Detection and Molecular Characterization of Phytoplasma Associated with Coconut Yellow Decline
Nejat, Naghmeh (2009) Detection and Molecular Characterization of Phytoplasma Associated with Coconut Yellow Decline. PhD thesis, Universiti Putra Malaysia.
Phytoplasmas have been detected and characterized by molecular methods in coconut palm (Cocos nucifera L.) for the first time in Malaysia. Polymerase chain reaction (PCR) assays were used to determine whether a phytoplasma is associated with a yellow decline disease in different coconut ecotypes including Malayan Red Dwarf (MRD), Malayan Yellow Dwarf (MYD) and Malayan Tall (MT) palms. No amplification products were visible from symptomatic samples in first round PCR using phytoplasma universal primer pair P1/P7, but nested PCR with primer pairs R16F2n/R16R2 and fU5/rU3 resulted in amplification of products of approximately 1.2 kb and 890 bp respectively, from 8 out of 20 MRD, 9 out of 12 MYD and 12 out of 12 MT symptomatic palms tested. Sequence analysis of the 16S rDNA PCR products determined that the phytoplasma strain associated with coconut yellow decline (CYD) in MRD and MT ecotypes belongs to the ‘Candidatus Phytoplasma cynodontis’ (16SrXIV) group of phytoplasmas. The phytoplasma derived from MYD presented high levels (97%) of homology with the sequences of the ‘Candidatus Phytoplasma trifolii’ (16SrVI) group. The virtual RFLP analyses also confirmed that MRD and MT CYD belongs to the ‘Ca. Phytoplasma cynodontis̕ group (16SrXIV), whilst MYD CYD does not belong to the identified groups based upon 16S rDNA virtual RFLP analysis. Nested R16F2n/R16R2 PCR products from 6 spear leaves and 2 inflorescences from MRD palms showed high sequence similarity to the 16S rRNA gene from coconut chloroplasts, with a similar size (approximately 1.3 kb), and a further 5 R16F2n/R16R2 PCR products from MRD infloresences showed high sequence similarities to Bacillus spp. and Bacillus megaterium 16S rRNA gene sequences. These Bacillus PCR products also showed a similar RFLP profile to that obtained from the CYD phytoplasma when the restriction enzyme EcoRI was used. Trunk borings were the most reliable source of DNA for phytoplasma detection in coconuts using 16S rRNA gene primers, since there is less co-amplification of PCR products from other organisms when compared to spear leaves and inflorescences. Real-time PCR using TaqMan probe was developed for sensitive, quantitative and rapid detection of coconut yellow decline (CYD) phytoplasma which is not related to the identified phytoplasma groups. Primers and probe were designed from the highly conserved 16S rRNA gene of CYD phytoplasma. The CYD primers were designed to amplify CYD phytoplasmas in genomic DNA extracts prepared from symptomatic MYD and MRD coconut ecotypes. The selected primers amplify specifically a target 112-bp fragment from the 16S rRNA gene region. The real-time PCR assay reliably detected the CYD phytoplasma in DNA from symptomatic MYD and MRD coconut palm ecotypes with the qCYD 16S probe. The result also shows that the concentration of the pathogen is typically low.