrus sinensis (L.) Osb.), Mexican lime (C. aurantifolia (Christm.) Swing.) and Arizona Etrog citron 861-Sl (C. medica L.) were cultured in vitro to induce shoots. Some virus and virus-like agents had a marked effect on bud development and further recovery of plantlets. The number and size of the shoots that developed from each bud were affected as a result of infection. The effect depended on the specific virus, the isolate and the host-disease combination. The possible implications of these results are discussed.     

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.     

Interference between viroids inducing exocortis and cachexia diseases of citrus

Systemic interactions of long duration among the Group I] Citrus Viroids, CVd-IIa and CVd-IIb were investigated by grafting buds from established citron (Citrus medica L.) sources containing single viroids to a common healthy seedling. In healthy tissues, the two viroids became established in approximately equal titres as a mixed infection. By contrast, tissues that grew from the CVd-IIa or CVd-IIb source buds contained only trace amounts of the heterologous invading viroids. This interference between CVd-IIa, a mild exocortis agent, and CVd-IIb, the cachexia agent, was also demonstrated in the presence of CEVd, the severe exocortis agent but not a Group II viroid. When a CVd-IIb bud was propagated on a citron containing CVd-IIa, a dramatic reduction in the titre of CVd-IIb was observed. The interference between CVd-IIa and CVd-IIb indicates that the mild and relatively innocuous isolate, CVd-IIa, can interfere with the replication and/or accumulation of the severe cachexia agent, CVd-IIb, in citrus. This offers a potential practical approach for the control of cachexia in commercial plantings by “viroid interference”.     

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。     

Genomic Structure of Three Phenotypically Different Isolates of Peach Latent Mosaic Viroid: Implications of the Existence of Constraints Limiting the Heterogeneity of Viroid Quasispecies

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.     

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.     

Genetic structure and diversity of natural and domesticated populations of Citrus medica L. in the Eastern Himalayan region of Northeast India

Citron (Citrus medica L.) is a medicinally important species of citrus native to India and occurs in natural forests and home gardens in the foothills of the eastern Himalayan region of northeast India. The wild populations of citron in the region have undergone rapid decline due to natural and anthropogenic disturbances and most of the remaining individuals of citron are found in fragmented natural forests and home gardens in the region. In order to assess the genetic structure and diversity of citron in wild and domesticated populations, we analyzed 219 individuals of C. medica collected from four wild and eight domesticated populations using microsatellite markers. The genetic analysis based on five polymorphic microsatellite loci revealed an average of 13.40 allele per locus. The mean observed and expected heterozygosity values ranged between 0.220–0.540 and 0.438–0.733 respectively among the wild and domesticated populations. Domesticated populations showed close genetic relationships as compared to wild populations and pairwise Nei's genetic distance ranged from 0.062 to 2.091 among wild and domesticated populations. Analysis of molecular variance (AMOVA) showed higher genetic diversity among‐ than within populations. The analysis of population structure revealed five groups. Mixed ancestry of few individuals of different populations revealed exchange of genetic materials among farmers in the region. Citron populations in the region show high genetic variation. The knowledge gained through this study is invaluable for devising genetically sound strategies for conservation of citron genetic resources in the region.