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 VIROIDS 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
The occurrence of Citrus exocortis viroid (CEVd), Citrus
bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus
dwarfing viroid (CDVd), Citrus bark cracking viroid (CBCVd) and Citrus viroid VI (CVd-VI) in Citrus spp. cultivated in six provinces of Uruguay was surveyed in 20082009 and 2009-2010 growing seasons using Northern blot
hybridization. Sixty two per cent of surveyed trees were
infected with either single or mixed viroid inocula, the
latter being more abundant. CBCVd and CVd-VI were not
detected in the samples analyzed. HSVd showed the highest prevalence (92%) among infected plants followed by
CDVd (50%), CEVd (23%) and CBLVd (19%). The most
frequently observed viroid combination was HSVd+CDVd.
Our results showed that CEVd, CBLVd, HSVd, CDVd are
widespread throughout citrus orchards in Uruguay and
highlight the presence of mixed viroid infections.
Key words: survey, citrus, CEVd, CBLVd, HSVd, CDVd,
Northern blot hybridization.
Seven viroids reported to infect Citrus spp. belong to
four genera of the family Pospiviroidae (Duran Vila et al.,
1988; Ashulin et al., 1991; Ito et al., 2001; Duran-Vila and
Semancik, 2003; Serra et al., 2008a): Citrus exocortis viroid (CEVd) (genus Pospiviroid), Hop stunt viroid (HSVd)
(genus Hostuviroid), Citrus bark cracking viroid (CBCVd)
(genus Cocadviroid), and Citrus bent leaf viroid (CBLVd),
Citrus dwarfing viroid (CDVd), Citrus viroid V (CVd-V)
and Citrus viroid VI (CVd-VI) (genus Apscaviroid). CEVd
is the causal agent of the exocortis disease. HSVd includes
variants that induce the cachexia disease as well as noncachexia inducing variants (Semancik et al., 1988; DuranVila and Semancik, 2003). The other viroids cause minor
effects (Vernière et al., 2004) with complex interactions
Corresponding author: G. Pagliano
Fax:+598.2.23562037
E-mail: [email protected]
27. JPP1650SC (Pagliano).indd 631
when co-infecting the same tree (Vernière et al., 2006).
CEVd, CBLVd, HSVd and CDVd are widely distributed
worldwide (Singh et al., 2003) whereas CBCVd has limited
distribution in citrus-growing areas (Malfitano et al., 2005;
Mohamed et al., 2009; Murcia et al., 2009; Cao et al., 2010).
CVd-V and CVd-VI are two newly reported viroid species
(Owen et al., 2011). CVd-V has been reported in California
(USA), Spain, Nepal, the Sultanate of Omán, Iran, China,
Japan (Serra et al., 2008b; Bani-Hashemian et al., 2010; Ito
and Ohta, 2010; Cao et al., 2010) and recently in Pakistan
(Cao et al., 2013), whereas CVd-VI seems to be restricted
to Japan (Ito et al., 2003).
Bark cracking and scaling followed by severe dwarfing and yield losses occur in viroid-infected citrus plants
when commercial species are grafted on sensitive rootstocks such as trifoliate orange (Poncirus trifoliata (L.)
Raf.), Rangpur lime (Citrus limonia Osb.) and citranges
(C. sinensis x P. trifoliata) and strain severity is high (BaniHashemian et al., 2009). Considering that more than 90%
of citrus plants in Uruguay are grafted on either trifoliate
orange or citranges, local citrus production may be at risk
(Bisio et al., 2004)
A procedure for viroid detection from field-grown citrus trees originally described by Murcia et al. (2009) and
successfully applied elsewhere (Mohamed et al., 2009) has
been found to be highly sensitive, specific and an efficient
alternative to biological indexing procedures because the
time required for diagnosis is markedly shortened. Briefly,
RNA preparations extracted from bark tissues of fieldgrown citrus trees were subjected to polyacrylamide gel
electrophoresis (PAGE) under non-denaturing conditions
and Northern blot hybridization analysis with viroid-specific digoxygenin (DIG)-labelled probes.
This work reports the results of a survey on the occurrence, prevalence and distribution of CEVd, CBLVd,
HSVd, CDVd, CBCVd and CVd-VI in commercial citrus
species in Uruguay using PAGE-Northern blot hybridization analysis.
A total of 84 mature plants of the genus Citrus and
Fortunella and trifoliate orange seedling trees were surveyed in commercial orchards located in six provinces
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632
Journal of Plant Pathology (2013), 95 (3), 631-635
Citrus viroids in Uruguay
Fig. 1. Map of Uruguay showing the location of Canelones,
Colonia, Montevideo, Río Negro, Salto and San José provinces where field-grown citrus plants were surveyed for citrus viroids infection during 2008-2009 and 2009-2010 growing seasons. Circles show the sampling areas. Further details about
the surveyed materials are shown in Table 1.
of Uruguay during the 2008-2009 and 2009-2010 growing seasons (Fig. 1; Table 1). In each orchard, plants were
randomly selected and sampling included both symptomatic and symptomless trees. Arizona 861-S1 Etrog citrons
(C. medica L.) grafted onto rough lemon (C. jambhiri
Lush.) and infected with different combinations of four
viroid species (CEVd, CBLVd, HSVd, CDVd) were used as
a source of leaf/bark tissue for positive controls. Uninfected and micrografted citron plants were used as negative
controls. Tissues or nucleic acid preparations, kindly provided by colleagues, were used as additional negative and
positive controls. As a whole, 17 positive and 6 negative
control plants were included in this study. In order to get
high viroid titers, infected citron plants, were maintained
at 28-32°C at 16 h light/8 h dark photoperiod. CBCVd and
CVd-VI controls were obtained as monomeric transcripts
from cDNA sequences cloned into TS4.7 and S10S plasmids according to Umaña (2010).
Five grams of bark tissues per tree, collected from
shoots 30-50 cm long and 0.5-1.0 cm in diameter were
processed following the nucleic acid extraction protocol previously described by Semancik et al. (1975) with
minimal modifications reported by Murcia et al. (2009).
DIG-labelling of viroid-specific cDNA probes of CEVd,
CBLVd, HSVd, CDVd, CBCVd and CVd-VI) was carried
out by PCR. In some instances the reverse transcription
PCR (RT-PCR) protocol described by Bernard and Duran-Vila (2006) was used as an additional procedure to
confirm the presence of viroids in nucleic acid samples.
27. JPP1650SC (Pagliano).indd 632
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 membrane (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 silverstained to verify that RNAs had been completely electrotransferred. 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
Applied Science, Germany).
An example of the results obtained by Northern hybridization analyses is shown in Fig. 2, in which RNA
preparations from bark tissues of six citrus varieties were
independently hybridized with four DIG-labelled probes,
each specific for a single viroid. This autoradiography
provided evidence of two-viroid mixed infections in lanes
3 (CBLVd+HSVd) and 4 (HSVd+CDVd) and four-viroid
Table 1. Citrus type, variety and sampling location (province)
of the 84 citrus plants surveyed for viroid infection during
2008-2009 and 2009-2010 growing seasons in Uruguay.
Cultivar
Plants
analyzeda
Locationb
Washington
Navel
19
CA, MO, SJ,
SA
Valencia
late
10
CA, CO, SA
Murcott
 3
CA, SA
Ellendale
 2
SA
C. deliciosa
Common
 7
CA, SA
C. unshiu
Satsuma
14
CO, RN, SA
Lemon
(C. limon)
Lisbon
16
CA, CO,
MO, RN, SJ
Marsh
Seedless
 4
CA
Duncan
 1
CA
Star Ruby
 2
CO, RN, SA
Nagami
 2
CO, MO
Volkamer lemon
(C. volkameriana)
 2
SA
Trifoliate orange
(Poncirus trifoliata)
 2
SA
Citrus type (species)
Orange
(Citrus sinensis)
Mandarin
C. sinensis x C. reticulata
Grapefruit
(C. paradisi)
Kumquat
(Fortunella margarita).
a number
of plants.
bsurveyed provinces: CA: Canelones, CO: Colonia, MO: Montevideo, RN:
Río Negro, SJ: San José, SA: Salto.
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Journal of Plant Pathology (2013), 95 (3), 631-635
Pagliano et al. 633
Fig. 2. Northern blot hybridization analysis of RNA preparations extracted from field-grown citrus plants in Uruguay
hybridized with single viroid-specific DIG probes: CEVd,
CBLVd, HSVd or CDVd (A, B, C, D, rispectively). Lanes 1 to 7: 1, uninfected citron (negative control); 2, common mandarin; 3 Valencia sweet orange; 4, Lisbon lemon;
5, Washington navel orange (sample 1); 6, Star ruby grapefruit; 7, Washington navel orange (sample 2).
mixed infections (CEVd+CBLVd+HSVd+CDVd) in lane
5. In addition to lane 1 (negative control), viroids were
undetectable in the remaining three lanes. No single viroid
infection was found in the samples shown in this figure.
The results were confirmed by RT-PCR for some fieldgrown samples (results not shown).
From the 84 field-grown citrus trees analyzed, 62%
(52/84) were infected with at least one viroid or combinations 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, respectively. 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 frequently detected viroid either in single or mixed infections with 92% (48/52) of the total number of infected
plants, while CDVd, CEVd and CBLVd were detected in
50% (26/52), 23% (12/52) and 21% (11/52) of the positive
samples, respectively. Noteworthy, HSVd+CDVd were
detected in 27% (14/52) of infected plants.
With the exception of grapefruit cv. Star Ruby, viroid infection was found in all the commercial varieties
tested: lemon (81%), grapefruit (71%), mandarine (62%)
and sweet orange (55%). Viroids were also detected in
trifoliate orange but neither in Volkamer lemon (C. volkameriana Ten. & Pasq.) nor in Nagami kumquat (Table
2). Since the sample size for some of these citrus types was
rather small, further sampling efforts would be necessary
to confirm these findings.
This survey complements previous data regarding viroid occurrence, prevalence and distribution in commercial
NDb
HSVd
CBLVd
CEVd +CDVd
CEVd +HSVd
HSVd +CBLVd
HSVd +CDVd
CEVd+HSVd+CDVd
CBLVd+HSVd +CDVd
CEVd+CBLVd +HSVd
CEVd+CBLVd
+HSVd +CDVd
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.
14
10
3
3
2
0
2
32
(38.0)
6
10
4
0
0
1
0
21
(25.0)
0
2
0
0
0
0
0
2
(2.4)
1
0
0
0
0
0
0
1
(1.2)
0
0
0
1
0
0
0
1
(1.2)
1
0
0
0
0
0
0
1
(1.2)
3
4
6
0
0
1
0
14
(16.6)
1
0
2
1
0
0
0
4
(4.8)
2
0
1
0
0
0
0
3
(3.6)
1
0
0
0
0
0
0
1
(1.2)
2
0
0
2
0
0
0
4
(4.8)
na
Citrus species
Orange
Mandarine
Lemon
Grapefruit
Volkamer lemon
Trifoliate orange
Fortunella sp.
29
26
16
7
2
2
2
Total (%)c
84
a
total number of sampled plants.
of plants with non-detectable infection.
c in parentheses: number of infected plants/total number of sampled plants x 100.
b number
27. JPP1650SC (Pagliano).indd 633
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Citrus viroids in Uruguay
citrus orchards in Uruguay (Pagliano et al., 1998) as also reported in other citrus-growing countries (Japan, Italy Cuba,
Brazil, Spain and Sudan) (Malfitano et al., 2005; Murcia
et al., 2009; Mohamed et al., 2009). Our results point out
that viroid infections occur (62%) in many citrus orchards
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
six provinces sampled in agreement with previous preliminary surveys conducted in Uruguay (Pagliano et al., 1998;
Pagliano, 2000). It is also confirmed in other reports that
there is no presence of CBCVd and CVD-VI in any of the
samples analyzed (Umaña, 2010). The failure to detect CBCVd and CVd-VI, confirms other reports indicating a more
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
confirms its high prevalence and worldwide distribution
(Ito et al., 2002). HSVd was found at a slightly lower frequency in single than mixed infections and it was detected
not only in plants older than 40 years but also in young
(three-year-old) symptomless trees.
No attempts were made in this study to differentiate
between cachexia and non-cachexia variants of the viroid.
The occurrence of mixed viroid infections was 20%
higher than single infections (Table 2). Malfitano et al.
(2005) also reported a high frequency of mixed viroid
infections in surveys conducted in southern Italy and
suggested that a high frequency of single and mixed infections in propagation material probably occurred long
time ago and that the use of infected budwood, contaminated tools and top-grafting may have been responsible
for viroid spread and accumulation in individual trees.
Overall, the prevalence of viroid-infected citrus trees in
commercial orchards grafted onto susceptible rootstocks
in Uruguay poses a threat to the current and future citrus production in the country. Therefore, actions against
further viroid dissemination to new plantations should
be undertaken by propagating viroid-free materials under
appropriate certification programs.
Northern blot hybridization analysis has been recently
highlighted as a useful method for viroid detection due
to its high specificity, sensitivity, repeatability and diagnostic efficiency (Mohamed et al., 2009; Murcia et al.,
2009). In the present work, we were able to survey viroid occurrence and distribution from a large number of
sampled field-grown citrus trees in a short time (three
days). Moreover, the results reported here expand those
of earlier surveys performed by using other techniques
(Pagliano et al., 1998; Pagliano, 2000) and update the occurrence and distribution of citrus viroids in the most
important citrus-growing areas of Uruguay. Northern
blot hybridization allowed the analysis of large numbers
of samples that could not be handled using more laborious, time-consuming analysis and/or biological indexing
methods. Consequently, as a molecular diagnostic tool,
27. JPP1650SC (Pagliano).indd 634
Journal of Plant Pathology (2013), 95 (3), 631-635
the Northern blot hybridization analysis meet diagnostic requirements for quarantine and sanitation programs
for propagating materials in Uruguay, either as a primary
screening method or as an additional tool to further support results from indexing tests on indicator plants.
ACKNOWLEDGEMENTS
The authors acknowledge funding provided by BIDCONICYT (Project PDT 74/19). Dr. Takao Ito (NIFTS,
Japan) for providing TS4.7 and S10S plasmids. Jacques
Borde (DGSSAA) and Ana Bertalmío (INIA-Salto
Grande) for providing citron controls and samples.
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Received March 22, 2013
Accepted July 3, 2013
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