Identification of natural host range of coconut cadang-cadang viroid and characterization of its small RNAs from oil palm
Coconut cadang-cadang viroid (CCCVd), a viroid from the genus Cocadviroid in the family Pospiviroidae, is the causal agent of orange spotting disorder in oil palm and variants of CCCVd have been detected and characterized from commercial oil palm plantations in Malaysia. It is considered as a pot...
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| Format: | Thesis |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/76097/1/ITA%202014%208%20-%20IR.pdf |
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| Summary: | Coconut cadang-cadang viroid (CCCVd), a viroid from the genus Cocadviroid in
the family Pospiviroidae, is the causal agent of orange spotting disorder in oil palm
and variants of CCCVd have been detected and characterized from commercial oil
palm plantations in Malaysia. It is considered as a potential threat to oil palm
industry while epidemiological aspects of the viroid infection in oil palm are
unknown. In order to investigate natural host range of the viroid, 64 leaf samples
from 25 plant species from various locations in Selangor state and Kuala Lumpur
were collected and tested by Reverse Transcription-Polymerase Chain Reaction (RTPCR)
assay. Based on amplification of DNA molecules of ca. 250 bp representing
basic monomeric form of the viroid (CCCVd246), CCCVd was detected in 14 plant
species including 7 palm, 3 monocotyledonous and 4 dicotyledonous plant species.
From the palm species, CCCVd was detected from Cocos nucifera, Corypha utan,
Pritchardia pacifica, Ptychosperma macarthurii, Livistona chinensis, Saribus
rotundifolius and Wodyetia bifurcata but not from Hyophorbe lagenicaulis,
Cyrtostachys renda, Veitchia merrillii, Rhapis excels, Dypsis lutescens and Ravenala
madagascariensis. Fourteen out of 16 collected samples from coconut palms (Cocos
nucifera) were found to contain CCCVd molecules. From other monocotyledonous
and dicotyledonous plant species CCCVd was detected from Heliconia sp., Maranta
arundinacea, Etlingera elatior, Pseuderanthemum reticulatum, Codiaeum
variegatum, Osmoxylon sp. and Carica papaya but it was not detected from
Calathea luthea, Canna sp., Dracaena surculosa and Bauhinia sp. Sequence
analysis of 10 isolated CCCVd variants from 9 hosts revealed that there was no
sequence variation among the variants. The consensus sequence of the variants was
246 nt in size with 100% homology to oil palm variant (CCCVd-OP246) and
exhibiting similar substitutions of C31 by U and G70 by C in P and CCR domains
respectively compared to CCCVd246 from coconut palms from the Philippines.
Validation of results from RT-PCR was carried out through Two Dimensional-
Polyacrylamide Gel Electrophoresis (2D-PAGE) analysis of nucleic acid extract of
the samples and by hybridization assay. In 2D-PAGE analysis, bands representing circular viroid RNAs were detected in the samples that were positive for CCCVd in
RT-PCR and not in CCCVd negative samples. In hybridization assay these bands
were hybridized with high stringency to full length digoxigenin (DIG) labeled cRNA
probe and colorimetric signal was produced in their positions on membrane. In order
to investigate the occurrence of Post-Transcriptional Gene Silencing (PTGS) and
CCCVd small RNAs accumulation in oil palm, total small RNAs of 20-30 nt from
two CCCVd infected asymptomatic and symptomatic oil palms were isolated and
cloned. Small RNAs of full homology to CCCVd sequence were not detected among
the sequenced clones. An attempt was made to establish Next Generation
Sequencing (NGS) for detection of CCCVd variants from oil palm. Analysis of data
obtained from deep sequencing of RNAs from a CCCVd infected oil palm showed
that populations of CCCVd variants or other novel viroids were not detected but a
365 nt plant rRNA sequence was detected that was erroneously deposited in
GenBank as Yucatan isolates of Citrus exocortis viroid (CEVd). |
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