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

Collect complete leaflets exhibiting symptoms of CCCVd.

Samples from plants with atypical or no symptoms can also be collected since the disease is difficult to recognize based on symptoms alone, because symptoms develop in stages depending upon the age of the palm.

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.

Real-time polymerase chain reaction (qPCR) kits for primary screening are available from Norgen Biotek Corp.: https://norgenbiotek.com/product/cccvd-detection-kits.

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

Fresh palm samples brown and degrade quickly during transport. To ensure sample quality, dry the palm samples over a drying agent, like calcium chloride or silica gel, prior to shipping to Dr. Hammond. To dry and ship samples we recommend:

1. cutting the leaf tissue into strips,


2. placing the strips into sealable tubes (e.g. 15 ml plastic test tube) containing a small amount of drying agent (do not overfill tubes with drying agent or leaves to allow for air circulation), and


3. seal the tube and prepare it for shipment.

If drying is not possible, DO NOT freeze the leaves. Instead, keep the leaves cool by placing the samples into an insulated box with lid and add freezer bags/cold packs. Tape the box shut and package it for shipment.

For shipment, package samples according to APHIS regulations. Place all leaflets from a single plant into a plastic bag and seal the bag. If samples are in tubes, place the tubes containing leaves from a single plant together in a plastic bag and seal the bag. Label the plastic bag with the following information:

• name of plant species and cultivar (if known),


• date sample was collected,


• location of plant sampled (coordinates when possible),


• and the name and institution of collector.

Send package by overnight delivery service. Notify Dr. Hammond"s lab prior to mailing samples. Avoid shipping samples on Fridays since samples are not delivered on weekends.

Send samples to Dr. Rosemarie Hammond. Notify Dr. Hammond and include an explanation of what you are sending, a PDF file of your completed PPQ Form 391, and the tracking number. If sending fresh leaflets, ship the package overnight. Do not ship samples on Fridays because package will not be received until Monday.

Dr. Rosemarie W. Hammond
USDA ARS NEA BARC Molecular Plant Pathology Laboratory
Building 004 Room 214 BARC-West
10300 Baltimore Avenue
Beltsville, MD 20705
(Phone) 301-504-5203
(Fax) 301-504-5449
rose.hammond@usda.gov

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.