Journal of Plant Pathology (2013), 95 (3), 631-635 Edizioni ETS Pisa, 2013 Pagliano et al. 631

Short Communication

OCCURRENCE, PREVALENCE AND DISTRIBUTION OF CITRUS IN URUGUAY

G. Pagliano1, R. Umaña1, C. Pritsch1, F. Rivas2 and N. Duran-Vila3

1Laboratorio de Biotecnología, Dpto. Biología Vegetal. Facultad de Agronomía, Universidad de la República, Montevideo, Uruguay 2Programa Nacional de Investigación Producción Citrícola. Instituto Nacional de Investigación Agropecuaria (INIA), Salto, Uruguay 3Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain

SUMMARY when co-infecting the same tree (Vernière et al., 2006). CEVd, CBLVd, HSVd and CDVd are widely distributed The occurrence of Citrus exocortis (CEVd), Citrus worldwide (Singh et al., 2003) whereas CBCVd has limited bent leaf viroid (CBLVd), (HSVd), Citrus distribution in citrus-growing areas (Malfitano et al., 2005; dwarfing viroid (CDVd), Citrus bark cracking viroid (CB- Mohamed et al., 2009; Murcia et al., 2009; Cao et al., 2010). CVd) and Citrus viroid VI (CVd-VI) in Citrus spp. culti- CVd-V and CVd-VI are two newly reported viroid species vated in six provinces of Uruguay was surveyed in 2008- (Owen et al., 2011). CVd-V has been reported in California 2009 and 2009-2010 growing seasons using Northern blot (USA), Spain, Nepal, the Sultanate of Omán, Iran, China, hybridization. Sixty two per cent of surveyed trees were Japan (Serra et al., 2008b; Bani-Hashemian et al., 2010; Ito infected with either single or mixed viroid inocula, the and Ohta, 2010; Cao et al., 2010) and recently in Pakistan latter being more abundant. CBCVd and CVd-VI were not (Cao et al., 2013), whereas CVd-VI seems to be restricted detected in the samples analyzed. HSVd showed the high- to Japan (Ito et al., 2003). est prevalence (92%) among infected plants followed by Bark cracking and scaling followed by severe dwarf- CDVd (50%), CEVd (23%) and CBLVd (19%). The most ing and yield losses occur in viroid-infected citrus plants frequently observed viroid combination was HSVd+CDVd. when commercial species are grafted on sensitive root- Our results showed that CEVd, CBLVd, HSVd, CDVd are stocks such as trifoliate orange (Poncirus trifoliata (L.) widespread throughout citrus orchards in Uruguay and Raf.), Rangpur lime (Citrus limonia Osb.) and citranges highlight the presence of mixed viroid infections. (C. sinensis x P. trifoliata) and strain severity is high (Bani- Hashemian et al., 2009). Considering that more than 90% of citrus plants in Uruguay are grafted on either trifoliate Key words: survey, citrus, CEVd, CBLVd, HSVd, CDVd, orange or citranges, local citrus production may be at risk Northern blot hybridization. (Bisio et al., 2004) A procedure for viroid detection from field-grown cit- rus trees originally described by Murcia et al. (2009) and Seven viroids reported to infect Citrus spp. belong to successfully applied elsewhere (Mohamed et al., 2009) has four genera of the family (Duran Vila et al., been found to be highly sensitive, specific and an efficient 1988; Ashulin et al., 1991; Ito et al., 2001; Duran-Vila and alternative to biological indexing procedures because the Semancik, 2003; Serra et al., 2008a): Citrus exocortis vi- time required for diagnosis is markedly shortened. Briefly, roid (CEVd) (genus Pospiviroid), Hop stunt viroid (HSVd) RNA preparations extracted from bark tissues of field- (genus ), Citrus bark cracking viroid (CBCVd) grown citrus trees were subjected to polyacrylamide gel (genus Cocadviroid), and Citrus bent leaf viroid (CBLVd), electrophoresis (PAGE) under non-denaturing conditions Citrus dwarfing viroid (CDVd), Citrus viroid V (CVd-V) and Northern blot hybridization analysis with viroid-spe- and Citrus viroid VI (CVd-VI) (genus Apscaviroid). CEVd cific digoxygenin (DIG)-labelled probes. is the causal agent of the exocortis disease. HSVd includes This work reports the results of a survey on the oc- variants that induce the cachexia disease as well as non- currence, prevalence and distribution of CEVd, CBLVd, cachexia inducing variants (Semancik et al., 1988; Duran- HSVd, CDVd, CBCVd and CVd-VI in commercial citrus Vila and Semancik, 2003). The other viroids cause minor species in Uruguay using PAGE-Northern blot hybridiza- effects (Vernière et al., 2004) with complex interactions tion analysis. A total of 84 mature plants of the genus Citrus and Corresponding author: G. Pagliano Fax:+598.2.23562037 Fortunella and trifoliate orange seedling trees were sur- E-mail: [email protected] veyed in commercial orchards located in six provinces

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Aliquots (20 μl) of nucleic acid preparations (about 333 mg of fresh weight tissue) were electrophoresed in 5% non-denaturing polyacrylamide gel for 3 h at 60 mA as described by Murcia et al. (2009). Following migration, a gel section (2 cm wide) corresponding to the region where the signal recognition particle 7S RNA migrates, was sliced and electrotransferred to a positively-charged nylon mem- brane (Roche Applied Science, Germany) using 1× TBE buffer (90 mM Tris, 90 mM boric acid, and 2 mM EDTA) at 400 mA for 1.5 h. After transfer, gel slices were silver- stained to verify that RNAs had been completely electro- transferred. For nucleic acid fixation, membranes were exposed to 70,000 μJoules/cm2 using an UV-crosslinking oven (Hoefer-Uvc500, Amersham Biosciences, USA). Pre-hybridization (42ºC for 2 h) and hybridization with viroid-specific DIG-labelled probes (60ºC overnight) were performed as previously described (Murcia et al., 2009). Chemiluminescence signals from DIG-labelled hybrids were detected by autoradiography using anti-DIG-alkaline phosphatase conjugate and the CSPD substrate (Roche Fig. 1. Map of Uruguay showing the location of Canelones, Applied Science, Germany). Colonia, Montevideo, Río Negro, Salto and San José provinc- An example of the results obtained by Northern hy- es where field-grown citrus plants were surveyed for citrus vi- bridization analyses is shown in Fig. 2, in which RNA roids infection during 2008-2009 and 2009-2010 growing sea- sons. Circles show the sampling areas. Further details about preparations from bark tissues of six citrus varieties were the surveyed materials are shown in Table 1. independently hybridized with four DIG-labelled probes, each specific for a single viroid. This autoradiography provided evidence of two-viroid mixed infections in lanes of Uruguay during the 2008-2009 and 2009-2010 grow- 3 (CBLVd+HSVd) and 4 (HSVd+CDVd) and four-viroid ing seasons (Fig. 1; Table 1). In each orchard, plants were randomly selected and sampling included both symptom- Table 1. Citrus type, variety and sampling location (province) atic and symptomless trees. Arizona 861-S1 Etrog citrons of the 84 citrus plants surveyed for viroid infection during (C. medica L.) grafted onto rough lemon (C. jambhiri 2008-2009 and 2009-2010 growing seasons in Uruguay. Lush.) and infected with different combinations of four Plants Citrus type (species) Cultivar Locationb viroid species (CEVd, CBLVd, HSVd, CDVd) were used as analyzeda a source of leaf/bark tissue for positive controls. Uninfect- Washington CA, MO, SJ, 19 ed and micrografted citron plants were used as negative Orange Navel SA (Citrus sinensis) Valencia controls. Tissues or nucleic acid preparations, kindly pro- 10 CA, CO, SA vided by colleagues, were used as additional negative and late positive controls. As a whole, 17 positive and 6 negative Mandarin Murcott 3 CA, SA control plants were included in this study. In order to get C. sinensis x C. reticulata high viroid titers, infected citron plants, were maintained Ellendale 2 SA at 28-32°C at 16 h light/8 h dark photoperiod. CBCVd and C. deliciosa Common 7 CA, SA CVd-VI controls were obtained as monomeric transcripts C. unshiu Satsuma 14 CO, RN, SA Lemon CA, CO, Lisbon 16 from cDNA sequences cloned into TS4.7 and S10S plas- (C. limon) MO, RN, SJ mids according to Umaña (2010). Marsh 4 CA Five grams of bark tissues per tree, collected from Grapefruit Seedless shoots 30-50 cm long and 0.5-1.0 cm in diameter were (C. paradisi) Duncan 1 CA processed following the nucleic acid extraction proto- Star Ruby 2 CO, RN, SA Kumquat col previously described by Semancik et al. (1975) with Nagami 2 CO, MO minimal modifications reported by Murcia et al. (2009). (Fortunella margarita). Volkamer lemon 2 SA DIG-labelling of viroid-specific cDNA probes of CEVd, (C. volkameriana) CBLVd, HSVd, CDVd, CBCVd and CVd-VI) was carried Trifoliate orange 2 SA out by PCR. In some instances the reverse transcription (Poncirus trifoliata) PCR (RT-PCR) protocol described by Bernard and Du- anumber of plants. ran-Vila (2006) was used as an additional procedure to bsurveyed provinces: CA: Canelones, CO: Colonia, MO: Montevideo, RN: confirm the presence of viroids in nucleic acid samples. Río Negro, SJ: San José, SA: Salto.

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From the 84 field-grown citrus trees analyzed, 62% (52/84) were infected with at least one viroid or combi- nations of CEVd, CBLVd, HSVd and CDVd (Table 2). CBCVd and CVd-VI were not detected (Umaña, 2010). Positive samples comprised either single (23/84) or mixed (29/84) viroid infections. Single viroid infections were only observed for CBLVd and HSVd, with HSVd being the most commonly detected (40% of infected plants). Mixed infections with two, three and four viroids were found in 19%, 10% and 5% of the tested plants, respec- tively. In the case of mixed infections with either two or three viroids, different viroid combinations were detected. The most frequent mixed infection was HSVd+CDVd (14/84) followed by CEVd+HSVd+CDVd (4/84) and CBLVd+HSVd+CDVd (3/84). HSVd was the most fre- quently detected viroid either in single or mixed infec- tions with 92% (48/52) of the total number of infected Fig. 2. Northern blot hybridization analysis of RNA prepa- plants, while CDVd, CEVd and CBLVd were detected in rations extracted from field-grown citrus plants in Uruguay 50% (26/52), 23% (12/52) and 21% (11/52) of the positive hybridized with single viroid-specific DIG probes: CEVd, samples, respectively. Noteworthy, HSVd+CDVd were CBLVd, HSVd or CDVd (A, B, C, D, rispectively). detected in 27% (14/52) of infected plants. Lanes 1 to 7: 1, uninfected citron (negative control); 2, com- With the exception of grapefruit cv. Star Ruby, vi- mon mandarin; 3 Valencia sweet orange; 4, Lisbon lemon; 5, Washington navel orange (sample 1); 6, Star ruby grape- roid infection was found in all the commercial varieties fruit; 7, Washington navel orange (sample 2). tested: lemon (81%), grapefruit (71%), mandarine (62%) and sweet orange (55%). Viroids were also detected in trifoliate orange but neither in Volkamer lemon (C. vol- mixed infections (CEVd+CBLVd+HSVd+CDVd) in lane kameriana Ten. & Pasq.) nor in Nagami kumquat (Table 5. In addition to lane 1 (negative control), viroids were 2). Since the sample size for some of these citrus types was undetectable in the remaining three lanes. No single viroid rather small, further sampling efforts would be necessary infection was found in the samples shown in this figure. to confirm these findings. The results were confirmed by RT-PCR for some field- This survey complements previous data regarding vi- grown samples (results not shown). roid occurrence, prevalence and distribution in commercial

Table 2. Detection of single infection of HSVd or CBLVd and mixed infections of CEVd, CDVd, HSVd and/or CBLVd in different citrus species from commercial orchards in Uruguay by Northern-blot hybridization analysis.

Citrus species b a n ND HSVd CBLVd CEVd +CDVd CEVd +HSVd HSVd +CBLVd HSVd +CDVd CEVd+HSVd+CDVd CBLVd+HSVd +CDVd CEVd+CBLVd +HSVd CEVd+CBLVd +HSVd +CDVd Orange 29 14 6 0 1 0 1 3 1 2 1 2 Mandarine 26 10 10 2 0 0 0 4 0 0 0 0 Lemon 16 3 4 0 0 0 0 6 2 1 0 0 Grapefruit 7 3 0 0 0 1 0 0 1 0 0 2 Volkamer lemon 2 2 0 0 0 0 0 0 0 0 0 0 Trifoliate orange 2 0 1 0 0 0 0 1 0 0 0 0 Fortunella sp. 2 2 0 0 0 0 0 0 0 0 0 0

c 32 21 2 1 1 1 14 4 3 1 4 Total (%) 84 (38.0) (25.0) (2.4) (1.2) (1.2) (1.2) (16.6) (4.8) (3.6) (1.2) (4.8) a total number of sampled plants. b number of plants with non-detectable infection. c in parentheses: number of infected plants/total number of sampled plants x 100.

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citrus orchards in Uruguay (Pagliano et al., 1998) as also re- the Northern blot hybridization analysis meet diagnos- ported in other citrus-growing countries (Japan, Italy Cuba, tic requirements for quarantine and sanitation programs Brazil, Spain and Sudan) (Malfitano et al., 2005; Murcia for propagating materials in Uruguay, either as a primary et al., 2009; Mohamed et al., 2009). Our results point out screening method or as an additional tool to further sup- that viroid infections occur (62%) in many citrus orchards port results from indexing tests on indicator plants. in Uruguay, affecting all the commercial varieties tested and confirm the occurrence of CEVd, CBLVd, HSVd and CDVd. These viroids appear to be present throughout the ACKNOWLEDGEMENTS six provinces sampled in agreement with previous prelimi- nary surveys conducted in Uruguay (Pagliano et al., 1998; The authors acknowledge funding provided by BID- Pagliano, 2000). It is also confirmed in other reports that CONICYT (Project PDT 74/19). Dr. Takao Ito (NIFTS, there is no presence of CBCVd and CVD-VI in any of the Japan) for providing TS4.7 and S10S plasmids. Jacques samples analyzed (Umaña, 2010). The failure to detect CB- Borde (DGSSAA) and Ana Bertalmío (INIA-Salto CVd and CVd-VI, confirms other reports indicating a more Grande) for providing citron controls and samples. restricted distribution worldwide (Duran-Vila et al., 1988; Ito et al., 2001). HSVd was the most frequently detected viroid, which is in agreement with previous reports and REFERENCES confirms its high prevalence and worldwide distribution (Ito et al., 2002). HSVd was found at a slightly lower fre- Ashulin L., Lachman O., Hadas R., Bar-Joseph M., 1991. Nu- quency in single than mixed infections and it was detected cleotide sequence of a new viroid species, citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. 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Received March 22, 2013 Accepted July 3, 2013

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