Molecular hybridization of Northern blots of single (1D) and two-dimensional polyacrylamide gels (2D-PAGE) with a ³² P-labelled full length CCCVd₂ ₄ ₆ cRNA probe demonstrated the presence of Coconut cadang - cadang viroid (CCCVd)-like RNAs in nucleic acid extracts of both symptomatic (orange spotted) and asymptomatic oil palms in commercial plantations in Malaysia. Compared with CCCVd in coconut these CCCVd-like RNAs seemed to be present at low concentration in the oil palm samples as shown by the weak hybridization signals observed in the oil palm samples even when large amounts of nucleic acid extract (leaf fresh weight equivalent of 20-100 g) were loaded onto the gel. Ribonuclease protection assay (RPA) was found to be more sensitive in detecting low concentrations of the CCCVd-like RNAs in the oil palm samples than Northern blots as shown by the higher percentage of positive samples. RPA showed that 90 % of the symptomatic and 50 % of asymptomatic palms from Malaysia had RNAs which protected the ³²P-labelled full length CCCVd antisense probe and produced a similar RPA pattern to that of CCCVd. RPA results also indicated that there were mismatches in the sequence of the CCCVd-like RNAs in the oil palms compared to CCCVd from coconut. RT-PCR amplification of CCCVd-like RNAs from an asymptomatic palm was only successful when nucleic acids were partially purified using 1D or 2D-PAGE. RNAs eluted from the circular region of 2D-gels of the asymptomatic palm were amplified to a low concentration using CCCVd-specific primers but re-amplification of these first round RT-PCR products was needed for detection of the amplicons by ethidium bromide staining. No amplified product was obtained from a symptomatic palm. Cloning and sequencing of the RT-PCR products from the asymptomatic oil palm produced 20 clones of five sizes comprising 297 nt (OP₂₉₇), 293 nt (OP₂₉₃), 270 nt (OP₂₇₀), 232 nt (OP₂₃ ₂) and 165 nt (OP₁₆₅). 71 % of the clones were OP₂₉₇. Comparison of OP₂₉₇, OP₂₉₃, and OP₂₇₀ with genome database sequences showed high sequence similarity with CCCVd₂₉₆. OP₂₉₇. OP₂₉₃, OP₂₇₀ had 98 %, 97 % and 90 % sequence similarity with CCCVd₂₉₆ respectively. OP₂₃₂ and OP₁₆₅ also had high sequence similarity with parts of CCCVd ₂₄₆ with which they were aligned. Because an arbitrary level of 90 % sequence similarity is accepted as separating viroid species from variants, OP₂₉₇ , OP₂₉₃ and OP₂₇₀ can be considered as variants of CCCVd. No variants of the ' fast ' CCCVd ₂ ₄ ₆ form were obtained. The consensus OP₂₉₇ sequence had single base substitutions or additions at 5 sites, OP₂₉₃ had substitutions, additions or deletions at 8 sites, and OP₂₇₀ had substitutions at 4 sites as well as deletion of a 26 nt repeat at the right terminus, producing a predicted branched secondary structure. Compared with CCCVd₂₉₆ , all variants substituted (C-U) at nt 31 in the pathogenicity domain and (A-C) at nt 175 in the right hand terminal domain. The presence of sequences similar to OP₂₃₂ and OP₁₆₅ has not been reported for CCCVd. Analysis of DNA extracted from both symptomatic and asymptomatic oil palms from Malaysia by nested PCR using universal primers sets to amplify the 16S rRNA operon showed the presence of phytoplasma-like DNAs in both sets of samples. They were also detected in DNA extracted from oil palm seedlings maintained at the Waite campus but not in the other palm species maintained in the glasshouse. RFLP analysis of phytoplasma-like DNAs gave a different pattern than that expected for Australian grapevine yellows phytoplasma. The phytoplasma - like DNAs were also not related to lethal yellowing phytoplasma (LYp) as PCR analysis with LYp specific primers did not produce any amplicon. No association with OS was found and so they were not characterised further. CCCVd-infected coconut leaf collected in the Philippines contained two short interfering RNAs (siRNA) approximately 20 nt and 25 nt in size. A high stringency wash of the Northern blots failed to remove the hybridisation signal suggesting that these siRNAs had sequences closely similar to CCCVd. The siRNAs were present in all stages of the cadang-cadang and also samples with the 'brooming' symptom. siRNAs are regarded as a marker for post-transcriptional gene silencing (PTGS) in plants infected by viroids but the results obtained were insufficient to determine whether PTGS regulates the accumulation of CCCVd. This is the first report that a viroid closely related to CCCVd occurs in oil palm, and in a region outside the Philippines, the country where CCCVd is thought to be contained. The implications for quarantine matters are discussed. / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2005.
| Identifer | oai:union.ndltd.org:ADTP/263647 |
| Date | January 2005 |
| Creators | Vadamalai, Ganesan |
| Source Sets | Australiasian Digital Theses Program |
| Language | en_US |
| Detected Language | English |
Page generated in 0.0016 seconds