Coconut cadang-cadang viroid
M. J. B. Rodriguez
Philippine Coconut Authority, Guinobatan, Albay, Philippines
J. W. Randles
Waite Campus, The University of Adelaide, South Australia, Australia
The disease was first described in the Philippines by
and the viroid was first identified by
A viroid consisting of circular or linear single-stranded RNA with a basic size of 246 or 247
nucleotides, occurring as monomeric and dimeric forms in Cocos nucifera. Larger molecular
forms appear as the disease progresses. Mechanically transmissible under laboratory conditions using
nucleic acid inocula. Natural transmission occurs, possibly by mechanical damage and pollen.
Host range is limited to the Palmae. Found only in the Philippines, where the disease has
been responsible for lost production with the premature death of more than 40 million coconut palms.
Annual losses are 200,000-400,000 palms and cost the industry around $40 million
(Randles & Rodriguez, 2003)
Cadang-cadang is a lethal disease of coconut (Cocos nucifera
), recognised by a generalised
yellow-bronze coloration of the lower two-thirds of fronds in the crown, persistence of stipules at
the base of fronds, cessation of nut production, crown diminution and death
The third or fourth fronds below the spear leaf show small chlorotic leaf spots which expand and
coalesce during ageing of the frond to produce a chlorotic mottle. As the spots appear, the nuts
become smaller and rounded
(Mohamed et al., 1982
and many of them develop equatorial scarification. Inflorescences later become necrotic and infertile,
and nut production ceases. Frond production and size gradually decline
(Randles et al., 1977
leaflets become brittle, and death of the crown ensues. Palms die between 8 and 16 years after the
onset of symptoms
(Zelazny & Niven, 1980
Cadang-cadang differs from the coconut Tinangaja disease in both symptomatology
(Boccado et al., 1981
and in the nucleotide sequence of the associated Coconut tinangaja viroid
Naturally-infected oil palm (Elaeis guineensis) shows a progression from general chlorosis
on younger fronds to development of translucent orange leaf spots and cessation of inflorescence
(Randles et al., 1980).
Cadang-cadang disease is limited to the central eastern Philippines (Quezon, Bicol provinces,
Samar provinces and Biliran). Distribution is unaffected by water barriers, but incidence is
negatively correlated with altitude
It has a slow rate of spread with new sites of incidence occurring within the confines of the
above-mentioned areas (M.J.B. Rodriguez, E.P. Pacumbaba, J.C. Orense, A.R. Alfiler & G.G. Manalo, unpublished data).
Host Range and Symptomatology
Known hosts are confined to the Palmae
. It is transmitted experimentally by high pressure
injection of seedlings (two to three months old) with concentrated or partially-purified viroid,
combined with pricking
(Randles et al., 1977
For coconut, high pressure injection alone is more efficient when one to seven day old sprouts
(Imperial et al. 1986
- Diagnostic species
- Cocos nucifera (coconut). Inoculated seedlings show spotting one to two years after
inoculation, on leaflets of fronds that are at least three positions below the unexpanded spear
leaf. There is an associated reduction in growth rate, frond size and production and failure to bear
nuts at the expected time.
A more severe type of symptom first appeared in 1987, in a significant number of artificially
inoculated coconut palms
(Rodriguez & Randles 1993;
Its main feature is a greater reduction in the width of the leaf lamina, giving the palm a 'broomed'
appearance, and more rapid death
(Fig.3 & Fig.4).
Occasionally palms showing similar symptoms are observed in plantations, suggesting that 'brooming'
Chlorotic or orange spotting of leaflets is characteristic of other inoculated species of palm,
such as betel nut palm (Areca catechu), buri palm (Corypha elata),
Manila palm (Adonidia merrillii), oil palm (Elaies guineensis), palmera
(Chrysalidocarpus lutescens) and royal palm (Oreodoxa regia)
(Imperial et al. 1985)
- Propagation species
- Naturally-infected Cocos nucifera.
- Assay species
- Cocos nucifera seedlings inoculated between seven days to three months after germination
are used as the systemic assay host. Infection is confirmed by detection of the viroid in leaf
extracts by gel electrophoresis
or molecular hybridisation, one to two years after inoculation.
StrainsCoconut cadang-cadang viroid
shows only minor variation in the minimal nucleotide sequence
of 246 nucleotides
(Haseloff et al., 1982
Rodriguez & Randles, 1993
Modifications to the right terminus are related to stage of disease development
(Imperial et al. 1981
and are not strain differences because they revert to the minimal form when inoculated to new host
seedlings. Mutations at positions 87, 197 and 216 are correlated with the expression of the severe
lamina-depleting 'brooming' symptom
(Rodriguez & Randles, 1993
Transmission by Vectors
Natural transmission does occur, but no vector has been identified.
Transmission through Seed
Occurs at a low rate, with studies showing that only one out of 320 seedlings derived from infected
material tested positive for CCCVd
low rate of pollen transmission
(Pacumbaba et al., 1994
No viroid-specific antibodies were detected in rabbits injected with the viroid-containing
polyethylene glycol-insoluble fraction from coconut leaves
(J.W. Randles & O.W. Barnett, unpublished data).
RelationshipsCoconut cadang-cadang viroid
is the smallest known nucleic acid containing pathogen.
It is biologically and structurally distinct from all other viroids and is the type member of the
It has about 64% overall sequence identity
with Coconut tinangaja viroid
. In nucleic acid hybridisation assays, it shows some
cross-hybridisation with Coconut tinangaja viroid
Potato spindle tuber viroid
Citrus exocortis viroid
) but not with
Avocado sunblotch viroid
(Hanold & Randles, 1991
J.W. Randles, R.A. Owens & J.A. Daros, unpublished data).
Stability in Sap
No information on survival of infectivity in sap. The structure of the viroid is more stable than
that of cell RNA species in incubated polyethylene glycol concentrates of coconut leaf sap
Randles et al., 1976
Chop leaflets from fronds four or more below the spear leaf and blend with 3 volumes of
0.1 M Na2
. Filter the extract through cloth mesh, clarify by centrifugation
at 10,000 g for 10 min and add polyethlene glycol (PEG), M. Wt 6000, to a final concentration of 5%
(w/v). After 1-2 h incubation at 4°C, centrifuge at low speed and extract nucleic acids from the
precipitate by protease, phenol-sodium dodecyl sulphate, or chloroform procedures
Randles et al., 1976
The viroid may also be recovered directly from the PEG precipitate by resuspending it in 1 mM EDTA
and precipitating the viroid with 3 vol. ethanol (J.W. Randles & J.S. Imperial, unpublished data).
Subject extracts to electrophoresis in 5% polyacrylamide gels and detect the bands by staining in
aqueous toluidine blue or ethidium bromide. Excise the bands and lay them over a gel containing 8 M
(Randles et al., 1982
A second electrophoresis under these partially denaturing conditions separates circular and linear
forms of the viroid monomer and dimer. Again excise the bands and extract the RNA by maceration and
extraction of gel followed by sucrose density gradient centrifugation
(Randles & Palukaitis, 1979
or by preparative gel electrophoresis, or by electrophoresis into agarose gel
(Randles et al., 1982
followed by gel maceration and extraction. RNA obtained by either method is precipitated with ethanol,
dried, resuspended in 0.2 M NaCl, then reprecipitated with 1% cetyl trimethyl ammonium bromide to
remove gel residue which remains in solution.
Properties of Particles
Occurs as both linear and circular monomers and dimers, with sedimentation coefficients of 7 S
and 10 S
(Riesner et al., 1982
Monomers and dimers are infectious.
Monomer M. Wt is 83,000.
A260/A280 ratio: 2.1.
Buoyant density in Cs2SO4: approximately 1.60 g/cm3
(Randles et al., 1976).
Both the monomer and dimer are single-stranded RNAs, occurring as either covalently closed circular
molecules or as linear molecules
(Randles & Hatta, 1979
In the earliest stages of infection, CCCVd is detected as small forms, with monomeric sizes of either
(Accession No. J02049
or 247 nucleotides; the latter formed by the addition of a cytosine residue
(Haseloff et al. 1982
The 246 form is more frequently observed as the initial infecting agent
(Rodriguez et al., 1995
The dimer is detected simultaneously with the monomer. As infection develops these small forms are
replaced by larger forms which arise through reiteration of a sequence at the 'right hand' end of the
native molecule to give molecules 287-297 nucleotides in size
(Accession No. J02051
The native monomer shows two thermal transitions in 10 mM Na+, at 49° and 58°C; the first
represents cooperative melting of the double-stranded regions to allow the formation of a stable
intermediate, the second represents melting of the intermediate to yield a covalently linked open
(Randles et al., 1982).
The native viroid has 70% GC pairs, the half-width of the first thermal transition
(DT1/2) is 1.4°C
(Randles et al., 1982).
The reversible melting of the viroid to an open circle is the basis of the 2-dimensional
polyacrylamide gel electrophoresis assay used for diagnosis
(Schumacher et al., 1983).
Relations with Cells and Tissues
The viroid has been detected by molecular hybridisation in the inflorescence, in the meristems,
in fronds of various ages and in roots (J.W. Randles & G. Boccardo, unpublished data).
In infected oil palm, CCCVd is localised in the vascular bundles and in the nuclei of mesophyll cells.
Within the nucleus both the viroid and its minus sense form are mainly concentrated in the nucleolus
(Bonfiglioli et al. 1994
Light microscopy of yellow leaf spots shows hypoplasia and loss of chloroplast pigments, while
electron microscopy shows the accumulation of dark structures (which are presumed to be tannin
bodies), but no disease-specific cytoplasmic changes have been recognised
Ecology and Control
Disease boundaries are quite stable. Epidemics occur at different times in different places with
examples of formerly high incidence areas reverting to low incidence areas and new epidemics occurring
in formerly low incidence areas. Patterns of disease increase vary from site-to-site. Sources of
infection have not been identified. A negative correlation has been observed between incidence and
but a positive correlation has been observed between palm age and incidence. Thus, palms less than 10
years old are rarely affected and the incidence increases nearly linearly up to palm ages of about 40
years, thereafter remaining constant
(Zelazny et al., 1982
The beetles, Oryctes rhinoceros
, Plesispa reichei
sp. are more
abundant in areas of high incidence
(Zelazny & Pacumbaba, 1982
but no insects have been shown to transmit Cadang-cadang.
Eradication where only late stage palms were removed has failed to control the disease and in the
absence of other management strategies, it is recommended that losses can be minimised by removing and
replanting diseased trees as soon as early symptoms are recognised. To date, no resistance has been
(Orolfo et al., 2000).
Coconut cadang-cadang is the most serious of all the viroid diseases because of its lethality and has
not been found outside the Philippines. The viroid is not associated with Kerala wilt or Tatipaka
disease of coconut palm in India, nor with coconut foliar decay in Vanuatu or coconut rapid decline in
Sri Lanka (A.A.L. Perera & J.W. Randles, unpublished data). The similar but distinct Tinangaja disease
of coconut, which is associated with Coconut tinangaja viroid
infection, is reported only from
(Wall & Randles, 2003
- Anonymous, Cadang-cadang research, 1971-1982. Tech. Rep. F.A.O./U.N. Dev. Proj. AG:DP/ PH1/523, 75 pp., 1982.
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A stand of Cadang-cadang affected old palms in the Philippines together with a 10 year-old
infected palm (indicated by the arrow).
High pressure jet inoculation of a 3-day old coconut sprout with Coconut cadang-cadang viroid.
The unusual 'brooming' symptom observed in an inoculated coconut palm with severe stunting
and persistence of stipules at the base of leaf fronds.
In the background is a healthy palm of the same age.
Reduced laminae from a palm with 'brooming' (lower) compared with leaflets from typical
cadang-cadang infected (middle) and healthy palms (upper).
20% non-denaturing polyacrylamide gel electrophoresis-silver stain assay of leaf extracts from
palms showing varying intensities of the 'brooming' symptom (lanes 1-5 and a mixture of their
isolates in lane no. 6; M denotes Coconut cadang-cadang viroid marker consisting of the
commonly detected variants 246, 247, 296 and 297 nucleotides.
The primary and secondary structure of Coconut cadang-cadang viroid 246 with demarcation
according to the proposed terminal (T1 and T2), pathogenicity (P), central conserved (CC) and
variable (V) domains of
Keese et al. (1988).
The sites of mutations associated with the brooming symptom are denoted by red asterisks.
Mutations at positions 87, 197 and 216 and their putative effects on secondary structure.
The substituted or added nucleotides are shown in red letters.