Recent advances in Citrus psorosis virus

Abstract

Psorosis is a globally devastating disease of
citrus caused by an infectious filamentous ophiovirus, Citrus
psorosis virus (CPsV), which causes annual losses of about
5 % and a progressive decline of trees by affecting the
conductive tissues. The disease can be harboured asymptomatically
in many citrus species. In the field, the most
characteristic symptoms of the disease in adult trees are bark
scaling in the trunk and main branches and also internal
staining in the underlying wood. The virus has a tripartite
single-stranded RNA genome, and has been inadvertently
spread to most citrus growing areas through the movement
of citrus propagative material. No natural vectors have been
identified except in limited citrus areas in some cases.
Management strategies for CPsV involving shoot-tip grafting
and thermotherapy or somatic embryogenesis from
stigma and style cultures have been successfully used to
eliminate CPsV from plant propagating material. Molecular
pathogen-mediated strategies have been used to produce
citrus plants. Such a strategy protects against infections by
the virus from which the resistance gene and promising
resistance may emerge from trials. Certification programs
are among the best established means of increasing phytosanitary
health, and some of those for citrus are among the
oldest in the world. In conjunction with quarantine and cleanstock programs, they remain important weapons in the
ongoing fight against citrus diseases. One of the elements
essential for successful certification programs to produce
such propagation material is the availability of sensitive and
effective diagnostic methods. In this review, we discuss an
updated status of CPsV disease.

Full text

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VirusDisease
ISSN 2347-3584
VirusDis.
DOI 10.1007/s13337-014-0199-7
Recent advances in Citrus psorosis virus
Asmae Achachi, Essaïd Ait Barka &
Mohammed Ibriz

1 23
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REVIEW ARTICLE
Recent advances in Citrus psorosis virus
Asmae Achachi •Essaı
¨d Ait Barka •
Mohammed Ibriz
Received: 1 August 2013 / Accepted: 31 January 2014
ÓIndian Virological Society 2014
Abstract Psorosis is a globally devastating disease of
citrus caused by an infectious filamentous ophiovirus, Citrus
psorosis virus (CPsV), which causes annual losses of about
5 % and a progressive decline of trees by affecting the
conductive tissues. The disease can be harboured asymp-
tomatically in many citrus species. In the field, the most
characteristic symptoms of the disease in adult trees are bark
scaling in the trunk and main branches and also internal
staining in the underlying wood. The virus has a tripartite
single-stranded RNA genome, and has been inadvertently
spread to most citrus growing areas through the movement
of citrus propagative material. No natural vectors have been
identified except in limited citrus areas in some cases.
Management strategies for CPsV involving shoot-tip graft-
ing and thermotherapy or somatic embryogenesis from
stigma and style cultures have been successfully used to
eliminate CPsV from plant propagating material. Molecular
pathogen-mediated strategies have been used to produce
citrus plants. Such a strategy protects against infections by
the virus from which the resistance gene and promising
resistance may emerge from trials. Certification programs
are among the best established means of increasing phyto-
sanitary health, and some of those for citrus are among the
oldest in the world. In conjunction with quarantine and clean
stock programs, they remain important weapons in the
ongoing fight against citrus diseases. One of the elements
essential for successful certification programs to produce
such propagation material is the availability of sensitive and
effective diagnostic methods. In this review, we discuss an
updated status of CPsV disease.
Keywords Citrus psorosis virus Ophiovirus 
Segmented negative-stranded RNA virus Diagnostics 
Viral resistance Certification
Introduction
Psorosis is a graft-transmissible viral disease of citrus. The
most characteristic symptoms of the disease in adult field
trees are bark scaling in the trunk and main branches. Gum
may accumulate below the bark scales and may impregnate
the xylem producing wood staining and vessel occlusion
[54]. Other symptoms including chlorotic flecking or
spotting in young leaves, and ringspots in leaves or fruits
are sometimes observed, particularly in the spring flush
[33].The disease has been reported from many citrus-
growing areas all over the world [54]. Usually, infected
trees are not killed by the virus but slowly decline and
eventually become unproductive, causing damage to citrus
industry in the Mediterranean basin, and in some areas of
South America. In the 1980s, in Argentina and Uruguay it
was a serious disease causing annual losses of about 5 % of
trees [8] and the disease is still present as recently reported
by Zanek et al. [80]. Another earlier investigation of Paul
W. Moore in California, published in 1957, was conducted
in order to find out the approximate bearing efficiency of an
orchard in which psorosis appears to be the principal cause
of crop reduction. The yields-during the harvest season of a
A. Achachi (&)M. Ibriz
Laboratoire de Ge
´ne
´tique et Biome
´trie, De
´partement de
Biologie, Faculte
´des Sciences, Universite
´Ibn Tofaı
¨l, Ke
´nitra,
Maroc
e-mail: Asmae.Achachi@univibntofail.ac.ma
E. Ait Barka
Laboratoire de Stress, De
´fenses et Reproduction des Plantes,
Unite
´de Recherche Vignes et Vins de Champagne, UFR
Sciences, Universite
´de Reims Champagne-Ardenne,
Reims Ce
´dex, France
123
VirusDis.
DOI 10.1007/s13337-014-0199-7
Author's personal copy

good crop year-of 153 Valencia orange trees affected with
psorosis were compared with the yields of 153 normal
trees. The production of trees in progressive stage of pso-
rosis was 28 % of a normal crop. Although the number of
trees in the comparative study is relatively small and was
taken in only one crop year, the data clearly point out the
role that psorosis plays in reducing Valencia yields even in
favorable crop years.
The causal agent of the psorosis disease is the Citrus
psorosis virus (CPsV). CPsV is classified in the genus
Ophiovirus (Greek ‘ophio’ =snake), indicating the elon-
gated twisted and coiled appearance of the virus particles
[33,36,73].
The last Psorosis review appearing 20 years ago focused
on the identity of the pathogen, methods for indexing,
cross-protection, elimination of the pathogen from propa-
gative budwood, programs for certification and eradication
of psorosis and relation-ship of the citrus ringspot to pso-
rosis [54]. This summary of research may contribute to a
more detailed understanding of CPsV and its relationships
with other members of the genus Ophiovirus. In addition,
an overview of recent advances in the use of CPsV for
virus-induced gene silencing is presented.
Pathology and symptoms
The term ‘‘citrus psorosis group’’ has been used to refer to
several widespread graft-transmissible disorders of citrus
diseases that may not have a common causal agent. Names
for virus-like pathogens causing psorosis-like symptoms on
indicator plants include concave gum, impietratura and
cristacortis, leading to emergence of the idea of a non-
CPsV psorosis-like disease of unknown etiology [71].
Two types of psorosis has been described, psorosis A
(PsA), the more common disease, characterized by the
presence of bark-scaling in the trunk and limbs of infected
field trees (Fig. 1a) and staining of interior wood. The more
aggressive psorosis B (PsB), causes rampant bark scaling
(Fig. 1b, c) even in fine twigs in field trees, chlorotic
blotching in old leaves (Fig. 1d, f) with brownish gummy
pustules in the leaf underside (Fig. 1g), and sometimes
ringspots on fruits [33,54,77]. Bark scaling often appears
in 10–12-year-old trees. However, in the absence of the
severe B form of the disease, the bark-scaling symptom
may be delayed or even absent [53,54]. These symptoms
appear to be caused by CPsV but in the former ‘psorosis
group’ there are other non-CPsV diseases with symptoms
such as chlorotic leaf-flecking and oak-leaf patterns; while
graft-transmissible, these diseases have not yet yielded to
further analysis.
The citrus species most severely affected are sweet
orange, grapefruit, and mandarin. Certain other species
show typical leaf symptoms when infected, but remain free
of bark lesions without obvious ill effects. Sour orange and
lemon fall in this category [37,54].
Apart from CPsV, several other diseases can cause
psorosis-like bark scaling on citrus, particularly, leprosis
(most commonly caused by Citrus leprosis virus-C), Bahı
´a
bark scaling, foot rot caused by Phytophtora, Rio Grande
gummosis, and genetic disorders including lemon shell
bark and sunscald [33,37], and psorosis-like bark scaling
reported from Spain noted in some trees apparently free of
CPsV by serology and psorosis-free by biological indexing
[32].
Historical background
The literature on psorosis identification has become
noticeably confused over the years. Since bark scaling was
the first and characteristic symptom of the disease, some
psorosis reports were based on the simple observation of
this symptom without further testing for its transmissibility.
After observation that grafts from bark-scaled trees were
able to induce young leaf symptoms in sweet orange
seedlings, graft inoculation of this indicator plants was
used for quick psorosis diagnosis [53,54], although other
graft-transmissible diseases were later shown to induce
similar symptoms in the same test [54]. The discovery that
lesion bark inoculum was able to induce PsB in sweet
orange seedlings, and that only PsA infected seedlings
showed cross protection against PsB and they do not
develop leaf pustules or twig blisters characteristic of the
PsB, provided a specific diagnostic test [53,54]. However,
many subsequent reports on psorosis diagnosis failed to use
it. Additional confusion was added by the consideration of
ringspot-type psorosis as a different disease from psorosis
[54]. Citrus ringspot have been described as a different
bark scaling disease, inducing chlorotic flecking and
spotting of young leaves with later developed into yellow
blotches vein banding and/or distinct rings in mature leaves
of several indicators species. Particularly, it was associated
with severe PsB type bark scaling.
Later on the basis of cross-protection tests [10], the
similarity of symptoms and filamentous morphology
between ringspot and psorosis isolates [19,42], and dif-
ferences in number of particles of CPsV detected by
immunosorbent electron microscopy (ISEM) using an
antiserum to the isolate CPV-4 [43], it was suggested that
ringspot and psorosis are variants of the same disease. The
same conclusions were advanced by Da-Grac¸a et al. [7] and
A. Achachi et al.
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Fig. 1 Symptoms characteristic of the psorosis A (PsA) and psorosis
B (PsB) syndromes in field trees in Northeast and Central Morocco
(a–d) or in indicator plants in the greenhouse at the index facility at
Kenitra, Morocco (e–g). PsA symptoms include bark scaling in the
trunk of field trees (a), a shock reaction with leaf shedding of the first
flush, and transient chlorotic flecking in young leaves of the following
flushes (e) in indicator sweet orange seedlings. PsB symptoms in the
field include rampant bark scaling (b–c) and chlorotic blotching in old
leaves (d). In indicator sweet orange seedlings PsB isolates incite the
shock reaction observed with PsA isolates (e), chlorotic blotches in
old leaves (f) with gummy pustules on the underside (g)
Recent advances in Citrus psorosis virus
123
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Rouag et al. [56] respectively, who used serological and
molecular comparisons.
Geographical distribution
Psorosis is an ancient disease; bark-scaling of citrus was
first observed in Florida and California in the 1890s [54].
The disease has been brought under control in most
advanced citrus-growing countries due to rigorous indexing
and quarantine [71]. Differences in the extent of infection
are found between countries. For instance, In Italy, the
spread of the disease is higher compared to other Medi-
terranean countries [2]. Nevertheless, these data may be
explained by a lack of data in other Mediterranean coun-
tries, particularly those in North Africa.
Host-virus relationships
Histology
CPsV particles occur in phloem and parenchyma cells of
citrus. No virus particles or any specific inclusion or
cytopathology have been detected in thin sections of
infected tissues [37]. However, a more recent study
detected the presence of inclusion bodies in the intercel-
lular spaces of infected leaves of grapefruit seedlings
(25 days post inoculation). These structures could contain
virus particles, but they are not the only structures to do so.
It should also be noted that the study of anatomical and
structural changes on grapefruit leaves infected with CPsV
have demonstrated that a set of alterations appear in the
microscopic structure of the leaves showing that CPsV
infection clearly affected the conductive tissues. The main
cytopathic changes in infected citrus cells were the pre-
sence of large number of abnormal chloroplasts, mito-
chondria and cellular abnormality and the presence of
hypertrophied nuclei. Other morphological changes such as
the presence of an unstructured parenchyma in the upper
epidermis, reduction of the number of oil glands, the
appearance of secondary growth in midvein and major
lateral veins in smaller veinlets, can be attributed to dif-
ferent indirect effects occurring in the host plant, for
instance the lower auxin level resulting from virus infec-
tion and not attributed to the virus itself [62].
Distribution of CPsV in the host
The heterogeneity of CPsV was established by Barragan-
Valencia et al. [4]. An initial test was to determine the
concentration of viral samples from field and it turned out
that the presence of virus in the tree was not uniform since
it was detected in only 40 % of the analyzed samples. In a
subsequent test, two assays were made to determine viral
concentration in leaves of 10 Mars orange trees; in the first,
quantitative detections were made in three different sam-
ples of a single leaf from each tree, whereas in the second,
detection were made in three samples of different leaves
from each tree. In the first assay, the virus was detected in
four trees but in the second assay, the virus was detected in
nine of the trees evaluated. Therefore, it is considered that
the distribution of the virus in field trees is not uniform
confirming thus the first observation. However, the distri-
bution of virus in the greenhouse plant shows a greater
homogeneity of viral concentration in the analyzed
samples.
Virus transmission
The virus is graft-transmitted, but symptoms rarely appear
before trees are 10 years old; consequently, growers often
propagate psorosis infected buds from symptomless trees,
explaining partly the high disease incidence in some areas
[32]. Some virus isolates are, though sometimes not easily,
mechanically transmissible to Chenopodium quinoa and
produces necrotic local lesions, and systemically infected
Gomphrena globosa [37,54]. More recent tests of seed
transmission provided no experimental evidence for the
transmission of CPsV through the seed in the mandarin and
sour orange [13].
However, several reports on the increase in incidence of
citrus psorosis symptoms in the field suggested a possible
natural spread of the disease. For instance, observation in
Texas, in nucellar Redblush grapefruit trees and in origi-
nally virus-free Rio Red grapefruit trees supports the
hypothesis that natural transmission occurs [8,21,54,61].
Spatial and spatio-temporal analyses of data from surveys
over a period of 3 years in four citrus orchards in Texas
containing psorosis-infected trees were consistent with
disease spread via vectors [21]. This information changed
considerations in the context of phytosanitary controls,
because, for the moment, psorosis was not considered as a
quarantine pest by any regional organization for protection
of plants [16].
At the present stage of research, it was suggested that
the vector is a species of the Oomycete genus Olpidium
[44]. Focusing on the Oomycete, Palle et al. [44] showed
that an Olpidium-like species is associated with citrus roots
(the study involved the grapefruit), and that CPsV could be
detected in zoospores from infected roots. Further work is
required to confirm if this Oomycete is able to transmit the
virus to healthy citrus. In 1998, T. Natsuaki and T. Mor-
ikawa have indicated that the vector of Tulip mild mottle
mosaic virus (TMMMV) is Olpidium brassicae (personnel
A. Achachi et al.
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communication). Lettuce big-vein associated virus
(LBVaV) (genus Varicosavirus) and Mirafiori lettuce big-
vein virus (MiLBVV) (genus Ophiovirus) are transmitted
by Olpidium virulentus, a noncrucifer strain of O. brassicae
[60], and Lettuce ring necrosis virus (LRNV) (genus
Ophiovirus) is also transmitted by O. brassicae [64]. A new
soil-transmitted ophiovirus associated with freesia leaf
necrosis disease [68], Freesia sneak virus (FreSV) is likely
involved [69,72]. In lettuce big-vein disease it has been
shown that zoospores of Olpidium released from sporangia
contain the virus in their protoplast and transmit to the
roots. The virus is also found in dormant spores, which
survive for years in the absence of host [30].
Diagnosis
Detection, identification and differentiation of viruses and
viroids infecting various crops constitute the basic steps for
the development of effective crop disease management
systems. The tools used to detect CPsV in citrus trees are
mainly based on using indicator plants and on laboratory
tests including enzyme-linked immunosorbent assay
(ELISA) [20,33,80]. After CPsV sequences became
available, molecular tests based on RT-PCR and dot blot or
tissue-print hybridization were also developed for CPsV
detection [5,20,26,28,32,55,57,67].
Biological indexing
Psorosis is currently diagnosed by biological indexing on
sensitive indicator plants, usually sweet orange seedlings
(Citrus sinensis) growing in a temperature-controlled
(18–26 °C) greenhouse [53,54]. Characteristic symptoms
include a shock reaction with leaf fall and shoot necrosis in
the first flush and/or chlorotic leaf flecking and spotting in
the following flushes (Fig. 1e). Since the latter symptoms
are also induced by other graft-transmissible diseases,
specific diagnosis of psorosis additionally requires a cross
protection test using PsB [53,54]. In this test, sweet orange
seedlings pre-inoculated with the candidate psorosis isolate
are challenge-inoculated with PsB. The latter psorosis type
additionally incite chlorotic blotching in old leaves with
gummy pustules on the leaf underside (Fig. 1f, g), similar
to symptoms observed in field trees, and blisters on the
stem and twigs that usually evolve to produce twig necrosis
[53,54]. It is important to state that sweet orange varieties
will differ in their ability to show shock or leaf symptoms
[53]. Psorosis symptom expression is affected by temper-
ature, with cool temperatures favouring appearance of the
shock reaction in the first flush after inoculation, while
warm temperatures often inhibit shock and mask leaf
symptoms [42,76]. Virus titre estimated by ELISA and by
northern and dot blot hybridization paralleled symptom
intensity, with significantly higher virus accumulation in
plants incubated at 26/18 °C[76]. The amount of CPsV-
derived small RNAs (CPsV-sRNAs) slightly increased at
32/26 °C, with the ratio of CPsV-sRNA/vRNA being
higher at 32/26 °C than at 26/18 °C. These results suggest
that CPsV infection induces RNA silencing in citrus plants,
that symptom intensity is associated with virus accumula-
tion, and temperature increase enhances the RNA silencing
response of citrus plants and decreases virus accumulation
[76].
Immunological-protein based methods
Rapid tests for detecting CPsV incidence and severity have
been developed using serology, and tested during CPsV
regional, country and state surveys. Availability of an
improved antiserum [20] and of monoclonal antibodies
(MAbs) to the coat protein (CP) [1,46], has allowed
detection of CPsV by double and triple antibody sandwich
(DAS- and TAS-) ELISA, in biologically characterized and
non-characterized psorosis sources [2,12,32]. Monoclonal
antibodies (24 MAbs) raised to the southern Italian CPsV
isolate IAM-191Xa have been found to be useful to dis-
tinguish between CPsV isolates. These MAbs were tested
singly by ELISA against a panel of 40 psorosis sources
from different geographical areas (Italy, Lebanon, Spain,
and USA). Sixteen different epitopes were identified and
considerable serological variability, apparently associated
with the geographical origin of the isolates was found [11],
but no data are available on the sensitivity of detection
achieved. Variation in the epitopes present in the CP of
different CPsV isolates [3,11] makes it advisable to use a
mixture of MAbs [32].
The antisera produced against recombinant viral CP
form an additional or alternative tool for manufacturing
standardized kits for serological detection of viruses like
CPsV that are difficult to purify. Further, in the indirect
DAS-ELISA format, the polyclonal antibodies (PAb)
developed against the recombinant CP protein was effi-
ciently and successfully employed in place of monoclonal
antibody used earlier for the detection of CPsV [27].
Alternative enzyme
Apart from AP and penicillinase (PNC), the enzymes
horseradish peroxidase (HRP) and urease are also suitable
for use in ELISA. Of these, HRP is the most widely used.
Its substrate is the chromogenic 3, 30,5,5
0-tetramethyl
benzidine dihydrochloride (TMB) or o-phenylenediamine
(OPD). Zanek et al. [80] describe the use of HRP for
detecting the presence of the virus in the field samples and
Recent advances in Citrus psorosis virus
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in the transgenic sweet oranges expressing very low
amounts of viral CP. HRP is more reliable, faster and more
sensitive than AP with no significant variability and has a
lower detection limit, since the AP reaction takes from 17
to 24 h instead of 30 min for the TAS-ELISA-HRP. Using
this improved method, the presence of psorosis has been
confirmed in the northwest region of Argentina.
Tissue blot immunoassay
Tissue-blot immunoassay (TBIA) is a variant of ELISA has
been developed for the detection of CPsV. Localization of
plant viruses has differentiated the types of tissues that
support virus replication/accumulation. Direct TBIA tech-
nique was employed for the detection of CPsV in the
ovaries of infected flowers [15]. Interestingly, CPsV was
detected in the ovary explants from closed flower more
than ovary from open flowers. DTBIA also represents a
very convenient and safe system for shipping blotted
membranes from one place to another [15].
The comparative efficacy of DAS-ELISA, TAS-ELISA
and DTBIA in detecting CPsV was assessed [32]. All the
three assay procedures detected CPsV readily in young
shoots and leaves. However, DTBIA was less efficient in
detecting the virus in old leaves. The presence of CPsV in
nine different citrus varieties was efficiently detected by
ELISA formats and DTBIA, indicating that CPsV accu-
mulated to equivalent levels in the varieties tested. There
was good correlation between results of immunoassays and
that of biological indexing [32]. The results showed that
CPsV in infected trees was clearly detected in old hardened
leaves in winter [32]. DTBIA is simpler, less expensive and
faster than ELISA providing the same level of specificity,
if young leaves are selected for testing. Furthermore, tissue
prints can be prepared in the field and stored for long
periods without loss of reactivity, making DTBIA more
useful and convenient technique for epidemiological stud-
ies. In addition, several membranes can be printed with the
same shoots for future processing with new antibodies and
tissue-printed membranes may be failed to a laboratory
where the required antibodies are available [32].
Transversely cut tender shoots, or whole flowers, or
rolled old and young leaf blades of citrus trees infected by
CPsV were gently pressed onto nitrocellulose membranes.
After adopting standard step of TBIA, the blotted mem-
branes were incubated with alcaline phosphatase conju-
gated As-Ps.Rc1 IgGs at a dilution of 1:500. Membranes
were incubated in substrate solution of BCIP-NBT Sigma
fast in distilled water for the development of purple color.
CPsV was readily detected in tissue samples from tender
shoots, flowers and young leaves collected during spring,
but not from hardened old leaves. Similar results were
observed with the various ELISA protocols and immuno-
sorbent electron microscopy. All these tests showed that
As-Ps.Rc1 antiserum was specific in detecting CPsV in
citrus samples, irrespective of the technique applied [27].
Immunosorbent electron microscopy
Antisera have been used to detect CPsV particles using
immunosorbent electron microscopy (ISEM) [9,10,19,36,
43]. ISEM technique was also applied for the efficient
detection of CPsV in cross-protected citrus plants [33].
Recently, CPsV was extracted from infected leaves of C.
quinoa. Electron microscope grids were sensitized by
floating them on drops of crude antiserum and then they
were floated on plant extracts at 4 °C overnight. The
adsorbed virus particles were stained with 2 % uranyl
acetate and examined under the electron microscope. The
CPsV particles trapped from crude plant extracts were
observed in infected leaves of C. quinoa [27].
Molecular-nucleic acid based methods
Nucleic acid-based virus detection systems make use of
cloned DNA probes in a dot-blot assay or specifically
designed primers in PCR-test. Dot-blots are unlikely to
differentiate by a single nucleotide and PCR may fail to
detect an isolate where the 30nt of the primer mismatches
the template, thus failing to detect rather than differenti-
ating isolates. These two approaches will be summarized in
the following paragraphs.
Dot-blot assay
This development in nucleic acid hybridization technology
offers a great potential for virus detection. CPsV can be
detected by dot blot or tissue print hybridization using full-
length
32
P-labelled riboprobes of the CPG [5]. Positive
sense probes hybridized to several isolates in tissue print
and dot-blots at 55 °C. At this temperature, non-homolo-
gous isolates could be detected without any appreciable
hybridization to host RNA. However, only the homologous
isolate CPV-4 was detected when hybridization tempera-
tures was at or above 60 °C. Some isolates of CPsV that
could be detected by RT-PCR could not be detected by
hybridizations assays due to low titre of the virus. Never-
theless, these isolates were not detected at hybridization
temperatures lower than 55 °C due to non-specific reac-
tions with host nucleic acids [5].
Recently, a dot blot hybridization protocol using dig-
oxigening-labelled riboprobes was finalized for the detec-
tion of CPsV and Citrus variegation virus (CVV) [28]. In
screenhouse analysis, CPsV detection rate in young leaves/
flowers was similar by ELISA and dot blot hybridization
A. Achachi et al.
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during spring. The higher sensitivity of the molecular with
respect to the serological assay was more evident in aged
leaves tested in winter, since CPsV was detected in 40
samples instead only 22 samples by ELISA. The higher
efficiency of the dot blot hybridization assay over ELISA
was confirmed by the results obtained over a 9 month
period, testing different tissues (aged leaves, young leaves,
flowers, fruit, peel and flavedo, juice and seeds) from
selected field-grown CPsV infected plants, molecular
detection was possible throughout the year from one organ/
tissue or the other, although the highest detection rates
were in spring from flowers and young leaves. It can be
concluded that dot blot hybridization is more sensitive than
ELISA and can be usefully applied for routine large-scale
diagnosis, thus constituting a useful alternative to sero-
logical assays.
RT-PCR methods
The CPsV genome consists of three single-stranded RNAs
of negative polarity. RNAs 1, 2 and 3 of the isolate CPV-4
have been sequenced [5,39,58,59]. RNA extracted from
citrus infected tissue is assayed for the presence of CPsV
sequences by RT-PCR, cDNA copies are made and
amplified for analyses. Based on increasing sensitivity
criteria, RT-PCR methods are classified as conventional,
nested, and real-time modes. Primer design must thus take
into account both the mode (conventional, nested, or real-
time) and the range of specificity of the RT-PCR to be
applied.
Portions of RNA1 have been cloned and a consensus
sequence was obtained from which primers were designed
for RT-PCR [20]. The CPG has been used to develop RT-
PCR [5]. In both cases, the RT-PCR was able to detect
CPsV at concentrations substantially lower than those
required for detection by immunoassays and electron
microscopy [20]. However, the sensitivity has not been
sufficiently high to detect the heterologous psorosis isolates
using primers designed from CPV-4. Nested RT-PCR is
usually applied when RNA sample concentration is low; it
is based on the use of the first PCR product as starting
material for the second PCR round. In some instances, a
hemi-nested RT-PCR is used, where one of the second-
round primers, usually the forward one, is the same as in
the first-round RT-PCR. For the CPsV, first step of the
hemi-nested RT-PCR is carried out as described above.
The second step (hemi-nested) is carried out with internal
forward (primer 6) and reverse (primer 7), yielding a
195-bp amplified fragment. This test is able to detect CPsV
in an amount as small as an equivalent of 10
10
of the ori-
ginal sample. This represents an improvement of 10
6
-fold
from previously reported sensitivity [26]. Disadvantages of
nested PCR include additional time and cost associated
with the two stages of RT-PCR, and the increased risk of
contamination incurred during transfer of first-stage
amplification products to a second tube.
Simplex PCR detects one target per reaction and
requires different PCRs to detect multiple targets. Multi-
plex PCR (mPCR) is a useful technique since different
RNA and DNA viruses may infect a single host like citrus.
Seven viruses infecting citrus viz., Citrus leaf rugose virus
(CLRV), C. psorosis virus (CPsV), C. tatter leaf virus
(CTLV), C. tristeza virus (CTV), C. variegation virus
(CVV), C. yellow mosaic virus (CYMV), and Indian citrus
ringspot virus (ICRSV) belonging to six different virus
genera were detected simultaneously by the multiplex PCR
(mPCR) assays [57]. Degenerate primers were designed
based on the sequences of respective virus isolates. The
cDNA fragments (245-942 bp) specific to the viruses were
simultaneously amplified using mPCR and they were
identified on the basis of their molecular sizes. The con-
sistent results of the mPCR were compared with simplex
PCR for detection of each virus pathogen. In most cases the
detection sensitivity for the mPCR at equivalent concen-
trations was less than for the simplex PCR assay. The
detection sensitivity of multiplex PCR decreased for
CLRV, CPsV and CTLV, but increased in case of CVV and
CYMV detection. The reason for this decrease may be that
in the multiplex assay the cocktail primers compete for all
the seven viral templates rather than for one and therefore
the detection end point is lower. This could pose a problem
for some assays, the use of the mPCR system needs testing
with naturally infected materials in conjunction with bio-
indexing methods. The diagnostic technique reduces the
risk of contamination, saves time and reduces the cost as
compared to other conventional methods for citrus virus
detection. Furthermore, the mPCR provides a useful rapid
method for detecting multiple virus infections in citrus
plants that will aid in the production of virus-free citrus
plants for certification programs [57].
An improved one-step RT-PCR was also developed for
the detection of CPsV in citrus trees. The test was more
sensitive than the ELISA and as accurate as bioasays [55].
Real time RT-PCR
This approach has provided insight into the kinetics of the
PCR reaction and it is the foundation of ‘real time’ PCR.
According to recent research from Bari, Italy, rapid and
reliable methods for detecting multiple pathogens are
important for routine diagnosis by reducing time, labour
and costs. To this end, primers and TaqMan probes for
CPsV and CVV detection by singleplex realtime (q) RT-
PCR were initially designed. Further optimizations inclu-
ded the development of a multiplex (m) RT-qPCR assay to
detect simultaneously CPsV, CVV, and CTV in a single
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reaction. When tenfold serial dilutions prepared using total
RNAs from CPsV- and CVV-infected plants were tested,
RT-qPCR assays proved to be 100 and 1,000 times more
sensitive than conventional RT-PCR, respectively. The
target viruses were effectively identified by mRT-qPCR in
field infected clementine and sweet orange trees. A one
step real-time RT-PCR protocol has been successfully
developed and validated to detect CVV and CPsV in citrus
trees. Rapid and accurate multiple detection of CTV, CPsV
and CVV was achieved using the triplex one step real-time
RT-PCR protocol. These assays may prove useful as a tool
to support quarantine, eradication and certification pro-
grams, resulting faster, more sensitive, relatively easier to
perform and less cost effective than the conventional
detection procedures [29].
Single-strand conformational polymorphism (SSCP)
The Single-stranded conformation polymorphism (SSCP)
of sub-isolates of PsA and PsB of CPsV showed identical
SSCP profiles in homologous segments of the RNAs 1 and
3. Segment of the RNA2, representing distinct SSCP pro-
files. The SSCP analysis of RNA2 reveals two kinds of
variants of CPsV. Analysis of the RNA2 population
showed that PsA-inducing isolates contain PsB associated
sequence variants at low frequency, the PsB-associated
sequence variant is predominant in blisters twigs and
gummy pustules affecting old leaves, characteristic of PsB
isolates, and the PsB-associated sequence variant accu-
mulates preferentially in bark lesions of the trunk and
limbs. SSCP analysis of the RNA2 population also enabled
monitoring of interference between PsA and PsB associ-
ated variants in plants co-inoculated with both psorosis
types [77].
Citrus psorosis virus characterization
Many CPsV isolates have been described. The original
isolate CPV-4 produces necrotic local lesions when upon
mechanical inoculation of the older, fully expanded leaves
of C. quinoa [53,54]. Another factor is that CPV-4 can
reach relatively high concentrations in infected tissues [20].
Ophiovirus taxonomy
Ophiovirus (Family: Ophioviridae) have single stranded
negative/possibly ambisense RNA genome (11.3-12.5 kb
size) divided into 3-4 segments, each encapsidated in a
single CP (43- 50 kDa) forming filamentous virions about
3 nm in diameter, in shape of kinked (probably internally
coiled) circles of at least two different contour lengths
[19,73]. Since only one genus is currently recognized, the
family description corresponds to the genus description.
The genus Ophiovirus is currently comprised of five rec-
ognized species: CPsV, TMMMV [38], Ranunculus white
mottle virus (RWMV) [66], MiLBVV [52], LRNV [63,64]
and FreSV [68]. The first ophiovirus described was dis-
covered in citrus, but most of them has been found in
ornemantal plants as ranunculus (dicotyledonous), freesia,
tulips and lachenalia (monocotyledonous), and lettuce
(dicotyledonous). The true fungi of the species O. brassi-
cae transmit viruses of the genera Ophiovirus and Vari-
cosavirus. These viruses are transmitted in a mode referred
to as ‘in vivo’, which resembles circulative transmission
because the viruses enter into the cytoplasm of the vector,
mostly zoospores from where they are released, presum-
ably during germination [51].
Assessment of the biological activity
A comprehensive review of the conditions, husbandry,
tools, grafting techniques, assessment of symptoms and
indexing methods used for CPsV has earlier published by
Roistacher [53]. Briefly, lateral buds or ‘‘blind buds or chip
buds’’ from the donor plant are graft-inoculated onto the
stem of a receptor or indicator plant, and usually this is
repeated in quadruplicate. Uninoculated receptors are kept
under the same conditions as control plants. Well charac-
terized CPsV isolates can be used also as controls in tests.
A panel of six citrus cultivar or species combinations is
generally used for the strain characterization. Visual
assessments of the subsequent growth from the receptor
plants are noted into two flushes after inoculation for each
of the six citrus cultivar or citrus species combinations.
Symptom expression in each host-isolate combination was
quantified by a pathogenicity index (PI) that considered
symptom intensity and the number of plants showing each
symptom. A general pathogenicity index (GPI) was defined
for each isolate as a weighted mean of the different PI.
Generally, the higher the cumulative GPI score, the more
severe the CPsV isolate [42].
The Citrus psorosis virus genome
CPsV, the type species of the genus Ophiovirus, is the
presumed causal agent of psorosis disease [33]. CPsV vi-
rions are kinked filaments, 3–4 nm in diameter [19] that
separate into two components during sucrose gradient
centrifugation [9,18]. They consists of three single-stran-
ded RNA (ssRNA) of negative polarity (vRNA) and a coat
protein (CP) of *48 kDa [5,58]. RNA 1 is *8,184 nt in
size and its viral complementary strand (vcRNA) has two
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open-reading frames (ORFs), The first ORF potentially
encodes a 24 kDa protein of unknown function and, sep-
arated by an intergenic region of 109 nt, a second ORF
potentially coding for a 280 kDa protein contains between
positions 635 and 874 the core polymerase module with the
five conserved motifs proposed to be part of the RNA
dependent RNA polymerase (RdRp). vcRNA 2 (*1,644 nt)
carries one ORF coding for a 54 kDa protein of unknown
function, which includes a motif resembling a nuclear
localization signal (NLS), and vcRNA 3 (*1,454 nt) codes
for the CP of 48, 6 kDa [5,39,58,59].
In the 30-terminal untranslated region of RNA2 there is a
putative polyadenylation signal [59], was also found in the
30UTR of the positive strand of RNA3 [58] and RNA1 [39].
The 30-terminal regions of the three CPsV RNAs are very
similar, while their 50-terminal regions in contrast, differ
considerably in sequence.
Genetic variability of the coat protein gene
Variability of the CP gene of CPsV was assessed sero-
logically and by sequence analysis of two genomic regions
located in the 30(region C) and 50(region V) halves of the
gene [3]. Serological analysis of the 53 CPsV field sources
from Campania with 23 MAbs revealed nine reaction
patterns (serogroups) and at least ten different epitopes.
Reaction pattern was not strictly associated to location of
the field trees or citrus cultivar. Phylogenetic analysis of
the V and C regions showed that the CPV-4 isolate was
clearly separated from Campania sources. Furthermore, a
low value of ratio between nonsynonymous and synony-
mous substitutions (d
N
/d
S
) was estimated for all CPsV
isolates, indicating negative selective pressure for amino
acid changes, more intense in the C region [3].
The genetic variations of distinct populations of CPsV
for 22 isolates from Argentina, California, Florida, Italy
and Spain were studied and compared with the control
isolate CPV-4 from Texas [35]. Sequence variability was
studied using cDNA of regions R1, R2 and R3, located on
genomic RNAs 1, 2 and 3, respectively. The three regions
selected represented about 12 % of the genome and vari-
ations internal to each population were minimal, suggesting
a genetic variation among three population groups. Group 1
is the group of isolates from Spain, Italy, Florida and
California, group 2 including the population of Argentina,
which form a genetically homogeneous population and
clearly separated from other populations, and group 3,
including the isolate CPV-4 from Texas was distant from
both groups. Incongruent phylogenetic relationships in
different genomic regions suggested that exchange of
genomic segments may have contributed to CPsV evolu-
tion [35]. In general, genetic variation of plant negative-
stranded RNA viruses have been less studied than those of
positive-strand RNA viruses. Knowledge of the structure
and variation of viral populations is often necessary for
diagnostic, epidemiological and control purposes [35].
These results point to the need to consider the research
programs and control specifically regional.
Insights into virus biology
The molecular and cellular aspects of the infection cycle
of ophioviruses have not been investigated yet. To gain
insight into CPsV biology, Pen
˜a et al. [45] have studied
the subcellular localization and functions of the proteins
24 K, 48 K and 54 K. The subcellular localization of the
CP and 54 K proteins was analyzed by subcellular
fractionation of infected C. quinoa and by confocal laser
scanning microscopy (CLSM) using GFP fusion proteins
expressed transiently in Nicotiana benthamiana. The
54 K protein was found in the nuclear, cytoplasmic, cell
wall and microsomal fractions. By CLSM, the 54-GFP
fusion protein was found in the nucleus, cytoplasm and
in the plasmodesmata. Applying the same strategies, the
CP was only localized to the cytoplasm but not in other
subcellular fractions. The 54 K is probably involved in
virion movement and suppression of post transcrip-
tional gene silencing (PTGS), the antiviral defence
mechanism of the plant. These two mentioned functions
seem to be shared with the 24 K protein of CPsV, but
not CP, have shown systemic suppressor activity [31,
45]. The results indicate that CP is limited to encapsi-
dation while the 24 K and 54 K proteins may be
involved in suppression of systemic RNA silencing and
viral movement [31,45].
Relationships with other members of the genus
Ophiovirus
CPsV is the type species of the genus Ophiovirus, Family
Ophioviridae [73]. The other species of this genus include
TMMMV [38], (RWMV) [66], MiLBVV [30,52,74],
LRNV [63,64] and FreSV [68]. All these viruses have
similar morphology: circular, filamentous, naked nucleo-
capsids of variable lengths and about 3 nm in diameter.
RWMV has at least three RNAs of approximately 7.5, 1.8
and 1.5 kb coated by a 43 kDa protein [66], and its RNA1
has been partially sequenced. TMMMV has a 47 kDa CP
[38], and MiLBVV contains four RNAs of 7.8, 1.7, 1.5 and
1.4 kb and a 48 kDa CP with similarity to the CP of CPsV
[52,74]. The complete sequence of MiLBVV RNAs,
potentially encoding seven ORFs, has been determined
[74]. LRNV contains four RNAs of 7.6, 1.8, 1.5 and 1.4 kb
and a 48 kDa CP [64], RNA 2 is about 1.7 for FreSV and
the RNA3 is 1.5 kb with a 48.4 kDa CP [70]. Negative and
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positive stranded RNAs of ophioviruses are encapsidated in
a single CP of 43-50 kDa [5,18,66].
The 30–terminal regions of the three CPsV RNAs are very
similar. In contrast, their 50- terminal regions differ consid-
erably in sequence. The circular morphology of CPsV viri-
ons suggests the possibility of a panhandle structure formed
by base pairing between the two terminal regions of each
RNA [19]. The first 75nt of the CPsV RNA1 terminal
regions show 41 % complementarity, a value similar to that
described for the RNAs of MiLBVV and of the lettuce big-
vein varicosavirus [39]. However, Naum-Ongania et al. [39]
observed that secondary structure predictions do not support
significant complementary between the termini of the viral
RNAs to afford panhandle formation, or of a ‘‘Corkscrew’’
structure as proposed for Influenza A virus and MiLBVV.
None of these structures were predicted by the MFOLD
program for any of the CPsV RNAs [39]. On the contrary, in
MiLBVV, the terminal sequences do carry partial inverted
repeats, potentially allowing panhandles to form; the ter-
minal sequences also contain palindromes potentially able to
fold into stem-loops, not found for CPsV sequences [74].
Alternative explanations for the observed circular structures
of CPsV are required [39].
At their 30termini, the three CPsV viral RNAs have
a conserved 12 nt sequence, A
7
GUAUC, similar to
the A
4-6
UAAUC found in MiLBVV RNAs [74], and
A
7
GUAUCA and A
3
UA
3
GUAUCA for LRNV [67]; which
might be involved in the recognition of the RNAs by the
RdRp [39].
Sequence analysis of CPsV RNA1 showed in its com-
plementary strand two ORFs potentially encoding proteins
of 24 kDa and 280 kDa, separated by an intergenic region.
Downstream the 50-UTR, the first ORF potentially encodes
a 24 kDa protein of unknown function is encoded. The
109-nt intergenic region following this ORF, (isolate CPV-
4 from Florida), is rich in A ?U (88.3 %) and contains an
18-nt sequence (UUAAAA)
3
that could form a hairpin
loop. Near the end of the intergenic region, a typical
AAUAAA polyadenylation signal is found 12 nt upstream
of the CA start polyadenylation site [39]. However, these
sequences were not found for the CPsV Spanich isolate
P-121 [34], neither for MiLBVV (intergenic region of 147
nt, 66 % of A ?U), and LRNV (intergenic region of 80 nt,
65%ofA?U) [67,74]. A second ORF potentially
coding for a 280 KDa protein of 2416 amino acid residues
was found after the intergenic region [39].
Interestingly, RNA1 of MiLBVV, the other ophiovirus
sequenced, also has two separate ORFs of similar size in its
complementary strand [74]. This is a taxonomically sig-
nificant feature because none of the segmented negative-
stranded RNA viruses possess such a genomic organization
[39]. The 24 KDa protein has no significant similarity with
other entries in data banks, including the 25 kDa ORF
encoded in the complementary strand of MiLBVV RNA1,
whereas the 280 KDa protein, which contains the five
conserved motifs proposed to be part of the RdRp, shows a
significant similarity with the 263 KDa protein also enco-
ded in the complementary strand of MiLBVV RNA1 [39].
These RdRps contain the SDD sequence which is a sig-
nature for segmented negative-stranded RNA viruses
(Orthomyxoviridae,Arenaviridae and Bunyaviridae fami-
lies) as opposed to the GDNQ characteristic of negative-
stranded non segmented RNA viruses (Rhabdoviridae and
Paramyxoviridae families). These data support grouping
both viruses in the same genus Ophiovirus [39] previously
advanced on the basis of their similar virion morphology
and other molecular characteristics [36]. Vaira et al. [67]
designed primers for an-ophiovirus-specific RT-PCR test
based on sequence similarities in conserved portions of the
ophiovirus RNA-1 polymerase encoding region. Phyloge-
netic studies based on comparisons of amino acid
sequences of the conserved portions of the polymerase of
MiLBVV, as well as two other ophioviruses, RWMV and
CPsV, suggest that ophioviruses represent a monophyletic
group that is separated from the other negative-stranded
RNA viruses, reinforcing the taxonomic relatedness of the
group [39]. Furthermore, RNA1 s of MiLBVV, CPsV and
RWMV are similar in size and those of MiLBVV and
CPsV also in genomic organization and sequence [39].
A protein about 50-55 kDa of unknown function is
encoded by RNA 2 in the positive strand of the ophiovi-
ruses CPsV, MiLBVV and LRNV [59,64,74]. The
54 KDa protein of CPsV has been detected in infected
tissue confirming its size and coding assignment [45]. It is
probably involved in virion movement and suppression of
post transcriptional gene silencing (PTGS), the antiviral
defence mechanism of the plant [31,45]. These two
mentioned functions seem to be shared with the 24K pro-
tein of CPsV.
On the other hand, the nuclear localization signal (NLS)
present in the 280 kDa protein was also found in the
54 kDa protein encode in the complementary strand of
CPsV RNA2 [59]. Naum-Ongania et al. [39] have observed
that a similar motif is present in the 263 kDa and 55 kDa
proteins of MiLBVV (between amino acids 222-238 and
275-291, respectively), and in the RWMV amino acid
sequence (positions 368-384), suggesting that in Ophiovi-
ruses, as in plant nucleorhabdoviruses, replication could
occur in the nucleus. In any case, gold immunolabeling and
electron microscopy of RWMV-infected tissues showed
accumulation of CP in the cytoplasm [66].
In the viral complementary RNA 2 of MiLBVV an
additional minor ORF encodes a putative protein of about
10 kDa [74], but its function is unknown. In the RNA 2 of
CPsV and LRNV this small protein is absent, and so far, it
is unknown whether this putative protein is present in
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RWMV and FreSV genomes. In case the 10 kDa poly-
peptide were not present in these viruses, MiLBVV would
be the unique ophiovirus with ambisense RNA 2 [74].
RNA3 contains one ORF in the vcRNA in all three
viruses which has been identified as coding for the CP [38,
58,64,66,70,74]. Although the morphology of ophiovi-
ruses resembles that of the nucleocapsids of bunyaviruses
and tenuiviruses, their RNAs 3 and 2 have no sequence
homology with them or with any other negative or ambi-
sense viruses and their CP have no serological relationship
[59,66]. CPsV is serologically unrelated to other ophi-
oviruses, but RNA3 and its product, the CP, share 46,5 %
and 30,9 % nucleotide and amino acid sequence identities,
respectively, with a Japanese isolate of MiLBVV [22].
FreSV CP protein (Freesia isolate) shows 48.8 % identity
(70.5 % similarity) with MiLBVV CP, 49 % identity
(68.5 % similarity) with LRNV CP and 30.6 % identity
(53.9 % similarity) with CPsV CP [70].
The CPs of MiLBVV and TMMMV are closer with
80 % homology. Attempts to find similarities, with the
exception of CPsV, some serological relationship between
TMMMV and MiLBVV, and between RWMV and MiL-
BVV, have been found indicating that some epitopes in the
CP among most of the ophiovirus are conserved [52]. In
general, different isolates of the same ophiovirus species
showed highly conserved amino acid sequences in the CP
as showed for CPsV [35] and MiLBVV [40], and less
conserved among the different ophiovirus species.
MiLBVV and LRNV present a fourth RNA of negative
polarity [64,65,74]. The RNA 4 of LRNV encodes a
potential protein of 38 kDa, and the RNA 4 of MiLBVV
one of 37 kDa (p37). MiLBVV has an additional ORF of
10.6 kDa with a 38 nt overlapping sequence with the p37.
This second ORF is proposed to be expressed by a ?1
translational frameshift of p37, but lacks an initiation
codon [74]. So far, the functions of these putative proteins
are unknown.
Control methods
Healing methods
Since the establishment of control programs based on the
elimination of sources of infection is primordial, the fight
against CPsV when the tree is infected is not a priority.
Indeed, preventing infection seems more appropriate that
eliminating the virus when the tree is already infected.
Sanitation technique based on the heat treatment com-
bined with the culture of meristems or grafting onto healthy
rootstocks can be adopted. Nevertheless, Roistacher [54]
indicated that shoot-tip-grafting (STG) or thermotherapy,
alone or in combination, is no guarantee of the elimination
of psorosis. On the other hand, Carvalho et al. [6] showed
that STG associated to thermotherapy are necessary to
assure a complete virus-free condition. In this experiment,
nursery trees were maintained in a climatic chamber at
38 °C under 16 h of light and 32 °C under 8 h of darkness
for 60 days, and the meristematic apices were used to shoot
tip graft. After the shooting of the meristematic part, the
shoot tip graft was over-grafted on 7-month-old Rangpur
lime (Citrus limonia) seedlings, and developed under
greenhouse conditions. Shoot tip grafted clones of sweet
orange (C. sinensis) cultivars Viz., Lima, Rubi, Piralima,
Salustiana, Joao Nunes, Rosa and Pera Caire, subjected to
thermotherapy, had 100 % success in elimination of pso-
rosis virus as reported by biological index using cv. Do Ceu
sweet orange as indicator plant.
Somatic embryogenesis was used to eliminate CPsV
from three citrus species (common mandarin, sweet orange
and Dweet tangor), all of which regenerated somatic
embryos with different embryogenic potential from stigma
and style explants. CPsV was detected by DAS-I-ELISA in
explants and embryogenic callus, but was not detected in
any plants obtained from somatic embryos, even
24 months after regeneration. Loss of juvenile characters
(disappearance of thorns) was observed in the first year of
growth and was retained in plants propagated by grafting
from thornless stems. Somatic embryogenesis appears to be
a very promising technique for the production of healthy
citrus stocks [14].
Cross protection
The test of cross-protection is an alternative method of
indirect biological indexing in the case of infection by
latent or moderate strains. This is a double inoculation of
indicator plants with a virulent strain and the inoculum to
be tested. The absence of the pathogen in question is
confirmed by the development of symptoms due to the lack
of cross-protection. Thus, it was determined that lesion
bark inoculum was able to induce PsB in sweet orange
seedlings, and that only PsA-infected seedlings showed
cross protection against PsB [54]. This feature is exploited
as a method of disease control [79], consisting to inocule
the plant with a less virulent strain to induce the protection
against severe strains. In the case studied, the severe strain
is PsB and moderate strain is PsA, which causes less severe
symptoms and leads to a slower decline of the tree.
Genetic engineering and disease control in citrus
Because of its economic impact, disease control is often the
objective of plant improvement programs. Hence, resis-
tance and defence genes isolated from well studied plant
species have been successfully incorporated into other
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species to generate pathogen-resistant plants. Recent
advances in genomics in citrus and other species have
made available an abundance of genes that can be easily
cloned and used in transformation process [17].
Whilst retaining desirable characteristics of yield and
quality, genetic crossing methods to incorporate CPsV
resistance genes into citrus cultivars have proved to be
very lengthy and difficult in citrus. Several citrus species
have been transformed with the CPG of a severe strain of
CPsV but presently there is little published information on
the resistance of such plants to CPsV infection [23,48,
49,50,81].
Transgenic lines are propagated by bud grafting onto
seedlings used as rootstocks generating replicates of each
line. For challenge, transgenic scions are infected by
grafting using infective tissue. With respect to CPsV,
Kayim et al. [23] have obtained transgenic citrus plants
expressing the CP of CPsV. In this study, Grapefruit
(Citrus paradisi Macf. cv Duncan), sweet orange (C. sin-
ensis Osbc. cv Hamlin) and Carrizo citrange [C. sinensis
(L.) Osbc. x Poncirus trifoliata (L.) Raf.] plants were
transformed with the CPG of a severe strain of CPsV.
Southern, Northern and Western blot analyses of transgenic
plants demonstrated that stably transformed grapefruit,
sweet orange and Carrizo citrange plants were obtained and
that all transgenic plants had at least one copy of the
transgene and showed transgene expression. Individual
transgenic lines from ‘‘Duncan’’grapefruit and ‘‘Hamlin’’
sweet orange were propagated for virus challenge by
budwood grafting onto Carrizo. Ten plants from each
transgenic line of Hamlin sweet orange and Duncan
grapefruit were graft inoculated with a severe strain of
CPsV. One transgenic line from Hamlin sweet orange was
resistant for the virus although two different transgenic
lines of Duncan grapefruit showed shock symptoms.
The same strategy was used by Zanek et al. [81] pro-
ducing 21 independent lines of transgenic sweet orange
(CP-lines) expressing low and variable amounts of CPsV
CP (isolate CPV-4). In these lines no correlation was found
between copy number and transgene expression and no
significant differences were observed in the response to
virus challenge among the lines or among the replicates.
Although two different viral loads were evaluated to
challenge the transgenic plants, no resistance or tolerance
was found in any line after 1 year of observations. On line
‘‘23’’ containing three copies of the transgene, only small
amounts of its mRNA is expressed and negligible amounts
of CP accumulated. Although the transformant failed to
protect against the viral load used, southern blot indicating
methylation of the ORF origin, suggests PTGS mechanism
of the CP transgene [81].
Recently, Reyes et al. [48] showed that the resistance
mechanism is the post transcriptional gene silencing
(PTGS). A mechanism of RNA degradation involved in
plant protection against viruses and can be induced by
transgenic expression of pathogen-derived sequences
encoding hairpin RNAs. Using this strategy, a herbaceous
model plant, Nicotiana benthamiana was transformed with
hairpin constructs obtained from two different genome
regions of the Argentine CPsV 90-1-1 isolate, the CP from
RNA3 and the 54K gene from RNA2. The degree of
resistance obtained varied according to the chosen viral
sequence. The analysis of the levels of small interfering
RNA (siRNA) accumulation by Northern blot and viral
RNAs analysis by RT-PCR indicated that the construct
derived from CP gene was better at inducing PTGS than
that originating from the 54K gene. The dependence of
PTGS induction on the degree of identity between the
target and the inducer transgene sequences was tested using
sequences derived from CPV-4, a more distant isolate of
CPsV, as PTGS targets. Efficient silencing induction was
also obtained to this isolate through the expression of the
CP-derived hairpin. Furthermore, alignment between CPV-
4 and the RNA2-hairpin fragment (54K-fragm) of CPsV
90-1-1 sharing 81 % identity, a good degree of gene
silencing is achieved by means of genes sequences having
less than 100 % identity between the inducer siRNA seg-
ment and the target sequence making unlikely the expla-
nation of the limited resistance observed in these intron-
hairpin-54K (ihp54K) lines by means of lack of sequence
identity between isolates [48].
Transgenic sweet orange plants transformed with hair-
pin constructs corresponding to viral CP, 54K or 24K
genes has been also reported [49]. After challenge with the
virus, the CP transgenic plants were more effective in
controlling the CPsV and consistently showed lower virus
levels and no symptom development compared to 54K and
24K transgenic plants. The study reported that the
observed CPsV resistance was due to preactivated RNA
silencing rather than the siRNA accumulation levels in the
ihp-CP transgenic sweet orange plants prior to virus
challenge [49]. Virus induced gene silencing (VIGS) is
evidenced by the presence of specific siRNA molecules for
RNA1, RNA2, and RNA3 of CPsV in infected plants [76].
Reyes et al. [49] observed also VIGS induction through CP
specific siRNA accumulation in the inoculated plants.
From sweet orange transgenic lines previously obtained,
fourteen lines were selected containing 1, 2 or 3 copies of
the transgenes. These plants were evaluated for their
acquired resistance against two isolates, PsA (CPV-4) and
PsB (CPsV 189-34), which differ in symptoms severity.
These lines were susceptible to both isolates when graft-
infected, although one of the lines carrying the CPG (CP-
96 line) containing two copies of the transgene and
expressing a low level of the CP showed a delay in
symptom expression when inoculated with the PsB isolate
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[50]. These studies represent a first step to a possible
control of CPsV, but the implementation of the technique
requires time consuming in transformation procedure and
multiplication. To date, control methods do not appear to
be effective solutions or sustainable and only the preven-
tion could contain the dispersion of the virus, through
certification programs which are crucial to update.
Certification program
Certification programs introduce regulatory or legal
requirements and restrictions that enforce to some degree
the use of clean material. These regulations are based upon
local conditions and may not regulate all pests and patho-
gens [25,41]. Certification programs are among the best
established means of increasing phytosanitary health, and
some of those for citrus are among the oldest in the world.
In conjunction with quarantine and clean stock programs,
they remain important weapons in the ongoing fight against
citrus diseases [24].
In California, the Citrus Clonal Protection Program
(CCPP) carries out a comprehensive biological indexing
program to detect graft-transmissible diseases that may
arrive in an imported budline. The tissue of the imported
budline is grafted onto citrus indicator seedlings, used to
detect specific diseases. The indicator varieties are selected
because of their sensitivity to diseases and their ability to
express symptoms. Laboratory tests are also used for dis-
ease diagnosis. If any disease or pathogen is found, various
therapies are used and if successful, the budline enters a
full-scale variety introduction index [78]. Other well-
established program includes those of Spain, Australia,
South Africa and Florida [24,75].
A successful certification programs actually comprises
three linked sub-programs: a quarantine programs, a clean
stock programs and a certification programs [75]. The
quarantine programs are designed to ensure the safe
introduction of new types of germplasm, while clean stock
programs have the aim of producing and maintaining
healthy, true-to-type local cultivars, and the certification
programs ensures that the already quarantined and tested
plants are distributed and tracked after distribution.
The most important components of certification pro-
grams are the sources of propagative material. The bases
for all further propagations are foundation blocks. These
include both protected foundation blocks of initial material
and field-planted or protected foundation blocks usually
located at the nurseries [24]. Certification programs may be
either mandatory or voluntary. Mandatory programs create
precise requirements and obligations to prevent the intro-
duction and spread of the relevant diseases. By distributing
and requiring the use of pathogen-tested and pathogen-free
budwood, mandatory certification decreases the ability of
pathogens to infiltrate citrus crops [75]. In voluntary pro-
grams, the initially higher investment in certified materials
may lead some nurserymen to use non-certified materials
despite the long-term negative economic impact of using
this inferior material [24].
Germplasm banks may have a very important role in
certification programs by supplying the initial materials for
propagation, particularly if they maintain healthy plants of
commercial varieties [24].
Almost every country facing the threat of citrus disease
has adopted a certification program to rebuild or preserve
its citrus industry [75], but they do not yet take in account
the recent data on the natural spread of the viruses men-
tioned above. The need for the updating the criteria is
evident as shown by the following examples.
The occurrence of CPsV in Campania, southern Italy,
was studied by Alioto et al. [2]. The results show that CPsV
is widespread in Campania with an especially high inci-
dence in younger plantings, suggesting that infected
propagating material has been increasingly used in this
area, in the absence of certification.
A study conducted by Powell et al. [47] showed that for
the variety of grapefruit ruby red, 79 % trees of orchards
were infected with psorosis and 31 % trees used to prop-
agation were contaminated (in seven centers of propaga-
tion). The fact that there are fewer individuals affected in
the center of propagation than in orchards could indicate a
natural spread. These results were quite unintended,
because California has developed a program in 1952 (based
on the first program «Psorosis Free Citrus») to eliminate
CPsV of the region. The development of a control program
should consider the scientific advances and technical on
knowledge of the virus and the detection means.
Concluding remarks
CPsV is the causal agent of old disease of citrus. Interac-
tions between the different CPsV strains and their citrus
hosts assembled a complicated plant pathosystem. Most of
the ophioviruses are soil-transmitted by a root-infecting
Oomycete genus Olpidium, and in the case of CPsV an
aerial vector is also suspected, although there are no evi-
dences so far. The virions are circles with 3-4 nm in
diameter of at least two different contour lengths, particles
can form pseudolinear duplex structures, and the coiled
filamentous are about 9–10 nm in diameter [36]. The 30-
terminal regions of the three CPsV RNAs are very similar.
In contrast, their 50-terminal regions differ considerably in
sequence and they do not anneal to perfect panhandle
structures, which is expected to form according to the
circular morphology and a ‘‘corkscrew’’ conformation was
not found. Although a remarkable advance in the
Recent advances in Citrus psorosis virus
123
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knowledge of CPsV genetics and the diversity of CPsV
viral populations have been achieved. Therefore, further
attention needs the study of the interactions between viral
products, the different citrus hosts and the vector trans-
mission factors, which are the basis of pathogenicity, host
resistance and viral epidemiology. The success of the citrus
management strategies depends on a deep understanding of
these interactions.
Recently, biotechnological approaches of viral control,
which exploit virus plant-host interactions for viral control,
such as sequence-based control strategies were developed.
Resistant transgenic plants based on PTGS, against specific
viral sequences, are already developed and promising
resistance may emerge from trials. This strategy retains
high possibilities of success in the proper management of
CPsV disease.
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