Coconut cadang-cadang viroid
J. W. Randles
Waite Agricultural Research Institute, The University of Adelaide, South Australia, Australia
J. S. Imperial
Philippine Coconut Authority, Guinobatan, Albay, Philippines
Disease first described by
the viroid 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 using nucleic acid inocula, but mode of natural transmission unknown.
Detected only in Palmae. Distributed in the Philippines and Guam.
Cadang-cadang is a lethal disease of coconut (Cocos nucifera
by a generalised yellow-bronze coloration of the lower two-thirds of fronds in
the crown, 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
As the spots appear, the nuts become smaller and rounded
(Mohamed et al., 1982
and many of them develop equatorial
scarifications. 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
Palms less than 10 years old are very rarely affected, and the incidence
increases nearly linearly up to palm age of about 40 years, thereafter remaining
(Zelazny et al., 1982
Total losses since recognition of the
disease are estimated at c.
30 million palms
(Zelazny et al., 1982
Tinangaja is a similar disease, but differs in that the nuts are characteristically
small and elongated, and lacking a kernel, i.e. mummified
Boccardo et al., 1981
Naturally infected oil palm (Elaeis guineensis) shows a progression
from general chlorosis on younger fronds to development of translucent orange
and cessation of inflorescence production
(Randles, Boccardo & Imperial, 1980).
Cadang-cadang disease is recognised in the central Philippines (southern
Luzon, Samar, Masbate and smaller islands within a zone about 600 km x 300 km).
Tinangaja disease occurs on Guam (Marianas Islands). Distribution is unaffected
by water barriers, but incidence is negatively correlated with altitude
Host Range and Symptomatology
Known hosts are confined to the Palmae. Transmitted experimentally by high
pressure injection combined with pricking
(Randles et al., 1977
- Cocos nucifera (coconut). Inoculated seedlings show spotting on leaflets
of fronds that are at least three positions below the unexpanded spear leaf
together with reduction in palm growth, frond size and production, and failure
to bear nuts at the expected time.
- Chlorotic or orange spotting of leaflets is characteristic of other inoculated
species of palm such as betel nut palm (Areca catechu), bori palm (Corypha
elata), Manila palm (Adonidia merrillii), oil palm, palmena
(Chrysalidocarpus lutescens) and royal palm (Oreodoxa regia).
- Naturally infected Cocos nucifera.
- Cocos nucifera is used as a systemic assay host; the viroid is detected
by gel electrophoretic or molecular hybridisation assay 1-2 years after inoculation.
All sequenced isolates of the coconut cadang-cadang viroid have the same
minimal nucleotide sequence
(Haseloff, Mohamed & Symons, 1982
to the structure are not strain differences but are related to stage of disease
(Imperial, Rodriguez & Randles, 1981
The symptoms of tinangaja
disease differ somewhat from those of cadang-cadang disease and the causal
viroid is regarded as a strain, although the nucleotide sequence is not available.
Transmission by Vectors
Mode of natural transmission is unknown.
Transmission through Seed
Very low, e.g.
1 out of 320 of seedlings tested
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).
The coconut cadang-cadang viroid is the smallest known pathogen, and is
biologically distinct from other viroids. It has two partially complementary
regions of about 20 nucleotides (I,
that are almost identical to regions
potato spindle tuber
chrysanthemum stunt and
but no other regions are homologous. It shows no significant nucleotide sequence
avocado sunblotch viroid
which is only one nucleotide
larger than the smallest form of coconut cadang-cadang viroid.
The viroid associated with tinangaja disease is similar in size to the
cadang-cadang viroid and has nucleotide sequence homology with it. The extent
of the differences between these two viroids has yet to be determined
(Boccardo et al., 1981).
Stability in Sap
No information on survival of infectivity in sap.
Chop leaflets from fronds four or more below the spear leaf, blend with 3
vol. 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 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 and J. S. Imperial, unpublished data).
Subject extracts to electrophoresis in 5% polyacrylamide gels, and detect the
bands by staining in aqueous toluidine blue. Excise the bands and lay them over
a gel containing 8 M urea
(Randles, Steger & Riesner, 1982
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
(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.
Viroid samples are assayed by electrophoresis in polyacrylamide gels
and the viroid is detected by staining with toluidine blue, ethidium bromide or
(Schumacher, Randles & Riesner, 1983).
Properties of Infective Nucleic Acid
Occurs as monomer and dimer, with sedimentation coefficients of 7 S and
(Riesner, Kaper & Randles, 1982
Monomer M. Wt is 83,000.
Buoyant density in
Cs2SO4: c. 1.60 g/cm3
(Randles et al., 1976).
Both monomer and dimer are single-stranded RNA occurring as covalently linked
or as linear molecules. The complete nucleotide
sequence is known
The smallest form comprises 246 nucleotides, but
addition of a cytosine residue (*,
) increases the size to 247
(Haseloff et al., 1982
The dimer is detected simultaneously
with the monomer. As infection develops these small forms are replaced by large
which arise through reiteration of a sequence at the right hand
end of the native molecule (region II,
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
(DT½) is 1.4°C
(Randles et al., 1982).
The structure of the viroid is more stable than that of cell RNA species in
incubated polyethylene glycol concentrates of coconut leaf sap
Randles, Rillo & Diener, 1976).
Relations with Cells and Tissues
The viroid has been detected by molecular hybridisation in the inflorescence
and in the meristems, in fronds of various ages and in roots (J. W. Randles and
G. Boccardo, unpublished data). Light microscopy of yellow leaf spots shows
hypoplasia, and loss of chloroplast pigments; electron microscopy shows accumulation
of dark structures which are presumed to be tannin bodies, but no disease-specific
cytoplasmic changes have been recognised
Coconut cadang-cadang is the most serious of the viroid diseases because of
its lethality. Its area of distribution in the Philippines has increased very
little in the last 26 years, but epidemics arise and decline within this zone
(Zelazny et al., 1982
The mode of natural transmission is unknown, and
eradication measures fail to control the disease. The viroid is not associated
with Kerala wilt or Tatipaka disease of coconut palm in India, nor with a lethal
disease of coconut palm in Vanuatu (J. W. Randles and M. Dollet, unpublished data).
- Anonymous, Cadang-cadang research, 1971-1982. Tech. Rep. F.A.O./U.N. Dev. Proj. AG:DP/PH1/523, 75 pp., 1982.
- Boccardo, Beaver, Randles & Imperial, Phytopathology 71: 1104, 1981.
- Haseloff, Mohamed & Symons, Nature, Lond. 299: 316, 1982.
- Imperial, Rodriguez & Randles, J. gen. Virol. 56: 77, 1981.
- Mohamed, Haseloff, Imperial & Symons, J. gen. Virol. 63: 181, 1982.
- Ocfemia, Philipp. Agric. 26: 338, 1937.
- Randles, Phytopathology 65: 163, 1975.
- Randles, in Subviral Pathogens of Plants and Animals: Viroids and Prions, ed. K. Maramorosch & J. J. McKelvey, New York, Academic Press, p. 39, 1985.
- Randles & Hatta, Virology 96: 47, 1979.
- Randles & Palukaitis, J. gen. Virol. 43: 649, 1979.
- Randles, Rillo & Diener, Virology 74: 128, 1976.
- Randles, Boccardo, Retuerma & Rillo, Phytopathology 67: 1211, 1977.
- Randles, Boccardo & Imperial, Phytopathology 70: 185, 1980.
- Randles, Steger & Riesner, Nucleic Acids Res. 10: 5569, 1982.
- Riesner, Kaper & Randles, Nucleic Acids Res. 10: 5587, 1982.
- Schumacher, Randles & Riesner, Analyt. Biochem. 135: 288, 1983.
- Symons, Nucleic Acids Res. 9: 6527, 1981.
- Zelazay, Phytopathology 70: 700, 1980.
- Zelazny & Niven, Pl. Dis. 64: 841, 1980.
- Zelazny, Randles, Boccardo & Imperial, Scientia Filipinas 2: 45, 1982.
An area of high cadang-cadang incidence.
Nuts typical of cadang-cadang (left) compared with mummified nuts
associated with tinangaja. (Photograph courtesy of G. Boccardo, Istituto di
Fitovirologia Applicata, Turin.)
Scarified and rounded nuts (right) from a palm at an early stage
of infection, compared with normal nuts.
Chlorosis and yellow-orange spotting of two leaves (centre and
right) of infected oil palm compared with healthy leaf (left).
Leaf spot development on second youngest open frond (top) to
ninth frond of cadang-cadang infected coconut palm.
Polyacrylamide gel electrophoretic separation of small (cc-1f) and large
(cc-1s) monomeric forms of the viroid and the small dimeric form (cc-2f).
Denatured circles of monomeric (above) and dimeric forms
(below) of the viroid (Randles & Hatta, 1979). Bar represents 150 nm.
Primary sequence of the minimal infective form of coconut cadang-cadang
viroid (from data of Haseloff et al., 1982, sequence
J02049). Note the conserved sequences (I),
the sequences (II) that are reiterated in variant molecules produced at later stages
of infection, and the site (*) where cytosine occurs either as a single or double