Citrus Viroids: Symptom Expression and Performance of Washington Navel Sweet Orange Trees Grafted on Carrizo Citrange.

Citrus are natural hosts of several viroid species. Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are the causal agents of two well-known diseases of citrus, exocortis and cachexia. Other viroids have been found to induce specific symptoms and different degrees of stunting in trees grafted on trifoliate orange and trifoliate orange hybrids. A field assay was initiated in 1989 to establish the effect of CEVd, HSVd, Citrus bent leaf viroid (CBLVd), Citrus dwarfing viroid (CDVd), and Citrus bark cracking viroid (CBCVd) on Washington navel sweet orange trees grafted on Carrizo citrange rootstock. Here we report the effect of viroid infection on symptom expression, tree size, fruit production and quality evaluated from 2004 to 2007. Vegetative growth was affected by viroid infection with height and canopy volume being reduced. No bark scaling symptoms were observed in CEVd-infected trees albeit they presented lesions and blisters in the roots. Bark cracking symptoms were consistently observed in CBCVd-infected trees that were smaller with enhanced productivity and fruit size. No major effects were found as a result of infection with CBLVd, HSVd, or CDVd. The quality of the fruits was not affected by viroid infection, except for the low diameter of the fruits harvested from HSVd-infected trees. An interesting effect was identified in terms of tree productivity increase (yield/canopy volume) as a result of infection with CEVd, CDVd, and especially CBCVd.

First Report of Citrus viroid V in Moro Blood Sweet Orange in Iran.

Viroids are nonencapsidated, small, circular, single-stranded RNAs that replicate autonomously when inoculated in their host plants in which they may elicit diseases (sensitive hosts) or replicate as latent infections (tolerant hosts). Citrus viroid V (CVd-V) was initially identified in Spain (1) and later found to be present in the United States, Nepal, and the Sultanate of Oman (2). CVd-V is a member of the Apscaviroid genus within the Pospiviroidae family. Like other members of this genus, CVd-V has a restricted host range but it is able to infect a wide range of citrus and citrus related species (1,2). Within the framework of a comprehensive survey of the sanitary status of the citrus industry in Iran, a sample from a private orchard of symptomless Moro blood sweet orange (Citrus sinensis) trees grafted on Mexican lime (C. aurantifolia) located at Javanan in the southern inland region was found to be infected with CVd-V. Briefly, RNAs of nucleic acid preparations from bark tissues were separated by 5% polyacrylamide gel electrophoresis (PAGE), electrotransferred to positively charged nylon membranes, immobilized by UV cross-linking, and hybridized with a full length CVd-V specific digoxigenin (DIG)-labeled DNA probe (2). A positive identification of CVd-V was made in these extracts. This positive detection of CVd-V was confirmed by reverse transcription-PCR using CVd-V specific primers of opposite polarity (5'-GACGAAGGCCGGTGAGCAGTAAGCC-3') and (5'-GACGACGACAGGTGAGTACTTTC-3') corresponding to CVd-V positions 90 to 114 and 69 to 89, respectively. Analysis of the sequence of the 293-bp amplicon (Genbank Accession No. GQ466068) revealed 99% identity with the reference sequence (Genbank Accession No. NC010165) of CVd-V. The rod-like predicted minimum free energy secondary structure of this new variant has 68.3% paired nucleotides. The changes with respect to the reference CVd-V variant are: (i) a deletion (48→-U) located in a loop of the V domain; (ii) a substitution (155A→C) located in a loop of the TR domain of the viroid secondary structure; and (iii) two compensatory substitutions located in the upper (46A→G) and lower (244U→C) strands of the viroid secondary structure. As shown earlier, the genome of CVd-V allows little variation with a large loop located in the segment I of the secondary structure (2) being the most amenable for mutations/changes. Among the viroids that have been found naturally infecting citrus, the members of the genus Apscaviroid are not associated with specific diseases but they cause a reduction of tree size and fruit harvest (3), an effect that is enhanced when several viroids coinfect the same plant (4). Therefore, the presence of CVd-V should be considered in further indexing tests aimed at the production and distribution of pathogen-free plants in Iran. References: (1) P. Serra et al. Virology 370:102, 2008. (2) P. Serra et al. Phytopathology 98:1199, 2008. (3) C. Vernière et al. Plant Dis. 88:1189, 2004. (4) C. Vernière et al. Phytopathology 96:356, 2006.

A novel hybridization approach for detection of citrus viroids.

Citrus plants are natural hosts of several viroid species all belonging to the family Pospiviroidae. Previous attempts to detect viroids from field-grown species and cultivars yielded erratic results unless analyses were performed using Etrog citron a secondary bio-amplification host. To overcome the use of Etrog citron a number of RT-PCR approaches have been proposed with different degrees of success. Here we report the suitability of an easy to handle northern hybridization protocol for viroid detection of samples collected from field-grown citrus species and cultivars. The protocol involves: (i) Nucleic acid preparations from bark tissue samples collected from field-grown trees regardless of the growing season and storage conditions; (ii) Separation in 5% PAGE or 1% agarose, blotting to membrane and fixing; (iii) Hybridization with viroid-specific DIG-labelled probes and detection with anti-DIG-alkaline phosphatase conjugate and autoradiography with the CSPD substrate. The method has been tested with viroid-infected trees of sweet orange, lemon, mandarin, grapefruit, sour orange, Swingle citrumello, Tahiti lime and Mexican lime. This novel hybridization approach is extremely sensitive, easy to handle and shortens the time needed for reliable viroid indexing tests. The suitability of PCR generated DIG-labelled probes and the sensitivity achieved when the samples are separated and blotted from non-denaturing gels are discussed.

Effect of a Field-Source Mixture of Citrus Viroids on the Performance of 'Nules' Clementine and 'Navelina' Sweet Orange Trees Grafted on Carrizo Citrange.

A field-source mixture of citrus viroids was characterized and shown to contain Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), Citrus bent leaf viroid (CBLVd), and Citrus dwarfing viroid (CDVd). Sequencing results showed that: (i) CEVd contained the PL and PR characteristic of class A variants; (ii) HSVd was a noncachexia variant; (iii) CBLVd was related to CVd-Ia variants; (iv) CDVd was a mixture of two types (CVd-IIIa and CVd-IIIb) of variants. The presence of the same type of variants in inoculated clementine (Citrus clementina 'Nules') and sweet orange (C. sinensis 'Navelina') trees on Carrizo citrange (Poncirus trifoliata × C. sinensis) rootstocks was confirmed. The effect of infection was determined by assessing the performance of infected and noninfected trees growing in the field. Infection resulted in small trees with reduced canopy, yielding a reduced crop. Fruit characteristics were also affected: (i) clementine and sweet orange fruits from infected trees were larger than those from noninfected trees; (ii) clementine fruits from infected trees differed in shape from those of noninfected trees; (iii) sweet orange fruits from infected trees had maturity indexes and juice contents higher than those from noninfected trees; (iv) in both species, the density of the juice, the amount of soluble solids, and the acidity of the fruits from infected trees were lower than those of fruits from noninfected trees. Infected trees had a poorly developed root system with fibrous roots containing fewer amyloplasts than noninfected trees. The results of an in vitro assay on the induction and development of roots in cultured explants are discussed.

A single nucleotide change in Hop stunt viroid modulates citrus cachexia symptoms.

Cachexia disease of citrus is caused by Hop stunt viroid (HSVd). In citrus, pathogenic and non-pathogenic strains differ by a "cachexia expression motif" of five to six nucleotides located in the variable domain of the proposed rod-like secondary structure. Here, site-directed mutants were generated to investigate if all these nucleotides were required for infectivity and/or symptom expression. Specifically an artificial cachexia inducing mutant M0 was generated by introducing the six nucleotides changes of the "cachexia expression motif" into a non-pathogenic sequence variant and M0 was used as a template to systematically restore some of the introduced changes. The resulting mutants in which specific changes introduced to generate M0, were restored presented a variety of responses: (i) M1, obtained by introducing two insertions forming a base-pair, was infectious but non-pathogenic; (ii) M2, obtained by introducing an insertion and restoring a substitution, presented low infectivity and the resulting progeny reverted to M0; (iii) M3, obtained by restoring a single substitution in the lower strand of the viroid secondary structure, was infectious but induced only mild cachexia symptoms; (iv) M4, obtained by restoring a single substitution in the upper strand of the viroid secondary structure, was non-infectious. These results confirm that the "cachexia expression motif" plays a major role in inciting cachexia symptoms, and that subtle changes within this motif affect symptom severity and may even suppress symptom expression.

A set of novel RNAs transcribed from the chloroplast genome accumulates in date palm leaflets affected by brittle leaf disease.

Brittle leaf disease or maladie des feuilles cassantes (MFC) is a lethal disorder of date palms that has assumed epidemic proportions in the oases of southern Tunisia. After a prolonged period during which palms are declining, the disease ends with the death of the palms. Whereas no pathogen could ever be associated with the disease, leaflets of affected palms have been previously shown to be deficient in manganese. Analysis of RNA preparations from leaflets of MFC-affected palms revealed the presence of a set of novel RNAs (MFC-RNAs) of sense and antisense polarities, which are homologous to various regions of the date palm chloroplast genome, such as the regions containing genes rrn5S-trnR(ACG) and trnM(CAU)-atpE. In the RNA preparations obtained from leaflets of affected palms, some of these RNAs are present as double-stranded species (MFC-dsRNAs), as witnessed by results from cellulose chromatography, end labeling, RNase digestion, and northern hybridization with strand specific probes. These MFC-RNAs represent a novel type of host-derived RNAs, and their presence in MFC-affected date palms is of diagnostic value.

Citrus viroid V: occurrence, host range, diagnosis, and identification of new variants.

The recently described Citrus viroid V (CVd-V) has been proposed as a new species of the genus Apscaviroid within the family Pospiviroidae. Analysis of 64 samples from different citrus-growing areas has shown that CVd-V is present in the United States, Spain, Nepal, and the Sultanate of Oman. CVd-V found in six sweet orange sources from the Sultanate of Oman was identical to the reference CVd-V variant, whereas three new variants with sequence identities of 98.6% (CVd-VCA), 97.3% (CVd-VST), and 94.9% (CVd-VNE) were identified in sources from California, Spain, and Nepal, respectively. These results suggest that this viroid has not emerged recently and that it is relatively widespread. Transmission assays to sweet orange, mandarin, and mandarin hybrids, clementine, satsuma, lemon, sour orange, Tahiti lime, Palestine sweet lime, calamondin, bergamot, and kumquat have shown that all these citrus species and citrus relatives are hosts for CVd-V. Several indexing approaches, including slot blot, northern blot hybridization, and reverse transcription-polymerase chain reaction, have been evaluated for detecting CVd-V, either using Etrog citron as an amplification host or directly from commercial species and cultivars.

A Survey of Citrus Viroids in Campania (Southern Italy).

Within a certification program of citrus, a survey covering 14 citrus fields not previously screened were randomly selected in seven areas of the Campania Region of southern Italy to evaluate the presence and distribution of citrus viroids in the region. During the last 3 years, 112 trees were visually inspected and sampled including 14 'Navelina', 8 'Washington navel', 13 'Biondo Commune', 10 'Tarocco', 6 'Valencia late', 1 'Sanguinello', and 1 'Vanilla' sweet oranges (Citrus sinensis), four sour oranges (C. aurantium), 21 'Commune' and 7 'Monreale' clementines (C. clementina), 8 'Common' and 2 'Avana' mandarins (C. reticulata), 8 'Ovale di Sorrento' and 6 'Zagara' lemons (C. lemon), 2 Tangelos (C. reticulata × C. paradisi), and 1 grapefruit (C. paradisi). Nearly all (37 of 38) mandarin and clementine trees showed cachexia-like symptoms. Severe bark scaling that is characteristic of exocortis disease was observed on a single 'Ovale di Sorrento' lemon, the only tree incidentally found to be grafted onto the exocortis-sensitive trifoliate orange (Poncirus trifoliata) rootstock, since sour orange (C. aurantium) is the main rootstock used in Campania. Samples (104 of 112) that had been graft-inoculated into Arizona 861-S1 'Etrog citron' (C. medica L.) on rough lemon (C. jambhiri Lush.) rootstock showed typical viroid symptoms. Viroid infection was confirmed by sequential polyacrylamide gel electrophoresis analysis (2) and molecular hybridization of imprinted membranes (1). Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), and Citrus viroid III (CVd-III) were the most frequently detected, accounting respectively, for 67.9, 86.6, and 84.8% of the infected samples. Citrus viroid IV (CVd-IV) and Citrus bent leaf viroid (CBLVd) were found in 24.1 and 13.4% of the sources. CBLVd was detected in sweet orange ('Tarocco' and 'Biondo Commune') and lemon ('Ovale di Sorrento' and 'Zagara'), while CVd-IV was detected in sweet orange ('Tarocco', 'Biondo Commune', and 'Washington navel'), lemon ('Ovale di Sorrento' and 'Zagara'), clementine ('Commune'), and sour orange. Most trees (82.1%) were infected with more than one viroid species. The most frequent viroid combinations were CEVd + HSVd + CVd-III (40.2%), HSVd + CVd-III (16.1%), and CEVd + CBLVd + HSVd + CVd-III + CVd-IV (12.5%). To our knowledge, this is the first report of CEVd, CBLVd, HSVd, CVd-III, and CVd-IV in the Campania Region and the first report of CBLVd and CVd-IV in Italy. References: (1) A. Palacio et al. Eur. J. Plant Pathol.105:897, 2000. (2) R. F. Rivera-Bustamante et al. Anal. Biochem. 156:91, 1986.

Mechanical Transmission of Citrus Viroids.

Preliminary transmission assays conducted under greenhouse conditions demonstrated that Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) can be mechanically transmitted from citron to citron (Citrus medica) by a single slash with a knife blade. The impact of mechanical transmission of viroids by pruning and harvesting operations was also demonstrated in experimental and commercial field plots. Transmission efficiency under field conditions ranged from 4% in 'Nules' clementine to 10% in 'Navelina' sweet orange and 21% in 'Verna' lemon. Transmission efficiency varied only slightly with viroid and donor hosts. The impact of viroid transmission on tree height, canopy volume, and crop harvest was minimal. When the donor host was coinfected with several viroids, the viroids were not necessarily cotransmitted. Considerations regarding viroid transmission in other climates are discussed. Measures to control viroid spread in nurseries should be mandatory in certification programs.

Identification in eggplant of a variant of citrus exocortis viroid (CEVd) with a 96 nucleotide duplication in the right terminal region of the rod-like secondary structure.

Analysis of eggplants, kept for years under greenhouse conditions after having been mechanically inoculated with nucleic acid preparations from field-grown eggplants containing two viroids, Eggplant latent viroid (ELVd) and Citrus exocortis viroid (CEVd), revealed the presence of an additional larger viroid RNA. Molecular characterization of this RNA showed that it is a stable 467-nt variant of CEVd with a 96-nt duplication of the right terminal region (CEVd-D96) that preserves the rod-like secondary structure. The coexistence in eggplant of CEVd (371nt) and CEVd-D96, and the fact that they have an almost identical sequence, strongly suggests the emergence of the latter from the former through an internal recombination mediated by a jumping RNA polymerase with low processivity. CEVd-D96 from eggplant is similar to the CEVd-D92 variant characterized previously in a hybrid tomato, suggesting that certain hosts may play a critical role in selecting and replicating this class of enlarged variants.

Single-strand conformation polymorphism (SSCP) analysis as a tool for viroid characterisation.

The potential of routine single-strand conformation polymorphism (SSCP) analysis for viroid characterization has been evaluated. Electrophoresis of 311 cloned full length viroid DNA inserts recovered from a field isolate of citrus exocortis viroid (CEVd) showed shifts in the migration of the cDNA or/and hDNA strands in non-denaturing 14% polyacrylamide gels. Using a single set of electrophoresis conditions seven different groups of variants (containing one to six changes), which did not represent the overall variability among clones, were identified. The relationship between the different SSCP profiles observed among clones and the variation in their nucleotide sequences was confirmed by sequence analysis. Variations in the dimensions of the gel allowed higher resolution and therefore the detection of additional single nucleotide variations among clones initially clustered into the same group. The viroid region affected by specific changes could be established by SSCP analysis of partial viroid length DNA. The potential use of SSCP analysis as a tool to screen existing viroid populations in infected hosts prior to sequencing is discussed.

A simple imprint-hybridization method for detection of viroids.

An imprint-hybridization method has been designed to simplify the processing of samples during routine viroid indexing. The method requires minimal sample manipulation and has been evaluated for detection of viroids in 11 viroid-host combinations including 4 viroids (CEVd, CSVd, HSVd, ASBVd) and 7 hosts (chrysanthemum, citron, cucumber, Gynura, tomato, peach and avocado). The method is fast and sensitive, and provides additional information on the sites of viroid accumulation.

Cryopreservation and storage of embryogenic callus cultures of several Citrus species and cultivars.

Nucellus-derived embryogenic callus cultures of Salustiana sweet orange were subjected to cryoconservation assays. Cryoprotection with 10%(vol/vol) dimethylsulfoxide, freezing by slow cooling and thawing by fast warming was suitable to recover viable growing cultures and whole plants through embryogenesis. Evaluation of liquid phase R 1 and solid phase R 2 cooling rates using a programmable freezing unit indicated that 100% of embryogenic cultures survived when frozen using a range of cooling rates (R 1 not above 0.5°C min-1 and R 2 not above 1°C min 1) and thawed by fast warming. Storage up to 2 years in liquid nitrogen did not affect the growth of the cryopreserved cultures and the recovery of whole plants. Cultures of four cultivars of sweet orange (C. sinensis Osb.), three cultivars of grapefruit (C. paradisi Macf.), and one cultivar each of lemon [C. limon (L.) Burm. f.], Cleopatra mandarin (C. reshni Hort. ex Tan.), sour orange (C. aurantium L.) and Mexican lime [C. aurantifolia (Christm.) Swing.] have been successfully cryopreserved. Problems using a viability assessment using fluorescein diacetate staining are discussed.

Viroid Prevalence in Tunisian Citrus.

The citrus industry in Tunisia is based mainly on the production of local cultivars of sweet orange (Citrus sinensis), common mandarin (C. reticulata), clementine (C. clementina), and lemon (C. limon). Sour orange (C. aurantium) is the only rootstock presently being used in the major growing area located at Cap Bon where 80% of citrus is being produced. The presence of tristeza disease in the Mediterranean basin is a threat to the Tunisian citrus industry, and new rootstocks giving tristeza tolerant rootstock/scion combinations are urgently needed as an alternative to sour orange. Since some promising rootstocks are known to be sensitive to viroids (1), a survey was conducted to determine if the cultivars grown presently in Tunisia were infected with viroids. Following a preliminary report (2), an extensive survey was conducted from 1995-2001 that included 174 symptomless sources being grown at Cap Bon: 26 Maltaise demi-sanguine, 9 Maltaise sanguine, 20 Maltaise blonde, 4 orange doublefine, 16 Washington navel, 12 Valencia late, 29 common mandarin, 42 Cassar clementine, 5 Lunari lemon, and 11 Eureka lemon. These sources were graft-inoculated into Etrog citron that subsequently developed symptoms characteristic of viroid infection. Sequential polyacrylamide gel electrophoresis analysis and molecular hybridization using viroid-specific probes (1) revealed that all sources were infected with at least two viroids. Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd), and Citrus viroid III (CVd-III) were widespread and accounted for 68.4, 67.8, and 81.0% of the sources tested, respectively. Citrus bent leaf viroid (CBLVd) and Citrus viroid IV (CVd-IV) were only found in 32.7 and 2.3% of the sources. The most frequent viroid combinations were CEVd+HSVd+CVd-III (17.8%) and CEVd+CVd-III (17,2%), whereas HSVd+CVd-IV and CEVd+CBLVd+CVd-III+CVd-IV were found in a single source (0.6%). References: (1) N. Duran-Vila and J. S. Semancik. Pages 178-194 in: Viroids. CSIRO Publishing, Australia, 2003. (2) A. Najar et al. Pages 398-400 in: Proc. 15th Conf. Int. Org. Citrus Virol, 2002.

Rubber Tree (Hevea brasiliensis) Bark Necrosis Syndrome I: Still No Evidence of a Biotic Causal Agent.

The main constraint in rubber plantations worldwide is the cessation of latex production because of two syndromes: (i) tapping panel dryness (TPD), a reversible physiological response to overexploitation; and (ii) bark necrosis (BN), an irreversible syndrome spreading from the collar toward the tapping cut. Early BN symptoms develop in the inner phloem tissues but never affect the cambium. Necrotic patches appear in the outer phloem, inducing bark cracking and peeling, but these alterations never lead to tree death. BN spreads gradually to neighboring rubber trees, and evidence of linear disease centers suggest that a pathogen may be involved, possibly transmitted by the tapping knife. Several previous etiological investigations (fungi, phytoplasma, bacteria, and virus) were performed (3) on leaves, bark, and latex using different methods (e.g., isolation, transmission, chemical treatments, and optic and electron microscope studies). Recent works focused on mechanically transmissible pathogens, such as viroid (2) or virus/double strand RNA, using RNA extraction (nonionic cellulose and appropriate ethanol concentrations) and treatment with RNase A, followed with sequential polyacrylamide gel electrophoresis (s-PAGE), reverse transcription-polymerase chain reaction (RT-PCR), degenerate oligonucleotide primer-PCR (DOP-PCR), and cloning and sequence analysis. While numerous viroid-like (between 250 and 400 nucleotides) and double strand virus-like (1,800 bp) low-molecular-weight RNAs were observed, no definite correlation was found with the BN status of trees. Sequencing of the various isolated RNAs only identified plasmids, nonpathogenic bacteria and yeasts, but none of the suspected pathogens. In addition, previous and recent transmission trials (tapping knife disinfection, bud grafting, bark implantation, and etc.) failed to confirm the involvement of a biotic agent. In conclusion, since these etiological investigations were inconclusive, a physiological disease is now suspected that involves exogenous stresses, nonoptimal vascular relations at the rootstock/scion junction and impaired cyanide metabolism (1,4). References: (1) H. Chrestin et al. Plant Dis. 88:1047, 2004. (2) N. Duran-Vila et al. J. Gen. Virol. 69:3069, 1988. (3) D. Nandris et al. Eur. J. For. Pathol. 21:340, 1991. (4) D. Nandris et al. Plant Dis. 88:1047, 2004.

Sudden Death of Citrus in Brazil: A Graft-Transmissible Bud Union Disease.

Citrus Sudden Death (CSD), a new, graft-transmissible disease of sweet orange and mandarin trees grafted on Rangpur lime rootstock, was first seen in 1999 in Brazil, where it is present in the southern Triângulo Mineiro and northwestern São Paulo State. The disease is a serious threat to the citrus industry, as 85% of 200 million sweet orange trees in the State of São Paulo are grafted on Rangpur lime. After showing general decline symptoms, affected trees suddenly collapse and die, in a manner similar to trees grafted on sour orange rootstock when affected by tristeza decline caused by infection with Citrus tristeza virus (CTV). In tristeza-affected trees, the sour orange bark near the bud union undergoes profound anatomical changes. Light and electron microscopic studies showed very similar changes in the Rangpur lime bark below the bud union of CSD-affected trees: size reduction of phloem cells, collapse and necrosis of sieve tubes, overproduction and degradation of phloem, accumulation of nonfunctioning phloem (NFP), and invasion of the cortex by old NFP. In both diseases, the sweet orange bark near the bud union was also affected by necrosis of sieve tubes, and the phloem parenchyma contained characteristic "chromatic" cells. In CSD-affected trees, these cells were seen not only in the sweet orange phloem, but also in the Rangpur lime phloem. Recent observations indicated that CSD affected not only citrus trees grafted on Rangpur lime but also those on Volkamer lemon, with anatomical symptoms similar to those seen in Rangpur lime bark. Trees on alternative rootstocks, such as Cleopatra mandarin and Swingle citrumelo, showed no symptoms of CSD. CSD-affected trees did recover when they were inarched with seedlings of these rootstocks, but not when inarched with Rangpur lime seedlings. These results indicate that CSD is a bud union disease. In addition, the bark of inarched Rangpur lime and Volkamer lemon seedlings showed, near the approach-graft union, the same anatomical alterations as the bud union bark from the Rangpur lime rootstock in CSD-affected trees. The dsRNA patterns from CSD-affected trees and unaffected trees were similar and indicative of CTV. CSD-affected trees did not react by immunoprinting-ELISA using monoclonal antibodies against 11 viruses. No evidence supported the involvement of viroids in CSD. The potential involvement of CTV and other viruses in CSD is discussed.

Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-dehydration technique.

Cryopreservation of ovules and somatic embryos from several genotypes of citrus was achieved using the encapsulation-dehydration technique. Survival of cryopreserved ovules was occasional and erratic after different pregrowth conditions in liquid medium with 0.75M, 1M or up to 1.25M sucrose. An efficient cryopreservation protocol was established for somatic embryos derived from two embryogenic sources (ovules and cut thin layer explants from stigma, style and ovaries). High survival rates (75-100%) were consistently obtained after 1 day pregrowth in 0.75M sucrose, desiccation down to 20-25% moisture content in the beads and direct immersion in liquid nitrogen. The histological study showed that embryos subjected to the encapsulation-dehydration, accumulated high sucrose levels which appear to ensure the recovery of the whole embryo after cryopreservation.

Citrus viroid V: molecular characterization and synergistic interactions with other members of the genus Apscaviroid.

Studies on Atalantia citroides, a citrus relative, revealed the existence of a viroid not described previously. The new viroid has a GC-rich genome of 293-294 nucleotides and contains the central conserved region characteristic of members of the genus Apscaviroid, and the terminal conserved region present in this and other genera of the family Pospiviroidae. The secondary structure of minimum free energy predicted for the new viroid is a rod-like conformation with 68.7% paired nucleotides and showing sequence identities with other viroids always lower than 90%, the conventional limit that separates different species within a given genus. Infectivity assays showed that the new viroid induces mild but characteristic symptoms on the indicator Etrog citron. Co-inoculation of CVd-V with either Citrus bent leaf viroid or Citrus viroid III, two other members of the genus Apscaviroid infecting citrus, disclosed synergistic interactions manifested in enhanced leaf symptoms and very pronounced dwarfing. Viroid titers, however, remained unaltered in co-infected plants. Possible mechanisms underlying the observed synergistic effects are discussed. According to its molecular and biological properties and its unusual ability to replicate in A. citroides, the new viroid, tentatively named Citrus viroid V (CVd-V), should be considered a new species of the genus Apscaviroid.

Molecular characterization of CEVd strains that induce different phenotypes in Gynura aurantiaca: structure-pathogenicity relationships.

Two Citrus exocortis viroid isolates (CEVd-s and CEVd-129) that induce severe and mild symptoms in Gynura aurantiaca, respectively, have been characterized. They present nucleotide sequences in the pathogenicity motifs P(L), C and P(R) similar to those of "Class A" and "Class B". Infectivity and symptom expression in G. aurantica and tomato were evaluated with a selection of sequence variants recovered from both isolates. As expected, the two variants selected from CEVd-s induced severe symptoms. The variants selected from CEVd-129 induced mild symptoms, except one of them, named MJ, that presented an unusual genotype and induced severe symptoms in G. aurantiaca. The biological properties of MJ show that the two nucleotide changes of the C domain normally associated with the P(L) and P(R) motifs of "Class B" strains are not implicated in symptom expression. The relationship between "Class A" and "Class B" strains with the symptoms induced in clementine trees grafted on trifoliate orange is discussed.

A novel RT-PCR approach for detection and characterization of citrus viroids.

Citrus plants are natural hosts of five viroid species and a large number of sequence variants. Because of their small size, viroids lend themselves to various RT-PCR approaches for their detection and further characterization. The one-step RT-PCR approach proposed here is based on the synthesis of viroid-cDNA by reverse transcription at 60 degrees C using a viroid specific 27-mer primer followed by standard second strand synthesis plus PCR amplification with various primer pairs. According to the primers used, full or partial length viroid-DNA is obtained. The technique avoids amplicon contamination in routine diagnosis. The suitability of the technique has been demonstrated using several nucleic acid extraction procedures and different viroid infected host species. The homogenization of tissue inside sealed plastic bags followed by nucleic acid extraction using a SDS/potassium acetate method is recommended because of its efficiency, simplicity and low cost. This extraction procedure, when coupled to the one-step RT-PCR approach, can be useful to avoid cross-contamination during routine diagnosis. A PCR strategy capable of discriminating between mild and severe strains of CEVd and identifying cachexia-inducing isolates of HSVd, is also described.