Rapid detection, accumulation and translocation of Coconut cadang-cadang viroid variants in oil palm
Coconut cadang-cadang viroid (CCCVd) is the causal agent of the lethal Coconut cadang-cadang disease in the Philippines. CCCVd variants were also found in Malaysian oil palm, but in low concentration and difficult to detect. Palms infected with CCCVd variants were associated with orange spotting...
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| Format: | Thesis |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/76104/1/ITA%202014%209%20-%20IR.pdf |
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| Summary: | Coconut cadang-cadang viroid (CCCVd) is the causal agent of the lethal Coconut
cadang-cadang disease in the Philippines. CCCVd variants were also found in
Malaysian oil palm, but in low concentration and difficult to detect. Palms infected
with CCCVd variants were associated with orange spotting (OS) disorder and it was
estimated that the average yield from the OS-affected palms was 25-50% lower than
the healthy palms. Existing diagnostic methods such as hybridization assay and
Reverse transcription polymerase chain reaction (RT-PCR) methods are able to
detect CCCVd variants in oil palms; however they are laborious, insensitive and
time-consuming. In addition, the relationship between CCCVd sequence variation
and viroid accumulation has not been studied. In view of this, the present research
was undertaken to produce a simple and rapid detection of CCCVd variants using
reverse transcription Loop-mediated isothermal amplification (RT-LAMP) method.
Besides that, CCCVd accumulation between two different variants together with
movement in inoculated oil palm seedlings was studied using real-time PCR.
Simultaneously, the pathogenicity of the two different variants was observed in the
inoculated seedlings. CCCVd variants were successfully detected in infected samples
as early as 60 min at 60 °C using the primer C2.1. Positive reactions of RT-LAMP
showed colour change from orange to green after addition of a fluorescent reagent.
The RT-LAMP products generated a laddering pattern on 2% agarose gel
electrophoresis with bands of different sizes. The optimal condition for RT-LAMP
amplification of CCCVd RNAs from oil palm was at 60 °C for 60 min with 6.0 mM
MgSO4, 0.8 M betaine, 2.4 mM dNTPs, 1.6 μM of each inner primer FIP and BIP,
0.4 μM each of outer primer B3 and F3 and 8U Bst DNA polymerase. The sensitivity
of both RT-PCR and RT-LAMP was found to be equal. The RT-LAMP primers were
specific in detecting CCCVd since they did not amplify other viroids that were used
in the specificity test. CCCVd were also detected in some of the random field
samples collected. The BLAST program results showed that the amplified product of
RT-LAMP was indeed a variant of CCCVd246OP. For viroid accumulation study, two
variants of CCCVd (CCCVd246OP and CCCVd293OP) were inoculated into 10 three month old oil palm seedlings each, with 10 control seedlings. A RT-PCR and
sequencing was carried out to characterise the CCCVd variants obtained from the
inoculated seedlings at different time intervals. CCCVd was detected three months
after inoculation in low concentrations. The viroid titre showed that the accumulation
of the CCCVd variant in the inoculated seedlings fluctuated without any distinct
increasing or decreasing pattern. The results of sequencing analysis showed that
CCCVd246OP (Genbank: HQ608513.1) was characterised from seedlings inoculated
with CCCVd293OP plasmids. In addition, a 246-nt sequence was also recovered from
the symptomatic (OS) seedling with 99% sequence similarity to CCCVd246OP, which
confirmed the pathogenicity of this 246-nt CCCVd variant. In viroid movement
study, the CCCVd246OP variant was inoculated into 12 seedlings. The seedlings were
sampled every three months with three replicates; separating the leaves, stems and
roots. CCCVd was detected in the leaves, stems and roots of the oil palm seedlings
after three, six, nine and twelve months of inoculation. The quantification showed
that CCCVd load varied in different parts of the seedlings, with higher
concentrations in the stems and leaves as compared to the roots. |
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