J. gen. Virol. (1988), 69, 3059-3068. Printed in Great Britain
3059
Key words: citrus cachexia viroid/exocortis disease/viroids
Citrus Cachexia Viroid, a New Viroid of Citrus: Relationship to Viroids of
the Exocortis Disease Complex
By J. S. S E M A N C I K , 1. C. N. R O I S T A C H E R , 1
R. R I V E R A - B U S T A M A N T E
1 AND N. D U R A N - V I L A 2
1Department of Plant Pathology and Cell Interaction Research Group, University of California,
Riverside, Calijbrnia 92521, U.S.A. and 2Instituto Valenciano de lnvestigaciones Agrarias,
46113 Moncada, Valencia, Spain
(Accepted 4 July 1988)
SUMMARY
Recovery of highly purified citrus cachexia viroid (CCaV) was accomplished by
serial elution following CF-11 cellulose chromatography of a 2 i-LiCl-soluble nucleic
acid preparation. The alternative herbaceous host, cucumber (Cucumis sativus cv.
Suyo), yielded greater quantities of the viroid than the highest yielding citrus host,
citron (Citrus medica cv. Etrog). A randomly primed c D N A probe to CCaV purified
from cucumber reacted positively to extracts from citron and cucumber inoculated with
the same isolate of CCaV. When tested against a broad range of other citrus viroids, the
CCaV c D N A hybridized to only one, CV-IIa, which has been identified as the causal
agent of a mild form of the citrus exocortis disease. Because of the apparent homology
between the nucleotide sequences of CV-IIa and CCaV, and a size difference of only
five to ten nucleotides, these RNAs can be considered as members of a common
subgroup of citrus viroids. These two viroids have been classified by bioassay reactions
as the causal agents of two distinct types of citrus disease, an 'exocortis-like' syndrome
and cachexia. The properties of and relationships between these two members of the
citrus viroid II group and the definition of the exocortis and cachexia (xyloporosis)
diseases are presented.
INTRODUCTION
The importance of viroid-induced diseases of citrus has been recognized since the citrus
exocortis viroid (CEV) was identified as a type example for this group of small, pathogenic
RNA molecules (Semancik & Weathers, 1972; Semancik, 1979). Until 1985, CEV remained the
only well described viroid known to affect citrus, although a viroid aetiology had been proposed
for the cachexia disease (Roistacher et al., 1983). A possible relationship between cachexia
(Childs, 1950) and the xyloporosis disease found in the Mediterranean region has been
speculated (Childs, 1952; Roistacher, 1988).
With the report of a new viroid distinct from CEV but inducing moderate to severe exocortislike symptoms on citron (Citrus medica) and consequently named the citron variable viroid
(CVaV) (Schlemmer et al., 1985), and the identification of a number of additional viroid RNAs
(Duran-Vila et al., 1986, 1988) previously described as mild strains of CEV, it now appears that
citrus harbours the largest collection of viroids of any single plant group. A proposal has been
made for the ordering of this collection of over 12 citrus viroids into five groups using the
parameters of molecular size, nucleotide sequence homology and host reaction (Duran-Vila et
al., 1988; N. Duran-Vila et al., unpublished data; Semancik, 1988).
Progress in the definition of this large number of citrus viroids has been greatly facilitated by
recognition of the ability of citron cv. Etrog to serve as a common host for all the citrus viroids. A
sensitive seedling selection of citron, Arizona 861-S 1, has functioned as the principal vehicle for
indexing exocortis disease for the past 10 years (Roistacher et al., 1977). This selection of citron
is also a symptomless host for the citrus cachexia viroid (CCaV) (Semancik et al., 1988).
0000-8284 © 1988 SGM
3060
J.s.
SEMANCIK AND OTHERS
C o m p a r a t i v e p r o p e r t i e s o f C C a V , t h e c a u s a l a g e n t o f t h e c a c h e x i a disease ( S e m a n c i k , 1986;
S e m a n c i k et al., 1988), a n d a c i t r u s viroid, C V - I I a , w h i c h i n d u c e s a m i l d f o r m o f t h e e x o c o r t i s
disease, are p r e s e n t e d here.
METHODS
Plant materials. The cachexia disease isolate, designated Ca 902, was derived from an old line navel orange field
source in 1963. The mild exocortis disease isolate, designated E 819, was derived from a Washington navel orange
field source in 1977. These isolates were chosen for investigation since each was shown to contain a single viroid
only. The causal agents of these diseases as well as all the other citrus viroids utilized in these studies were
maintained in symptomless sweet orange seedlings as part of the University of California (Riverside, Ca., U.S.A.)
citrus virus collection. These viroid reservoir hosts indexed negative for all other known citrus diseases.
The citrus viroids were transmitted by bud inoculation to Arizona 86 I-S1 citron. Bud inoculation of citron with
the mild exocortis source, E 819, results in a very mild leaf tip browning reaction and petiole wrinkling, while the
cachexia source (Ca 902) produces no visible reaction. Unless otherwise indicated, nucleic acid extracts and viroid
purifications were made from citron.
Cucumber (Cucumis sat&us cv. Suyo) was inoculated with a 2 i-LiCl-soluble nucleic acid preparation by needle
puncture of the hypocotyl through an inoculum drop as the first true leaves were emerging. After 3 weeks, the
plants were trimmed to two or three nodes and all foliage was removed. After a further 3 weeks, the regrowth was
collected for extraction. This process was repeated two or three times or until the new growth was severely
retarded.
Positive identification of the cachexia disease agent was by the characteristic browning reaction associated with
gumming induced in the stem and bark of Parsons Special mandarin tissues when forced on rough lemon rootstock (Roistacher et al., 1973). Infection of the bioassay plants was by bud inoculation of the rough lemon rootstock or by slashing the citron tissue with a razor blade moistened in inoculum. Bioassay plants are normally
monitored for 1 to 2 years for evidence of a positive reaction of cachexia.
Extraction of nucleic acids and viroid purification. Tip tissue consisting of young leaves and stems was collected
and either extracted as fresh tissue or frozen in liquid nitrogen, powdered and stored at - 20 °C until extraction.
Homogenization was performed in a medium containing water-saturated phenol, Tris-HC1 buffer pH 8.9, SDS,
EDTA and mercaptoethanol as previously reported (Semancik et al., 1975; Duran-Vila et al., 1986). Total nucleic
acid was concentrated by ethanol precipitation from the aqueous phase formed after centrifugation of the tissue
homogenate. A 2 M-LiCl-soluble fraction was then collected and concentrated by ethanol precipitation and
centrifugation.
Serial elution of the LiCl-soluble fraction from CF-11 cellulose was employed for separation and concentration
of some groups of viroids (Semancik, 1986). Viroids were isolated using the sequential 5 ~ PAGE system described
in the following section. The purified viroid RNA was visualized in the final denaturing gel after staining with
ethidium bromide rather than silver. Gel segments containing individual viroid bands were excised and the viroid
was recovered by electroelution using an IBI Model UEA unidirectional electroelutor (Semancik et al., 1988).
Analysis and characterization ofviroids. Detection of viroid RNA was by sequential 5 ~ PAGE under standard
and denaturing conditions as described by Semancik & Harper (1984). Following the first PAGE, a section of the
gel defining the viroid zone between CEV and 7S RNA (Rivera-Bustamante et al., 1986) was cut and placed on the
top of the second 5 ~ gel containing 8 M-urea for denaturing PAGE. Viroid RNA was viewed on the second gel
following silver staining (Igloi, 1983). This procedure has proved invaluable for the detection of citrus viroids
(Duran-Vila et al., 1986, 1988) and is employed in Fig. 2, 3 and 10.
For nucleotide homology studies, viroids were electrotransferred from denaturing gels onto nylon-based
membranes (Nytran) using an LKB Transphor apparatus. A randomly primed CCaV cDNA probe was made
essentially by the procedure of Maniatis et al. (1982) using cloned Moloney murine leukaemia virus reverse
transcriptase. Hybridization conditions were as reported by Garger et al. (1983).
RESULTS
Purification o f the citrus cachexia viroid
A l t h o u g h c i t r o n h a s b e e n r e c o g n i z e d as a c o m m o n h o s t in d e f i n i n g t h e c i t r u s v i r o i d g r o u p i n g s
( D u r a n - V i l a et al., 1988), s i g n i f i c a n t d i f f e r e n c e s c a n b e o b s e r v e d in t h e r a t e o f a c c u m u l a t i o n o f
the different viroids in infected citron. Whereas the bulk of citrus viroids can be readily detected
in e x t r a c t s f r o m i n f e c t e d c i t r o n ( D u r a n - V i l a et al., 1986), C C a V is v e r y difficult to resolve f r o m
t h e h e a l t h y p l a n t b a c k g r o u n d n u c l e i c a c i d a f t e r P A G E (Fig. 1). T h e f a c t t h a t C C a V is t h e o n l y
m e m b e r o f t h e c i t r u s v i r o i d f a m i l y t h a t is s y m p t o m l e s s i n c i t r o n m a y reflect a n i m p a i r e d r a t e o f
replication and/or accumulation.
Cachexia disease and the viroid complex
(a)
3061
(b)
1
3
2
---CEVc
7/
~CEV
CCaVc~
--7S
4-....--
RNA
CCaV 1 _
~.~; : ~ - - 7 S
RNA
Fig. 1
Fig. 2
Fig. 1. Polyacrylamide (5 ~) gel containing 8 M-ureaafter denaturing PAGE of 2 M-LiCl-solublenucleic
acid fractions from healthy (lane 1) and CCaV-inoculated (lane 2) citrons after silver staining. The gel
pictured is the second in the sequential PAGE procedure and results from removal of the section as
defined by CEV and 7S RNA from the first PAGE separation and subsequent analysis of its contents
by denaturing PAGE as depicted in Fig. 2.
Fig. 2. Non-denaturing PAGE (a) of the single electrophoretic species of purified CCaV (lane 1) and a
2 M-LiCl-solubleextract from CEV-infected citron (lane 2) used to define the section between arrows
that was removed and applied to a second gel and run under denaturing (8 M-urea)conditions (b). The
bands from the non-denaturing gel would normally be stained with non-immobilizing ethidium
bromide, but are stained with silver here for illustration purposes. The circular (c) and linear (1)forms of
CCaY (lane 3) and CEV (lane 4) were observed after silver staining of the denaturing PAGE gel.
Nevertheless, with repeated harvesting of new flush growth from the same CCaV-inoculated
citron plants maintained under warm glasshouse conditions, and combining viroid recovered
from several sets of denaturing gels, it has been possible to accumulate sufficient C C a V for
partial characterization.
The final purified viroid R N A species migrates as a single electrophoretic component under
standard conditions of P A G E (Fig. 2, lane 1), and separates into the characteristic circular and
linear forms of the viroid R N A (Fig. 2, lane 3) when subjected to denaturing P A G E in the
presence of 8 M-urea. A n estimated molecular size o f approx. 300 nucleotides for C C a V was
obtained by comparison of its migration relative to that of the 371 nucleotide C E V (Fig. 2, lanes
2 and 4).
The highly purified C C a V R N A has been demonstrated to be transmissible in the
symptomless carrier, citron, and to produce the characteristic cachexia disease reaction when
bud-inoculated to its bioassay host, Parsons Special m a n d a r i n (Semancik et al., 1988). The
viroid R N A accumulated to a much greater concentration in cucumber than in citron. Partially
purified nucleic acid extracts from cucumber were subjected to elution from CF-11 cellulose in a
graded ethanol series. The 25 ~ e t h a n o l - S T E buffer eluant was highly enriched in C C a V (Fig.
3). This manipulation greatly improved the recovery of viroid, resulting in quantities sufficient
to function as a template in the production of a c D N A probe.
3062
J.s.
(a)
1
2
3
4
5
6
SEMANCIK AND OTHERS
7
8
(---CEV
--7S
(.--
Fig. 3. Polyacrylamide (5~) gels after nondenaturing PAGE (a), stained with ethidium
bromide, and denaturing PAGE in the presence
of 8 M-urea (b) stained with silver. Fractions of a 2
M-LiCl-soluble nucleic acid preparation from
CCaV-inoculated citron were obtained by serial
elution from CF-I1 cellulose by a series of
buffered solutions with diminishing ethanol concentrations (lane 1, 30~; lane 2, 25~; lane 3,
20 ~ ; lane 4, 15 ~ ; lane 5, 10 ~ ; lane 6, 5 ~ ; lane 7,
0H). The section of gel (a) between the arrows as
defined by CEV and 7S RNA contained in the
standard lane (lane 8) was analysed by denaturing
PAGE (b). Elution of CCaV is observed in the
fractions containing 25 to 15 ~ ethanol (lanes 2 to
--CEV
4).
(a)
1
(b)
2
3
4
5
6
7
1
2
3
4
5
6
7
Fig. 4. A section of an ethidium bromide-stained polyacrylamide (5~) gel (a) containing 8 M-urea after
sequential PAGE and denaturing PAGE as in Fig. 2, with nucleic acid preparations from: citron
inoculated with the Ca 902 cachexia disease isolate (lane 1), CCaV inoculum purified from infected
cucumber (lane 2), healthy citron (lane 3), citron inoculated with purified CCaV inoculum as in lane 2
(lane 4), healthy cucumber (lane 5), cucumber infected with a purified CCaV inoculum as in lane 2
(lane 6) and a CEV standard (lane 7). Autoradiograph (b) of matching pattern (lanes 1 to 7) after
electrotransfer onto Nytran membrane and hybridization with a 3Zp-labelled cDNA probe to CCaV.
A r a n d o m l y p r i m e d C C a V c D N A p r o b e was constructed to verify that the viroid purified
f r o m c u c u m b e r was the s a m e as the original source o f c a c h e x i a disease a g e n t o b t a i n e d f r o m the
sweet o r a n g e reservoir. C o m p a r a b l e nucleic acid samples f r o m citron, b u d - i n o c u l a t e d w i t h the
sweet orange c a c h e x i a isolate, C a 902, as well as citron and c u c u m b e r i n o c u l a t e d w i t h purified
C C a V were electrotransferred directly f r o m gels c o n t a i n i n g 8 M-urea and h y b r i d i z e d w i t h 32p°
labelled c D N A to C C a V .
T h e a u t o r a d i o g r a p h p r e s e n t e d in Fig. 4 clearly confirms the identity in e l e c t r o p h o r e t i c
m i g r a t i o n a n d s e q u e n c e h o m o l o g y o f the purified C C a V used as i n o c u l u m for citron and
3063
Cachexia disease and the viroid complex
2
3
4
--CEV
(a)
1
(b)
2
3
4
1
2
3
4
CV-IIa
CV-IIb
--CCaV
CCaV)
Fig. 5
Fig. 6
Fig. 5. Polyacrylamide (5%) gel containing 8 M-urea, stained with silver after processing by sequential
PAGE as in Fig. 2. Nucleic acid extracts from citron infected with the mild exocortis isolate E 819
containing CV-IIa (lane 1), and cachexia isolate Ca 902 containing CCaV or CV-IIb (lane 3); identical
samples as in lanes 1 and 3 were mixed before electrophoresis (lane 2); CEV was used as a standard
(lane 4).
Fig. 6. Polyacrylamide (5%) gel containing 8 M-urea, stained with silver after processing by sequential
PAGE as in Fig. 2. Nucleic acid extracts from healthy citrons (lane 1) and citrons inoculated with
Ca 902 (lane 2) containing CCaV (CV-IIb), a mild 'exocortis' isolate E 819 (lane 3) containing CV-IIa,
and the 'citron variable' viroid source, E 821 (lane 4) containing CV-Ib, CV-IIa, CV-IIb and CV-IIIb,
were eluted from CF-11 cellulose by step gradients of STE buffer containing 25% (a) and 0% (b)
ethanol.
cucumber with the original cachexia disease isolate derived from the sweet orange reservoir.
Confirmation of the identity of the biological activity of the C C a V isolated from cucumber with
the cachexia agent found in Ca 902 has been accomplished by transmission o f the viroid purified
from cucumber to citron and subsequent positive bioassay on Parsons Special mandarin.
PAGE and chromatography analyses of CCa V and CV-IIa
According to the description of citrus viroids made by Duran-Vila et al. (1988) and Semancik
(1988), only two viroids were included in the G r o u p II class. These two viroids, designated CVI I a and CV-IIb, displayed very similar electrophoretic migration rates, corresponding to
molecules o f about 300 to 310 nucleotides, as well as a similar host range and similar properties
on elution from CF-11 cellulose.
W h e n individually purified sources of C V - I I a (Fig. 5, lane 1) and C C a V (Fig. 5, lane 3) were
subjected to coelectrophoresis as an artificial mixture under denaturing conditions in the same
channel (Fig. 5, lane 2) the doublet visible indicated a slight but clear distinction in the
migration rates and, accordingly, the molecular size of the two viroids. The position of C C a V
relative to that of C V - I I a was identical to that expected for C V - I I b under comparable
conditions.
This analysis indicated that C C a V and C V - I I b constitute the same molecular entity. The
strong homology reaction o f C C a V c D N A with C V - I I b presented later (Fig. 7) further supports
this conclusion. The two very similar viroids, C V - I I a and CCaV, have been implicated as the
causal agents of what are considered as two distinct diseases of citrus.
3064
J . S. S E M A N C I K
Ca)
1
AND
OTHERS
(b)
2
3
4
5
1
2
3
4
5
CCaV)
Fig. 7. A section of an ethidium bromide-stained polyacrylamide (5%) gel (a) containing 8 M-urea after
sequential PAGE and denaturing PAGE as in Fig. 2, with 2 M-LiCl-soluble nucleic acid fractions from
citron containing the CVs indicated; CEV and CV-Ia (lane 1), CV-Ib and CV-IIIb (lane 2), CV-/IIa
and CV-IV (lane 3), CV-IIa (lane 4), and CV-IIb or CCaV (lane 5). Autoradiograph (b) of matching
pattern after electrotransfer to Nytran membrane and hybridization with 3zp-labelled cDNA CCaV
probe.
To investigate further the extent of this relationship, pure viroid isolates containing only CVIIa or CCaV as well as an isolate, E 821, containing the citron variable viroid source (Schlemmer
et al., 1985), and now known to comprise a mixture of four viroids, were analysed by sequential
elution from CF-11 cellulose. This procedure has been utilized for the detection of viroid-like
RNA present in low concentrations as well as in distinguishing among different viroids
(Semancik, 1986).
When samples eluted from cellulose as a 25 ~ ethanol-STE buffer solution were compared in
denaturing P A G E with those eluted by STE buffer alone, both CV-IIa present in the E 819
source and CCaV present in the Ca 902 source were concentrated in the 2 5 ~ ethanol-STE
fraction (Fig. 6). In addition, a similar fraction from the mixed viroid isolate, E 821, contained
both Group II viroids. Particularly striking is the clear segregation of the Group II viroids from
the other viroids present in the E 821 isolate in response to analytical CF-11 chromatography.
Although both CV-IIa and CCaV can be eluted with 25 ~ ethanol-STE, CV-Ib as well as CVIIIb require a lower ethanol concentration for elution and are evident only in the STE buffer
fraction.
After this analysis, it was determined that the E 821 isolate is also positive in the Parsons
Special mandarin bioassay for the cachexia disease (unpublished data), thus confirming the
presence of CCaV in this mixed viroid source.
Nucleotide sequence homology between CCaV and other citrus viroids
Nucleotide sequence homology as determined by molecular hybridization with specific
c D N A probes constructed from citrus viroids has been essential in establishing the general
groupings of the citrus viroids (Duran-Vila et al., 1988; Semancik, 1988). When the CCaV
cDNA probe, as described above and employed in Fig. 4, was tested against a wide range of
citrus viroids, only CV-IIa (Fig. 7b, lane 4) and CV-IIb (Fig. 7b, lane 5) reacted positively and
with a similarly high intensity. Thus, what appears to be an exclusive and highly homologous
relationship exists between CV-IIa and CCaV (CV-IIb).
Possible biological relationships between CV-IIa and CCaV
With the physical characterization data presented above, an implicit structural and
conformational relationship between CV-IIa and CCaV (CV-IIb) can be proposed. Host range
specificity and symptom expression in the Etrog citron utilized in defining the biological activity
of citrus viroids (Duran-Vila et al., 1988) were tested to discriminate between members of the
same group. Table I summarizes some of the physical properties of the Group II citrus viroids as
well as the reactions on the two bioassay hosts for the exocortis and cachexia diseases.
Cachexia disease and the viroid complex
3065
Table 1. Comparative properties of the Group H citrus viroids
Isolate*
E 819
Ca 902
Primary source
Washington navel orange
Old line navel orange
PAGE
band
CV-IIa
CV-IIb
(CCaV)
CCaV
cDNA
+++
++++
Bioassay reactiont
r
~
Exocortis
Cachexia
+
++++
* E indicates an exocortis disease source; Ca indicates a cachexia disease source.
t Exocortis disease is bioassayed by leaf symptoms on citron (C. medica). Cachexia disease is bioassayed by
browning reaction associated with gumming induced in the stem and bark in Parsons Special mandarin when
forced on rough lemon root-stock.
Although the citron isolate E 819 which contains CV-IIa has been designated as an exocortislike disease isolate, the distinct tip-browning of leaves, petiole wrinkling and browning of the
citron bioassay host constitutes an unusual response for an exocortis source arid certainly the
mildest reaction of all known exocortis isolates (Roistacher et al., 1977). In fact, this reaction is
very difficult to reproduce unless ideal environmental conditions are available. Therefore, in
most cases, the response of citron to CV-IIa is exceedingly difficult to distinguish from its
response to CCaV, even though both viroids are present in similar detectable titres in this host.
Evaluation of the root-stock and scion components of the bioassay system for the cachexia
disease was accomplished by comparing a nucleic acid extract from infected citron with extracts
from new flush tissues from Parsons Special mandarin scion displaying a strong cachexia
reaction and from the rough lemon root-stock on which the mandarin was grafted. After
denaturing P A G E , the characteristic CCaV band was visible only in the extracts from infected
citron, suggesting an extremely low rate of replication and/or accumulation in mandarin and
rough lemon.
Therefore, the responses of the two indexing hosts to CV-IIa and CCaV are quite distinct.
Citron remains a host that can maintain detectable titres of both viroids and a weak symptom
expression to CV-IIa, while Parsons Special mandarin produces a strong response to CCaV, but
in the absence of detectable levels of the viroid.
Effect of temperature and mixed infection on CCa V replication
It has been recognized that temperature can dramatically influence the incidence of viroid
infection (S/inger, 1972) as well as the optimal conditions for the growth of viroid-containing
cells in culture (Marton et al., 1982). The viroid structure is not only resistant to thermal
inactivation, but it is also generally accepted that elevated temperatures favour viroid
replication. These properties have almost become diagnostic indicators of infection of plants by
viroids.
Observations of the effect of temperature on viroid accumulation have been made for the
most part with plant species which display a symptomatic response to viroid infection. Since
Etrog citron remains symptomless during CCaV infection, a study was initiated to determine
the influence of temperature on the accumulation of CCaV in this host as compared to the
accumulation of CEV, a viroid which induces a severe stunting response in citron.
Fig. 8 presents P A G E (a) and denaturing P A G E (b) patterns of extracts from CCaV- and
CEV-infected citrons grown in the glasshouse under relatively hot (22 to 38 °C) and cool (17 to
29 °C) conditions of minimum and maximum temperatures, respectively. The g r o w t h . o f
uninoculated citron was noticeably retarded (approx. 5 0 ~ ) by the low temperature regime.
Nevertheless, the nucleic acid preparations from plants grown under cool conditions displayed
less D N A polydispersity in the gel (Fig. 8a, lanes 2 and 4) than those from plants grown under
hot conditions (Fig. 8 a, lanes 1 and 3). Samples of CEV and CCaV were equalized as pairs on a
extracted fresh weight basis.
As anticipated, a greater concentration of CEV accumulated in plants grown under hot (Fig.
8, lanes 3) rather than cool (Fig. 8, lanes 4) conditions. This response is especially visible in the
3066
J . S. S E M A N C I K
AND
OTHERS
(a)
(b)
1
i
3
CEV
--CEV
jCCaV
-(---.
- 7 S RNA
CCaV
Fig. 8. Polyacrylamide(5 ~) gels after non-denaturing PAGE (a), stained with ethidium bromide, and
subsequent denaturing PAGE (8 M-urea)(b), stained with silver. The section of the gel (a) between the
arrows was analysed on the gel (b). Nucleic acid extracts (2 M-LiCl-solublefraction) were made from
cachexia (lanes 1, 2) and 'exocortis (lanes 3, 4) disease' citrons grown under high (22 to 38 °C; lanes 1
and 3) or low (17 to 29 °C; lanes 2 and 4) regimes of minimum-maximum temperatures.
ethidium bromide-stained gel (Fig. 8a). The concentration of CCaV under both conditions
appeared to be essentially equivalent or even slightly greater in plants grown under cool
conditions (Fig. 8b, lanes 1 and 2), a response uncommon in viroid infection. A direct
comparison cannot be made between the extracts from plants containing distinct viroids grown
at either high or low temperature, because the CEV-containing gel samples were made
approximately half as concentrated as the CCaV samples to avoid overstaining of the CEV band
in the denaturing gel.
DISCUSSION
With the recent advances in the detection of viroids and the increase in the reported numbers
of viroids described, the general grouping of citrus plant species has been found to harbour the
largest collection of naturally occurring viroids. However, only two diseases, exocortis and
cachexia (xyloporosis), have thus far been either defined or suggested to result from viroid
infection in citrus. From this, significant questions of major importance to citriculture present
themselves such as, what the implications of this large reservoir of more than 12 viroid species
are to plant development and productivity, and what physical and biological relationships exist
among the citrus viroids.
CEV, as the well characterized type citrus viroid, has long been recognized as the causal agent
of the exocortis disease. With this report, a second citrus viroid, the citrus cachexia viroid
(CCaV), has now been identified and demonstrated to be the causal agent of the cachexia
disease. Comparative properties including molecular size, conformation and nucleotide
sequence homology suggest that CCaV is closely related to CV-IIa and is probably identical to
CV-IIb in the scheme of Duran-Vila et al. (1988).
Rapid indexing techniques employing PAGE and molecular hybridization offer the potential
to shorten the existing 1 to 2 year cachexia disease bioassay procedure. It should be cautioned,
however, that most field samples analysed contained a mixture of viroids, and if CV-IIa is
Cachexia d&ease and the viroid complex
3067
present the similarity in size and sequence homology with CCaV could pose potential sources of
false positive reactions in rapid detection procedures such as dot blot hybridizations or P A G E
with crude samples. Therefore, the bioassay indexing procedure for the ultimate identification
of the cachexia disease should be retained to confirm the results of any rapid detection method.
Although exocortis and cachexia have been demonstrated as quite distinct diseases of citrus
based on distinct field symptomatology, the high degree of nucleotide sequence homology
between the causal agents, CV-IIa and CCaV, suggests the possibility of a much closer
relationship between these diseases. The basic question that remains is whether the responses of
bioassay plants are sufficient to establish the existence of distinct diseases and whether
fundamental relationships between diseases are minimized by the acceptance of apparently
different host reactions. In the case of CCaV and CV-IIa two very similar molecules have been
indicated as distinct pathogenic agents basically on the symptom reaction of citron for the
bioassay of the 'exocortis' disease and mandarin for the 'cachexia' disease.
Since the ethanol-dependent elution of viroids from CF-11 cellulose appears to be based on
molecular conformation (Semancik, 1986), it is possible to suggest also a similarity in this
parameter between CV-IIa and CCaV. Because viroid RNA sequences are not translated (Hall
et al., 1974; Semancik et al., 1977) and, thus, biological effects on the host are not exerted via
viroid-specified proteins, the property of molecular conformation is of special significance for
viroids.
It would not seem reasonable that CCaV (CV-IIb) is related to CV-IIa by being a deletion
variant which somehow acquired an additional capacity, i.e. the ability to cause cachexia in the
mandarin bioassay host, in spite of a net reduction in total genetic information. The precise
effect on conformation caused by the postulated loss of approx, five to ten nucleotides from CVIIa can only be conjectured at this time. Nevertheless, these data support the view that
molecular conformation and not the total genetic capacity as judged by nucleotide content may
be the more significant factor in the expression of biological activity of viroids.
With a total nucleotide range of about 300 to 305, both of these Group II citrus viroids are in
the size range of the hop stunt viroid (HSV) and cucumber pale fruit viroid (CPFV) and may be
similar to the HSV-related viroid of 302 nucleotides recently isolated from Etrog citron (Sano et
al., 1986, 1988). A further relationship can be suggested by the transmission and the symptom
expression of CV-IIa and CCaV in cucumber, a common host of HSV and CPFV.
The response of CCaV replication to temperature is unlike that normally expected for viroids.
No significant increase in the accumulation of CCaV in citron could be detected with increase in
growing temperatures, whereas CEV replication in the same host is enhanced by high
temperatures. Since CEV can be incorporated into the nucleic acid population of tomato cells in
culture (Lin & Semancik, 1985) and even enhance the viability of CEV-containing cells (Marton
et al., 1982), the absence of any demonstrable symptom expression in citron containing CCaV
may also reflect a significant level of coordinated synthesis of viroid with host nucleic acid.
The bioassay reaction for CCaV in the indicator, Parsons Special mandarin, is enhanced by
high temperatures, but only when CCaV is indexed as a pure viroid species (J. A. Pina, personal
communication). When a mixture of citrus viroids, including both the cachexia and those citrus
viroids producing a symptom response on citron and collectively known as exocortis disease
agents, is bioassayed on Parsons Special mandarin, a more complex picture emerges. Index
readings are more consistent in response to viroid mixtures containing CCaV when bioassay
plants are incubated at lower temperatures, whereas the CCaV detection rate is lower when
bioassay plants are maintained at higher temperatures.
This effect may result from an interference or competition by the additional viroid(s) in the
mixture with the cachexia bioassay. Temperature mediation of this effect might be explained if
CCaV replication is not seriously affected by temperature while the replication of the other
citrus viroids, as with CEV, is preferentially enhanced at higher temperatures. It can also be
predicted that the closely related Group II viroid, CV-IIa, would be probably the most effective
of the citrus viroids to accomplish the interference with the cachexia bioassay. It should be
cautioned that this interference phenomenon should not be equated with a classical crossprotection reaction since no report of this type of reaction exists for viroid infection of citrus.
3068
J. S. S E M A N C I K A N D O T H E R S
The excellent assistance of Mr J. Bash with the bioassay procedures and K a t h y Harper with illustration and
manuscript preparation are gratefully acknowledged. These studies were supported in part by a grant from the
U.S.-Spain Joint Committee for Scientific and Technological Cooperation.
REFERENCES
CHILDS, J. F. L. (1950). The cachexia disease of Orlando tangelo. Plant Disease Reporter 34, 295-298.
CHILDS, J. F. L. (1952). Cachexia disease: its bud transmission and relation to xyloporosis and tristeza.
Phytopathology 42, 265-268.
DURAN-VILA, N., FLORES, R. & SEMANCIK,I. S. (1986). Characterization of viroid-like R N A s associated with the
citrus exocortis syndrome. Virology 150, 75-84.
DURAN-VILA,N., PINA, J. A., BALLESTER,J. F., JUAREZ, J., ROISTACHER,C. N., RIVERA-BUSTAMANTE,R. & SEMANCIK,J. S.
(1988). The citrus exocortis disease: a complex of viroid R N A s . Proceedingsof the International Organization
of Citrus Virologists 10, 152-164.
GARGER, S. l., TURPEN, T., CARRINGTON, J. C., MORRIS, T. J., DODDS, I. A. & GRILL, L. K. (1983). Rapid detection of
plant viruses by dot blot hybridization. Plant Molecular Biology Reporter 1, 21-25.
HALL, T. C., WEPPRICH, R. K., DAVIES, J. W., WEATHERS, L. G. & SEMANCIK, J. S. (1974). Functional distinctions
between the ribonucleic acids from citrus exocortis viroid and plant viruses: cell-free translation and
aminoacylation reactions. Virology 61, 486-492.
IGLOI, G. L. (1983). A silver stain for the detection of n a n o g r a m amounts of t R N A following two-dimensional
electrophoresis. Analytical Biochemistry 134, 184-188.
LIN, J. J. & SEMANCIK,J. S. (1985). Coordination between host nucleic acid metabolism and citrus exocortis viroid
turnover. Virus Research 3, 213-230.
MANIATIS,T., FRITSCH,E. V. & SAMBROOK,T. (1982). Molecular Cloning: A Laboratory Manual, pp. 129-132, 230-234.
New York: Cold Spring Harbor Laboratory.
MARTON, L., DURAN-VILA,N., LIN, L-L & SEMANCIK, J. S. (1982). Properties of cell cultures containing the citrus
exocortis viroid. Virology 122, 229-238.
RIVERA-BUSTAMANTE,R., GIN, R. & SEMANCIK, J. S. (1986). Enhanced resolution of circular and linear molecular
forms of viroid and viroid-like R N A by electrophoresis in a discontinuous-pH system. AnalyticalBiochemistry
156, 91-95.
ROISTACHER, C. N. (1988). Cachexia and xyloporosis diseases. A review. Proceedings of the International
Organization of Citrus Virologists 10, 116-124.
ROISTACHER, C. N., BLUE, R. L. & CALAVAN,E. C. (1973). A new test for citrus cachexia. Citrograph 58, 261-262.
ROISTACHER, C. N., CALAVAN,E. C., BLUE, R. L., NAVARRO,L. & GONTALES, R. (1977). A new more sensitive citron
indicator for detection of mild isolates of citrus exocortis viroid (CEV). Plant Disease Reporter 53, 333-336.
ROISTACHER,¢. N., GUMPF, D. J., NAUER, E. M. & GONZALES,R. (1983). Cachexia disease: virus or viroid. Citrograph
68, 111-113.
S)~NGER, H. L. (1972). A n infectious and replicating R N A of low molecular weight: the agent of the exocortis
disease of citrus. Advances in Bioscience 8, 103-116.
SANO, T., HATAYA,T., SASAKI,A. & SHIKATA,T. (1986). Etrog citron is latently infected with hop stunt viroid-like
R N A . Proceedings of the Japan Academy 62, 325-328.
SANO, T., HATAYA,T. & SHIKATA,T. (1988). Complete nucleotide sequence of a viroid isolated from Etrog citron, a
new m e m b e r of hop stunt viroid group. Nucleic Acids Research 16, 347.
SCHLEMMER, A., ROISTACHER,C. N. & SEMANCIK,J. S. (1985). A unique, infectious R N A which causes symptoms in
citron typical of citrus exocortis disease. Phytopathology 75, 946-949.
SEMANCIK,J. S. (1979). Small pathogenic R N A in plants - the viroids. Annual Review of Phytopathology 17, 461-484.
SEMANClg, J. S. (1986). Separation of viroid R N A s by cellulose chromatography indicating conformational
distinctions. Virology 155, 39-45.
SEMANCIK, J. S. (1988). The citrus exocortis disease. A review 1976-1986. Proceedings of the International
Organization of Citrus Virologists 10, 136-151.
SE~t~NCIK, J. S. & HARPER, K. L. (1984). Optimal conditions for cell-free synthesis of citrus exocortis viroid and the
question of specificity of R N A polymerase activity. Proceedings of the National Academy of Sciences, U.S.A.
81, 4429-4433.
SEMANCIK,J. S. & WEATHERS,L. G. (1972). Exocortis disease : evidence for a new species of'infectious' low molecular
weight R N A in plants. Nature New Biology 237, 242-244.
SEMANCIK, J. S., MORRIS, r. L, WEATHERS, L. G., ROROORF, n. F. & KEARNS, D. R. (1975). Physical properties of a
minimal infectious R N A (viroid) associated with the exocortis disease. Virology 63, 160-167.
SEMANCIK, J. S., CONEJERO,V. & GERHART, J. (1977). Citrus exocortis viroid: survey of protein synthesis in Xenopus
laevis oocytes following addition of viroid R N A . Virology 80, 218-221.
SEMANCIK,J. S., ROISTACHER,C. N. & DURAN-VILA,N. (1988). A new viroid is the causal agent of the citrus cachexia
disease. Proceedings of the International Organization of Citrus Virologists 10, 125 135.
(Received 3 February 1988)