The viroid is thought to cause infection through feeding wounds of coleopteran insects, but this has not been confirmed

Method	Instructions	NAPIS Survey Method
Visual	Visual survey for symptomatic plants is the approved method for Coconut cadang-cadang viroid. Normally leaf (leaflet) tissue is used for diagnosis/identification, but the viroid is present in most host tissues.	3031 - General Visual Observation

No specific signs are present.

Although a range of symptoms are observed with CCCVd, the symptoms alone are not diagnostic. Look for a combination of symptoms when inspecting plant tissue of coconut and oil palms reoccurring in the same tree over time. Molecular testing is necessary to identify the viroid in symptomatic tissue. Most of the time, the lower two-thirds of the leaf crown is yellow; while the upper one-third is still dark green with CCCVd infection.

Some of the general symptoms of CCCVd include: reduced nut size and production, reduced husk production in nuts, reduced tree length/stunting, "genetic " orange spotting on fronds (especially in oil palms), and yellow spotting on leaves. All of these symptoms can eventually lead to plant death. If a palm patch is infected with CCCVd, death of the palms within the patch will continue until the patch is "bare".

The CCCVd-infected leaves in the palm crown are typically smaller than those observed in healthy palms. Leaf scarring is often found on the trunk of the infected palm. Palms that are infected with CCCVd before flowering have leaf scars that are spread farther apart than healthy plants. In contrast, plants infected with CCCVd after flowering have leaf scars that are closer together than healthy plants. Water-soaked leaf spots may appear, but this symptom is not unique to CCCVd. Another possible symptom (although rarely found in the field and in only about 3% of experimental inoculations) is lamina reduction. This symptom can lead to a "brooming symptom" where parts of the frond contain only the midrib.

The life span for a CCCVd-infected palm is about eight years for younger palms (22-year old palms) and 16 years for older palms (44-year old palms). The early stage of disease development, which usually lasts two to four years, is divided into three substages:

• In E1, the first early substage, the nuts usually "round out" with strong "equatorial scarification".



• In E2, the second early substage, yellowing occurs, "water-soaked leaf spots appear", round nuts with equatorial scarification are more frequent, necrosis appears at the tips of inflorescences, and male florets may be lost.



• In E3, the final early substage, nuts are usually not being produced, larger leaf spots appear, "new inflorescences [are] stunted and sterile" and fibrous tissue remains attached to petioles as "winging".

During the middle stage, which usually lasts two years, nut production slows down or completely stops, leaf spots grow and are abundant, and "inflorescences become necrotic (brown/dead)". During the final stage of disease development, usually lasting about five years, the crown is reduced, necrotic leaf spots are still present, and the palm is nearing death or dead.

ID/Diagnostic: Molecular

Reverse transcriptase polymerase chain reaction (RT-PCR) is the approved method for Coconut cadang-cadang viroid.

All palm viroid samples should be sent to Dr. Rosemarie Hammond for sample processing. Palm samples should be received as freshly harvested leaves (complete leaflets) exhibiting symptoms of CCCVd. Samples from plants with atyptical or no symptoms can also be included as the disease is difficult to recognize based on symptoms alone, because symptoms develop in stages depending upon the age of the palm.

Dr. Rosemarie Hammond, Ph.D.
Molecular Plant Pathology Laboratory
USDA-Agricultural Research Service
Building 004, Room 204, BARC-West
Beltsville, MD, USA 20705
Voice: (301) 504-5203
Fax: (301) 504-5449
Email: rose.hammond@ars.usda.gov

For shipment, leaves must be packaged according to APHIS regulations and under permit. Each sample, in a separate plastic bag, will be labeled according to cultivar (when known) and plant species, date sample was collected, location of plant sampled, and name and institution of sample collector; each bag should contain leaves collected from only a single plant. Samples must be shipped to the testing laboratory by overnight express service.

CCCVd is commonly confused with Coconut tinangaja viroid (CTiVd), which is found on Guam. CTiVd is 254 nucleotides in length and has 64 to 65% nucleotide sequence homology to CCCVd. CTiVd has similar visual disease symptoms as well. Both viroids are known to have three stages of infection (early, middle, late). Water-logging and nutrient deficiency due to CTiVd cause yellowing of the palm, showing similar symptoms to CCCVd. Symptoms of both viroids include: spotting on the leaves, reduced crown, and death. The main difference in symptomatology between the two viroids is the effect on the fruit. Plants infected with CCCVd have nuts that are greatly reduced and round. Plants infected with CTiVd have nuts that are small, elongated mummified husks with no kernel.

Electrophoresis: When cellular nucleic acids purified from approximately 1g of coconut tissue are analyzed on polyacrylamide gels, the molecular forms of CCCVd can be identified by their relative electrophoretic mobility.

Dot Blot Hybridization: CCCVd has been cloned and can also be amplified by polymerase chain reaction. The clones and PCR product can be used as templates for synthesis of radioactively labeled complementary RNA or DNA probes. These are used in hybridization assays to detect nucleotide sequences similar to CCCVd. Extracts to be tested are applied to a supporting membrane and the presence of the viroid is detected by its specific hybridization with the probe. The radioactive label causes darkening of exposed x-ray film, while samples without the viroid show no signal. This diagnostic method is called "dot blot hybridization". This method is more sensitive than electrophoresis and the viroid can be detected many months before symptoms appear.

Molecular hybridization of dot-blotted extracts has been used by collecting nuclei acid from chopped leaflets. The dot blots extracts are probed on nitrocellulose sheets with 32P labeled DNA probes complementary to CCCVd. An autoradiogram will reveal the resulting banding pattern (Imperial et al., 1985; Mohamed and Imperial, 1984).

A 3H-cDNA probe has been used for hybridization in solution (Mohamed and Imperial, 1984). The PAGE procedure uses nucleic acids from a leaflet to elute RNA from the gel (Imperial et al., 1985; Mohamed and Imperial, 1984; Mohamed et al.,1985).

PCR: RT-PCR and PCR for CCCVd used primer G2-1 for RT-PCR and primers G2-1 and D9-1 for conventional PCR. RT-PCR needs to be used to be able to separate CCCVd from CTiVd (Hodgson et al.,1998).

A commercial diagnostic kit is available: "Coconut cadang-cadang viroid (CCCVd) RT-PCR detection kit; Norgen Biotek Corp., Thorold, ON Canada, Cat # 47900." This kit is being tested for its sensitivity and specificity for CCCVd detection via a Farm Bill agreement with Dr. Hammond and USDA APHIS.

The pathogen is currently only known to occur in the Philippines.