Molecular Characterization of a Phytoplasma Associated with Coconut Yellow Decline (CYD) in Malaysia

K Sijam,S.N.A Abdullah,M Dickinson,G Vadamalai,N Nejat

doi:10.3844/ajassp.2009.1331.1340

Abstract

Problem statement: Molecular methods have been used to detect phytoplasma in Malayan Red Dwarf (MRD) ecotype of coconut for the first time in Malaysia. Detect phytoplasma from coconut showing yellowing symptoms by nested PCR and Classify phytoplasma associated with disease of coconut palms, based on analysis of 16S rRNA gene operon sequences and virtual RFLP. Approach: Twenty MRD symptomatic palms were tested. Leaf spear, inflorescence and wood shavings from trunks of coconut palms showing yellowing symptoms were harvested from Serdang located in Selangor state. PCR assays and sequence analysis were carried out. Results: 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 symptomatic palms tested. Sequence analysis of the 16S rDNA PCR products determined that the phytoplasma strain associated with Coconut Yellow Decline (CYD) in MRD ecotype belongs to the 'Candidatus Phytoplasma cynodontis' (16SrXIV) group of phytoplasmas. In addition, 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 inflorescences showed high sequence similarities to Bacillus spp. 16S rRNA gene sequences. Conclusion: These results indicate that sequencing is a reliable method for the detection. Furthermore, trunk borings are 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, nor from the spear leaves and inflorescences of MRD palms when primers fU5/rU3 were used in nested PCR.

Highlights

Coconut palm (Cocos nucifera L.) is a versatile tree, popularly know as 'King of Palms' and 'Kalpavriksha', which translates as "tree that gives all that is necessary for living." Considered the most useful tree in the world, the coconut palm provides sustainable income to millions who are directly and indirectly dependant on it, and provides highly nutritious food, drink, clothing, shelter, heirloom history, financial security, alleviate poverty and export earnings[48].Many historians believe that the origin of coconut was in the Asia- Pacific region, the Malayan Archipelago[32]
Sequence analysis of amplified products: Polymerase Chain Reaction (PCR) products obtained from the Malayan Red Dwarf (MRD) palms were cloned into the vector pCR2.1 and the sequences of the inserts determined using primer pairs R16F2n/R16R2 (1250 bp) and fU5/rU3 (890 bp)
Clones from the 8 symptomatic MRD samples that were tested were all identical. Comparisons of these 16S rRNA gene sequences of Coconut Yellow Decline (CYD) to other phytoplasma 16S rDNA sequences reported in Gen Bank, using the tool Basic Local Alignment Search Tool (BLAST) revealed that CYD belongs to the ‘Candidatus phytoplasma cynodontis’ group (16SrXIV) with 99% homology to the rRNA gene sequence of bermuda grass whiteleaf phytoplasma from Thailand (AF248961) and Malaysia (EU294011), members of the 16SrXIV

Summary

Introduction

Coconut palm (Cocos nucifera L.) is a versatile tree, popularly know as 'King of Palms' and 'Kalpavriksha', which translates as "tree that gives all that is necessary for living." Considered the most useful tree in the world, the coconut palm provides sustainable income to millions who are directly and indirectly dependant on it, and provides highly nutritious food, drink, clothing, shelter, heirloom history, financial security, alleviate poverty and export earnings[48].Many historians believe that the origin of coconut was in the Asia- Pacific region, the Malayan Archipelago[32]. The large number of coconut palms shows general yellowing symptoms of fronds. General yellowing and decline of plants are often considered as the symptoms of the presence of phytoplasmas, which are the causal agents of plants yellows diseases[23] and the symptoms of diseased palms show similarities to coconut lethal yellowing. Lethal Yellowing (LY) caused by phytoplasmas is a highly destructive disease on coconut and has been reported in the Caribbean, Florida and Mexico[38,39,40]. The first symptoms are yellowing of fronds followed by blackening of emerging inflorescences, premature nutfall and usually nut production effectively stops. Similar lethal-yellowing-like diseases caused by phytoplasmas have devastated palms in both west and east Africa[17]

Methods

Results

Conclusion