INTERACTIONS BETWEEN CITRUS CACHEXIA VIROID AND CLOSELY RELATED SEQUENCE VARIANTS MAY IMPAIR EXPRESSION OF CACHEXIA SYMPTOMS

We report the sequences of four variants of Hop stunt viroid (HSVd) isolated from a naturally infected citrus tree of the variety ‘Clementinier’ in Tunisia. One of the variants (HSVd Tun/cl3) had the characteristic changes of the cachexia-inducing variant CVd-IIb, whereas the others were more similar to variant CV-IIa not inducing cachexia. However, when material from this tree was bioassayed in the cachexia indicator ‘Parson’s special’, no symptoms were observed for two years after inoculation. This might result from the concurrent presence of non-cachexia inducing variants that could interfere with CVd-IIb. Our data also show the worldwide distribution of this viroid and the presence of mixture of sequence variants in natural infections. INTRODUCTION Xyloporosis/cachexia of citrus was considered for some time a virus disease but recent evidence suggests that it is caused by a viroid (Reanwarakron and Semancik, 1998). Although xyloporosis and cachexia were originally described in sweet lime and ‘Orlando tangelo’, respectively, both diseases are apparently identical. Cachexia is common in old-line trees and is a continuing problem where growers have top-worked these trees with sensitive cultivars such as mandarin hybrids. Cachexia may remain symptomless in old-line citrus cultivars whereas it is particularly pronounced in some mandarins, tangelos and rootstocks. It causes stunting, chlorotic foliage and decline symptoms that can be similar to those caused by other agents (Semancik et al., 1988). The definitive symptoms are stem pitting and gum impregnation of the bark in sensitive cultivars. Pitting and gum development usually start near the bud union and may spread to the sensitive portion of the tree. Pegs on the bark face usually contain gum. Once these symptoms become well developed, a flat cut about halfway into the bark will reveal gum pockets of varying size. Symptoms usually take 18-48 months to develop in highly susceptible cultivars such as ‘Orlando tangelo’ and Citrus macrophylla, and longer in less sensitive cultivars. Indexing is usually done by inoculating an indicator side graft of ‘Parson's Special’ mandarin on a vigorous rootstock such as ‘Rough lemon’. Cachexia can affect most tangelos, some mandarins, many mandarin hybrids, sweet lime and Citrus macrophylla. Infected rough lemon may also show mild symptoms. It is especially severe on Citrus macrophylla and ‘Orlando tangelo’. Oranges, grapefruit, lemons, acid limes, some mandarins, trifoliate orange and its hybrids are considered as susceptible hosts even if they usually do not show obvious symptoms. The disease is transmitted through infected budwood by budding and pruning equipment. Proc. XIX IS on Fruit Tree Virus Diseases Ed. G. Llácer Acta Hort 657, ISHS 2004 402 Cachexia is caused by one variant of Hop stunt viroid (HSVd) called CVd-IIb. Other citrus variants of HSVd are CVd-IIa and CVd-IIc. Sequence identity between variants CVd-IIa and CVd-IIb is 95%, with the principal nucleotide differences being located in the variable domain of the viroid (Reanwarakron and Semancik, 1998). In the present work we have cloned and sequenced HSVd variants from a single infected citrus tree. Sequence analysis showed that one has the characteristic changes of the cachexia inducing variant CV-IIb, whereas the others are more similar to the CVd-IIa variant. Because indexing of the source material did not promote any symptoms in the ‘Parson’s special’ indicator two years after inoculation, this might be interpreted as a result of interferences between CV-IIb and the other HSVd variants. MATERIALS AND METHODS Plant Material The vegetable material consisted on bark and leaves from a 4-years-old tree of the variety ‘Clementinier’ grafted on Citrus volkamerina. Bark was grafted on ‘Parson’s special’ grown on Citrus volkamerina. Symptoms of the cachexia disease were not expressed by the indicator two years after inoculation. RNA Extraction Total leaf RNA was isolated by phenol extraction and partially purified by chromatography on non-ionic cellulose (Whatman, CF11). The resulting RNAs were fractionated by lithium chloride precipitation, quantified by UV spectroscopy and their quality assessed by electrophoresis in 1% agarose gel. RT-PCR Amplification Template RNAs were denatured at 95°C for 5 min and chilled on ice for 2 min. First-strand HSVd-cDNA was synthesized using the antisense primer VP19 (5’GCCCCGGGGCTCCTTTCTCAGGTAAG3’) and avian myeloblastosis virus reverse transcriptase (AMV-RT) according to manufacturer’s instructions (Roche). The resulting cDNA was then amplified by the polymerase chain reaction (PCR) using the same antisense primer coupled with the sense adjacent primer VP20 (5’CCCGGGGCAACTCTTCTCAGAATCC3’) (Kofalvi et al., 1997). These primers correspond to the upper strand of the central conserved region (CCR) and its flanking sequence. The amplifications were performed with Pwo DNA polymerase (Roche) in a thermal cycler (Perkin-Elmer) according to the following program (30 cycles): 1 min at 94°C, 1 min at 52°C and 1 min at 72°C, followed by a final extension of 5 min at 72°C. PCR products were analyzed by electrophoresis on a 2.0% agarose gel. cDNA Purification, Cloning and Sequencing Full-size HSVd-cDNA was eluted from gel slices, precipitated with ethanol and resuspended. Using the Taq DNA polymerase, a 3’ adenylate residue was then added to the 3' ends of the PCR-amplified products so that they could be ligated into the linearized pCR 2.1 vector with overhanging 3’deoxythymidilates (T) (TA cloning kit, Invitrogen). Recombinant clones were identified by restriction analysis. Four clones were manually sequenced in both directions by the dideoxynucleotide chain termination method using the M13 universal and reverse primers (T7 DNA sequencing kit, United State Biochemical). Sequence Analysis Sequence analysis was carried out using Clustal X program (Thompson et al., 1994). The RNA secondary structure was predicted with the aid of the mfold program (http://mfold2.wustl.edu/mfold/rna/form).