Mechanical Transmission of Viroids

C. J. Barbosa, Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, Apartado Oficial, 46113-Moncada, Valencia, Spain; J. A. Pina, Servicio de Semillas y Plantas de Vivero, Valencia, Spain; J. Pérez-Panadés, Unidad de Biometría, Instituto Valenciano de Investigationes Agrarias, Apartado Oficial, 46113-Moncada, Valencia, Spain; and L. Bernad, P. Serra, L. Navarro, and N. Duran-Vila, Departamento de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigationes Agrarias, Apartado Oficial, 46113- Moncada, Valencia, Spain

Pospiviroidae family comprised of species ABSTRACT with a central conserved region (CCR) and Barbosa, C. J., Pina, J. A., Pérez-Panadés, J., Bernad, L., Serra, P., Navarro, L., and Duran-Vila, without hammerhead ribozymes (13). N. 2005. Mechanical transmission of citrus viroids. Plant Dis. 89:749-754. CBLVd and CVd-III are mild pathogens that may cause a reduction in tree size and Preliminary transmission assays conducted under greenhouse conditions demonstrated that Cit- fruit harvest yields (21,38,42). In addition, rus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus CVd-IV also causes severe bark cracking viroid III (CVd-III), and Citrus viroid IV (CVd-IV) can be mechanically transmitted from on susceptible species (42). Two additional to citron (Citrus medica) by a single slash with a knife blade. The impact of mechanical trans- viroid variants, CVd-I-LSS (LSS = low mission 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 sequence similarity) and CVd-OS (OS = ‘Nules’ to 10% in ‘Navelina’ sweet and 21% in ‘Verna’ . Transmission original sample), have been reported in efficiency varied only slightly with viroid and donor hosts. The impact of viroid transmission on Japan (23,24). Since all citrus viroids in- tree height, canopy volume, and crop harvest was minimal. When the donor host was coinfected duce symptoms on citron, biological with several viroids, the viroids were not necessarily cotransmitted. Considerations regarding indexing tests using this indicator are not viroid transmission in other climates are discussed. Measures to control viroid spread in nurser- specific for the exocortis agent as initially ies should be mandatory in certification programs. thought. The utilization of viroid indexing tests based on nucleic acid analysis have Additional keywords: cachexia, xyloporosis, exocortis shown that samples collected from natu- rally infected trees usually contain mix- tures of several viroids (1,9,11,25). Many field sources that were initially character- Exocortis disease of citrus was de- number of procedures to decontaminate ized as severe exocortis isolates based on scribed in 1948 as a bark shelling or scal- cutting tools were proposed to avoid the the symptoms induced in Etrog citron have ing disorder affecting trees grown on trifo- spread of the disease (17,34). since been shown to contain other viroids liate orange (Poncirus trifoliata (L.) Raf.) Cachexia disease was described in 1948 or viroid mixtures in addition to CEVd, rootstock (12), and soon afterward it was as discoloration, gumming, and browning whereas sources characterized as mild or demonstrated to be a graft-transmissible of phloem tissues, wood pitting and bark moderate isolates contain other viroids disease (4,5). The hybrids (Citrus cracking on Orlando (C. paradisi × or viroid mixtures but not CEVd sinensis × P. trifoliata) and C. reticulata) (8). Most commercial spe- (10,20,26,36). Similarly, cachexia isolates (C. limonia Osb.) used as rootstocks, and cies and cultivars are tolerant, but mandar- may contain other viroids in addition to the some , limes, and , are also ins, some mandarin hybrids ( and cachexia agent (39). Because assays con- sensitive to exocortis. The disease was ), and (Fortunella sp.) ducted on the transmission of what were considered to be induced by infection with are sensitive. Alemow (C. macrophylla initially considered exocortis and cachexia a virus until it was shown that Citrus exo- Wester) and Rangpur lime used as root- isolates most likely contained viroid mix- cortis viroid (CEVd) was the causal agent stocks are also sensitive. Because the dis- tures, the information generated needs to (41). Studies regarding the mechanical ease shares biological properties with exo- be reassessed according to the present transmission of the exocortis agent were cortis, including efficient transmission by knowledge of viroids infecting citrus. conducted before the agent of the disease slashing with a knife blade, it was sus- Assays demonstrating that exocortis and was established (17) and demonstrated that pected to be caused by a viroid infection cachexia were mechanically transmissible it could be transmitted from citron (C. (34). Following the discovery that citrus were mainly conducted from citron to medica L.) to citron with 100% efficiency are naturally infected with several viroids, citron under controlled greenhouse condi- simply by slashing with a knife blade the agent of the disease was identified (40) tions; however, the possibility that their (17,19), which retained viable inoculum and characterized as a specific sequence viroid agents might spread naturally in the for several days (2). As a consequence, a variant of Hop stunt viroid (HSVd) (30). field was considered. Additional transmis- Furthermore, its identity to a similar disor- sion assays, also conducted under green- der described earlier as xyloporosis in house conditions using species other than Corresponding author: Núria Duran-Vila Palestine sweet lime (C. limettioides Tan.) citron either as donor or as receptor hosts E-mail: [email protected] (32) was demonstrated (31). (18), further reinforced the risk that com- Current address of C. J. Barbosa: Embrapa- Viroids are small, covalently closed, mercial cultivars growing in the field could Mandioca e Fruticultura, Rua Embrapa, CP. 007, single-stranded RNAs (246 to 401 nucleo- become infected as a result of mechanical CEP : 44380-000, Cruz das Almas, Bahia, Brasil. tides) that replicate in their host plants. In transmission during pruning and harvest- addition to CEVd and HSVd, three addi- ing operations, as evidenced later in lemon Accepted for publication 10 March 2005. tional viroids were reported in citrus in- orchards (6,7). cluding Citrus bent leaf viroid (CBLVd), In many citrus-growing countries, trees DOI: 10.1094/PD-89-0749 Citrus viroid III (CVd-III), and Citrus are grown on viroid sensitive rootstocks © 2005 The American Phytopathological Society viroid IV (CVd-IV), all belonging to the such as Rangpur lime, P. trifoliate, citrange

Plant Disease / July 2005 749 Carrizo, citrange Troyer, and alemow. The IIc) (29–31), CVd-III (CVd-IIId) (14), and were propagated using viroid-free certified performance of trees grafted on these root- CVd-IV (CVd-IV-Ca) (15). budwood. Half of the trees of each species stocks relies on the propagation of virus- Mechanical viroid transmission was per- were graft inoculated with a field isolate of free certified budwood. However, newly formed in the spring by the slash inocula- citrus viroids containing CEVd, CBLVd, established viroid-free trees are subjected tion method using a sterile surgical blade. HSVd (CVd-IIa), and CVd-III. Trees were to the risk of mechanical infection from A single slash incision wound was made at planted in the field in such a way that in- symptomless infected trees growing in an angle of 45° into the bark of the stem of oculated trees were always located next to adjacent orchards or even within the same the actively growing donor plants; and noninoculated trees within rows as well as orchard. immediately thereafter, with the same between rows. The plants were subjected In the present work, we describe the re- blade, a similar single slash incision to normal pruning and harvesting (with sults of a preliminary assay conducted wound was made in the bark of the recep- clippers) operations without disinfesting under greenhouse conditions demonstrat- tor citron. The incision wound made in the the tools. In 2002, all plants were indexed ing the transmissibility of all citrus viroids. receptor stem was wrapped with Parafilm, on the 861-S1 selection of Etrog citron Subsequent assays in experimental and and the plants were maintained in the grafted on rootstock. Each commercial field orchards were designed greenhouse at 28 to 32°C. Three months indicator plant was graft inoculated with to evaluate transmission efficiency in the later, receptor citrons were cut back 40 cm three bark pieces collected from each of field and its impact on the performance of above the soil level, and the new shoots the main branches of the tree and main- trees under the growing conditions of east- formed were analyzed by sequential poly- tained in the greenhouse at 28 to 32°C. ern Spain. acrylamide gel electrophoresis (sPAGE) After 6 months, the citrons were analyzed analysis. by imprint hybridization (27,28). MATERIALS AND METHODS Experimental field plot. Following the Commercial field plot. A commercial Greenhouse transmission assays. Pre- demonstration that all citrus viroids were field plot suspected of having been ex- liminary experiments under greenhouse mechanically transmissible by knife posed to the mechanical transmission of conditions were conducted prior to field blades, three experimental field plots were citrus viroids was selected in the location experiments to verify that all citrus viroids established in 1988 to evaluate the impact of Benicarló (Castelló, Spain). The orchard described herein could be mechanically of mechanical transmission under field was established in 1985 with noncertified transmitted by slash wounding using a conditions. Trees of ‘Verna’ lemon (56 ‘Nules’ clementine trees grafted on sour knife blade. Plants of the 861-S1 selection trees), ‘Nules’ clementine (50 trees), and orange that were suspected to be infected of Etrog citron grafted onto Rough lemon ‘Navelina’ sweet orange (C. sinensis (L.) with viroids. In 1995, this plot was inter- (C. jambhiri Lush.) rootstock were used as Osb.) (60 trees) grafted on Carrizo citrange planted with ‘Nules’ clementine trees receptor plants when the scion reached at least 40 cm in height (4 to 6 months after ). Citrons infected with CEVd, CBLVd, HSVd, CVd-III, or CVd-IV were used as donor plants. Citron, ‘Nules’ clementine (C. clementina Hort. ex Tan.), and ‘Verna’ lemon (C. limon (L.) Burm. f.) infected with viroid mixtures were also used as donor plants. Citrons had been infected for several years and were peri- odically pruned to produce actively grow- ing shoots under greenhouse conditions (28 to 32°C and a relative humidity around 60%). Shoots of ‘Nules’ clementine and ‘Verna’ lemon were collected in early summer from inoculated plants actively growing in the field. Viroids used as single Fig. 1. Indexing by sequential polyacrylamide gel electrophoresis (sPAGE) analysis of a lemon donor and six receptor citron plants. Lane 1, lemon donor coinfected with Citrus exocortis viroid (CEVd), viroid sources or as mixtures were: CEVd Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), and Citrus viroid III (CVd-III); lanes 2, 3, (CEVd-117) (16), CBLVd (CVd-Ia-117) 7, three noninfected receptor citron plants; lanes 4, 5, 6, three infected receptor citron plants. The (14), HSVd (CVd-IIa-117, CVd-IIb, CVd- lemon donor was first graft inoculated on Etrog citron and subsequently analyzed by sPAGE.

Table 1. Mechanical transmission of citrus viroids by a single slash using a surgical blade under experimental greenhouse conditions Receptor citronsa Donor plant No. infected plants/no. slashed plantsb Species Inoculated viroidsc CEVd CBLVd HSVd CVd-III CVd-IV Citron None 0/8 0/8 0/8 0/8 0/8 Citron CEVd 2/8 0/8 0/8 0/8 0/8 Citron CBLVd 0/8 2/8 0/8 0/8 0/8 Citron HSVd (CVd-IIa) 0/8 0/8 4/8 0/8 0/8 Citron HSVd (CVd-IIb) 0/8 0/8 2/8 0/8 0/8 Citron HSVd (CVd-IIc) 0/8 0/8 6/8 0/8 0/8 Citron CVd-III 0/8 0/8 0/8 3/8 0/8 Citron CVd-IV 0/8 0/8 0/8 0/8 1/8 Citron CBLVd+HSVd+CVd-III 0/6 0/6 0/6 1/6 0/6 Clementine CEVd+CBLVd+HSVd+CVd-III 0/6 0/6 0/6 0/6 0/6 Lemon CEVd+CBLVd+HSVd+CVd-III 0/6 1/6 3/6 1/6 0/6 a Receptor plants were indexed by sequential polyacrylamide gel electrophoresis (sPAGE) analysis. b Gray boxes designate positive transmissions of each viroid. c Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV).

750 Plant Disease / Vol. 89 No. 7 grafted on Carrizo citrange purchased as dium (0.4 M Tris-HCl, pH 8.9, 1% excised and subjected to a second 5% certified virus-free plants. No measures [wt/vol] sodium dodecyl sulfate [SDS], 5 PAGE containing 8 M urea (33). Viroid were implemented to disinfect the tools mM EDTA, and 4% [vol/vol] mercap- bands were viewed by silver staining (22). during the pruning and harvesting opera- toethanol) (37). The total nucleic acids For Northern blot analysis, nucleic acids tions. were partitioned in 2 M LiCl, and the were transferred electrophoretically to A sample of 18 presumably infected soluble fraction was concentrated by etha- positively charged Nylon membranes at 80 trees grafted on sour orange were tested nol precipitation and resuspended in TKM V for 1 h directly from the urea-containing for viroid infection by biological indexing buffer (10 mM Tris-HCl, pH 7.4, 10 mM gel obtained after sPAGE using a modified on Etrog citron, sPAGE, Northern blot KCl, and 0.1 mM MgCl2). Aliquots of TBE buffer (40 mM Tris, 40 mM boric hybridization, and reverse transcription– these preparations were subjected to acid, 1 MM EDTA, pH 8.3) for blotting. polymerase chain reaction (RT-PCR). Sev- sPAGE, Northern blot hybridization For imprint hybridization analysis, stems enty trees grafted on Carrizo citrange were against viroid-specific probes, or RT-PCR. were freshly cut transversely and firmly biologically indexed on Etrog citron, and For sPAGE analysis, samples (20 µl pressed onto the surface of positively viroid infection was determined by imprint equivalent to 300 mg fresh weight) were charged Nylon membranes (27,28). In all hybridization of the inoculated citrons. initially subjected to nondenaturing 5% instances, the nucleic acids were immobi- Viroid indexing. Citron (5 g of young PAGE at 60 mA for 2.5 h. After ethidium lized by UV cross-linking. Prehybridiza- leaves and stems) samples were homoge- bromide staining, a segment (1.5 cm) of tion and hybridization were carried out in nized in phenol containing extraction me- the gel between CEVd and 7sRNA was 50% formamide using digoxigenin (DIG)- labeled DNA probes (27,28). Table 2. Mechanical transmission of citrus viroids by standard agronomic practices in ‘Nules’ RT-PCR was performed essentially as clementine, ‘Navelina’ sweet orange, and ‘Verna’ lemon trees growing adjacent to infected trees under described by Yang et al. (43) using two experimental field conditionsa contiguous primers of opposite polarity specific for each viroid (28). In order to b,c No. infected plants/no. tested plants increase the specificity of the reaction (L. Species CEVd CBLVd HSVd CVd-III Transmissiond Bernad, unpublished results), first strand ‘Nules’ clementine cDNA was synthesized at 60°C using 15 U – Infected donor 25/25 25/25 25/25 25/25 of ThermoScript RNase H Reverse Tran- Receptor 1/25 0/25 0/25 0/25 1/25 (4%) scriptase (Invitrogen, Carlsbad, CA). ‘Navelina’ sweet orange Tree growth and fruit yield. Tree Infected donor 30/30 30/30 30/30 30/30 height, canopy size, and fruit yield were Receptor 0/30 1/30 0/30 2/30 3/30 (10%) measured at the end of 2003. Canopy size ‘Verna’ lemon was calculated as Vc = 4/3πR3, where R is Infected donor 28/28 28/28 28/28 28/28 the radius of the canopy. R was estimated Receptor 5/28 0/28 1/28 0/28 6/28 (21%) as one-half the average of canopy height

Viroid transmissione 6/83 (7.2%) 1/83 (1.2%) 1/83 (1.2%) 2/83 (2.4%) and two perpendicular width measure- a All trees were subjected to standard fertilization and phytosanitary treatments. Pruning and harvest- ments. Data from each citrus species were ing were conducted without disinfecting the tools. analyzed by ANOVA. b Receptor plants were biologically indexed on Etrog citron and by imprint hybridization analysis. Gray boxes highlight positive transmissions of each viroid. RESULTS c Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), and Cit- Mechanical transmission under rus viroid III (CVd-III). greenhouse conditions. The results of a d Number of infected receptors over total number of receptor plants of each species. Percentages were compared using normal approximation and a t test. small-scale preliminary transmission assay e Number of receptor plants infected with each viroid over total number of receptor plants. Percentages using citron as the receptor host conducted were compared using normal approximation and a t test. under greenhouse conditions demonstrated

Fig. 2. Statistical analysis by ANOVA of height, canopy volume, and yield parameters collected in three experimental field plots of A, ‘Nules’ clementine, B, ‘Navelina’ sweet orange, and C, ‘Verna’ lemon grafted on Carrizo citrange. Bars show the mean standard error of infected (nonshaded) and healthy (shaded) trees. Asterisks (*) show values from individual receptor trees that succumbed to infection with the viroid indicated during the trial.

Plant Disease / July 2005 751 that all citrus viroids could be mechani- In fact, when lemon coinfected with CEVd, perimental plots of ‘Verna’ lemon, ‘Nules’ cally transmitted from citron to citron by CBLVd, HSVd, and CVd-III was used as a clementine, and ‘Navelina’ sweet orange slash inoculation with a knife blade. This donor host, three of the six citrons used as were designed to mimic the number of confirms that in addition to the exocortis receptor hosts became infected. These in- growers who renew their orchards by and cachexia agents, CBLVd, CVd-III, fected citrons presented mild and moderate planting young, certified virus-free plants CVd-IV, and non-cachexia variants of symptoms compared with the severe symp- in between old viroid-infected plants. The HSVd can also be transmitted to citron by toms observed in citrons that were graft results demonstrate that citrus viroids are a single slash (Table 1). inoculated from the donor lemon. The re- also transmissible under field conditions Transmission assays using citron, clem- sults of sPAGE analysis of donor and recep- (Table 2). The number of infected plants as entine, and lemon coinfected with several tor plants are shown in Figure 1. a result of mechanical transmission ranged viroids as donor hosts showed that viroids Mechanical transmission under ex- from 21% in lemon to only 4% in are not necessarily cotransmitted (Table 1). perimental field conditions. Three ex- clementine. Comparisons of transmission

Fig. 3. Indexing of 15-year-old ‘Nules’ clementine trees grafted on sour orange sampled in a commercial field plot. Plants were graft inoculated on Etrog citron. Figures show results of A, sequential polyacrylamide gel electrophoresis (sPAGE), B, Northern blot hybridization against viroid-specific probes, and C, reverse transcription–polymerase chain reaction (RT-PCR) using viroid-specific primers. Lane 1, positive control infected with Citrus exocortis viroid (CEVd) and Citrus viroid IV (CVd-IV); lanes 2 and 3, two of 16 indexed clementine trees; lane 4, positive control infected with Citrus bent leaf viroid (CBLVd) (variant CVd-Ib), Hop stunt viroid (HSVd), and Citrus viroid III (CVd-III).

752 Plant Disease / Vol. 89 No. 7 percentages in the three species were cal- analysis. As summarized in Table 3, 10% nor hosts are not necessarily mechanically culated using normal approximation and a of the trees succumbed to infection as a cotransmitted. Transmission assays con- t test. Differences in transmission were result of mechanical transmission. Recep- ducted by Roistacher et al. (35) showed found (10% significance level) between tor trees were infected either with a single that receptor citrons presented a great vari- lemon and clementine (P = 0.0856), but viroid or with combinations of two to three ability of symptoms, ranging from mild to not between clementine and sweet orange viroids. No significant differences were severe, when compared with the severe (P = 0.5378) or between sweet orange and found among the frequencies of transmis- reaction induced by the donor plant. Those lemon (P = 0.3194). sion of different viroids when comparisons observations were considered to be due to All transmissions were of a single vi- of transmission percentages were calcu- separation of strains differing in virulence roid, confirming that the four viroids coin- lated using normal approximation and a t contained in the severe donor isolate. The fecting the donor plants were not necessar- test. results reported here have demonstrated ily cotransmitted, results that were that the mild and moderate reactions consistent with greenhouse transmission DISCUSSION thought to be the result of infection with assays. Comparisons of transmission per- Early reports demonstrated that the exo- mild exocortis are in fact the result of dif- centages of different viroids were calcu- cortis and cachexia agents were mechani- ferent viroid species present as mixtures in lated as above. Differences in transmission cally transmitted by slashing with a knife field isolates. were only found between CEVd and blade (17,19,34). In spite of the small These results confirm that, in addition to CBLVd (P = 0.0822) and between CEVd number of receptor plants used in the pre- the exocortis and cachexia agents, all and HSVd (P = 0.0822). liminary greenhouse transmission study, known citrus viroids are mechanically Donor trees presented bark scaling the results showed that CBLVd, CVd-III, transmissible. Under the environmental symptoms that were only perceptible be- CVd-IV, and all strains of HSVd were also conditions in which the assay was per- low the soil level; however, none of the mechanically transmissible by a single formed, rate of transmission in the field trees infected as a result of mechanical slash. The apparent discrepancy between and impact on the performance of trees transmission showed symptoms. Infected the frequency of transmission from citron that became infected was lower than an- donor clementine trees were significantly to citron found in the present study and the ticipated. However, measures to control the smaller than the noninfected receptor trees near 100% transmission reported earlier spread of viroid agents should still be rec- in terms of height, canopy volume, and (17,19,34) was likely due to the fact that ommended in areas in which viroid-free yield (Fig. 2A). The size and yield of the we used a single slash versus 10 slashes in plants are grown close to infected plants. single receptor clementine tree that suc- earlier studies. These strategies should be reinforced in cumbed to CEVd infection was within the Viroid transmission has also been dem- those orchards planted with trees of which range of the noninfected trees, suggesting onstrated among trees growing in the field, either the rootstock, the scion, or both are a recent infection (Fig. 2A). No significant with rates lower than anticipated when viroid-sensitive species. Even though the differences were found between donor and taking into consideration the high number results of the present study indicate that noninfected receptor sweet orange trees of slashes performed during pruning and transmission rates in field grown trees are (Fig. 2B), whereas highly significant sta- harvesting operations without disinfecting fairly low, this information should not be tistical differences were found between the tools. Only slight differences in terms extrapolated to trees growing under other noninfected receptor and infected donor of transmission efficiencies of different environmental conditions. In temperate lemon trees (Fig. 2C). The height and can- viroids and within different host species climates such as those of Mediterranean opy volume of the six lemon trees that were found. These differences may result countries, harvesting and pruning opera- became infected as a result of mechanical from the differences in viroid titers found tions take place in late autumn and winter transmission ranged from 1.7 to 2.3 m among different infected species (3,28). when trees are dormant and contain low (height) and 6.46 to 16.80 m3 (canopy The results of the three assays have viroid titers (28), a situation that probably volume), falling within the range of in- demonstrated that viroids coinfecting do- does not favor mechanical transmission. fected as well as noninfected trees (Fig. 2C). These differences suggest that the trees became infected at different times. Table 3. Mechanical transmission of citrus viroids by standard agronomic practices in ‘Nules’ clementine trees growing adjacent to infected trees under commercial field conditionsa Transmission under commercial field conditions. Transmission results obtained No. infected plants/no. tested plantsb,c in the experimental field assay were fur- ‘Nules’ clementine CEVd CBLVd HSVd CVd-III Transmissiond ther confirmed by analysis of plants col- lected from a commercial orchard of non- Infected donor 18/18 18/18 18/18 18/18 Receptor 1/70 4/70 4/70 3/70 7/70 (10%) certified ‘Nules’ clementine trees grafted Viroids detected in receptor plants on sour orange that had been interplanted Receptor plants infected/ with young certified trees of the same receptor plants tested CEVd CBLVd HSVd CVd-III cultivar grafted on Carrizo citrange. Eight- 2/70 – + + – een samples of the old trees suspected of 1/70 + – – – being infected, were subjected to biologi- 1/70 – + + + cal indexing in Etrog citron and sPAGE 1/70 – – – + analysis. All trees were infected with the 1/70 – – + + same viroids (CEVd, CBLVd, HSVd, and 1/70 – + – – CVd-III) (Fig. 3), indicating that they had 63/70 – – – – e been propagated from the same scion Frequency 1.4% 5.7% 5.7% 4.3% source. Identities of the viroids identified a All trees were subjected to standard fertilization and phytosanitary treatments. Pruning and harvest- by sPAGE were confirmed by Northern ing were conducted without disinfecting the tools. hybridization analysis using viroid-specific b Receptor plants were biologically indexed on Etrog citron and by imprint hybridization analysis. DIG-labeled DNA probes (27) and RT- Gray boxes highlight transmission frequencies of each viroid. c Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), and Cit- PCR (Fig. 3). rus viroid III (CVd-III). Seventy clementine trees grafted on Car- d Number of infected receptors over the total number of receptor plants. rizo citrange were indexed biologically on e Number of receptor plants infected with each viroid over the total number of receptor plants. Per- Etrog citron and by imprint hybridization centages were compared using normal approximation and a t test.

Plant Disease / July 2005 753 However, the high relative humidity and Pages 202-211 in: Proc. Conf. Int. Org. Citrus 28. Palacio-Bielsa, A., Foissac, X., and Duran-Vila, elevated temperatures of tropical climates, Virol. 12th. IOCV, Riverside, CA. N. 2000. Indexing of citrus viroids by imprint 11. Duran-Vila, N., Roistacher, C. N., Rivera- hybridization. Pages 294-301 in: Proc. Conf. Int. similar to the greenhouse conditions where Bustamante, R., and Semancik, J. S. 1988. A Org. Citrus Virol. 14th. IOCV, Riverside, CA. our transmission assays were performed, definition of citrus viroid groups and their rela- 29. Palacio-Bielsa, A., Romero-Durbán, J., and prevent dormancy, and therefore trees may tionship to the exocortis disease. J. Gen. Virol. Duran-Vila, N. 2004. Characterization of citrus be more susceptible to infection. 69:3069-3080. HSVd isolates. Arch. Virol. 149:537-552. Control of viroid spread is critical in 12. Fawcett, H. S., and Klotz, L. J. 1948. Exocortis 30. Reanwarakorn, K., and Semancik, J. S. 1998. on . Citrus Leaves 28:8. Regulation of pathogenicity in hop stunt viroid nurseries where the unfortunate contami- 13. Flores, R., Randles, J. W., Bar-Joseph, M., and related group II citrus viroids. J. Gen. Virol. nation of a single mother tree may result in Diener, T. O. 2000. Subviral agents: Viroids. 79:3163-3171. the dissemination of many infected plants. Pages 1009-1024 in: Virus Taxonomy, Seventh 31. Reanwarakorn, K., and Semancik, J. S. 1999. For this reason, disinfection of tools used Report of the International Committee of Tax- Correlation of hop stunt viroid variants to in pruning and harvesting operation should onomy of Viruses. Academic Press, San Diego, cachexia and xyloporosis diseases of citrus. CA. Phytopathology 89:568-574. be mandatory in certification programs. 14. Foissac, X., and Duran-Vila, N. 2000. Charac- 32. Reichert, I., and Perlberger, P. 1934. Xylopo- ACKNOWLEDGMENTS terisation of two citrus apscaviroids isolated in rosis, the new citrus disease. Agric. Exp. Stn. This work was partially supported by grants Spain. Arch. Virol. 145:1975-1983. Rehovoth, Palestine Bull. 12. p. 44. SC97-108 and RTA01-119 from the Ministerio de 15. Francis, M., Szychowski, J. A., and Semancik, 33. Rivera-Bustamante, R. F., Gin, R., and Seman- Ciencia y Tecnología (Spain). The work was part of J. S. 1995. Structural sites specific to citrus vi- cik, J. S. 1986. Enhanced resolution of circular the Ph.D. thesis of C. J. Barbosa, who was the roid groups. J. Gen. Virol. 76:1081-1089. and linear molecular forms of viroid and vi- recipient of a fellowship provided by Fundação 16. Gandía, M., Palacio, A., and Duran-Vila, N. roid-like RNA by electrophoresis in a discon- CAPES. We thank Rosario Carbó for technical 1998. Variability of citrus exocortis viroid tinuous-pH system. Anal. Biochem. 156:91- assistance, J. M. Corvera and A. Lluch for their (CEVd). 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