Detection and identification of phytoplasmas associated with coconut wilt disease in derawan Island East Kalimantan

Abstract

Coconut as a main commodity in Derawan Island has two functions, i.e. farmer’s income source and beach plant aesthetics. The occurrence of coconut wilt disease resulted in eradication of about 10% of the palm in 2010. The objectives of this study were: (1) to describe morphological and histopathological symptoms of coconut wilt disease in Derawan island, (2) to detect the presence of phytoplasmas associated with coconut wilt disease in Derawan island using nested-PCR (n-PCR) technique, (3) to identify and classify phytoplasmas associated with coconut wilt disease based on 16S rRNA gene sequences, (4) to detect the presence of phytoplasmas from leafhoppers (Hemiptera: Cicadellidae) and love vein (Cassytha filiformis) (Laurales: Lauraceae), which expected to have ability to transmit the pathogen. The methods in this research included visual and microscopic observation, detection of phytoplasmas with n-PCR method, cloning of the nPCR products, sequencing, analysis of sequencing results using BLAST, construction of phylogenetic tree, and in silico RFLP analysis. The results showed that the suspected phytoplasmas reside in phloem tissues and were detected with both microscopic and molecular techniques. Phytoplasmas associated with coconut wilt disease in Derawan island belong to 16SrII (witches’ broom phytoplasma) and 16SrXI (Ca. Phytoplasma oryzae) groups. The use of nPCR employing R16F2n/R16R2 primer pair with expected amplicon at size of about 1.25 kb are positive not only for particular strain of phytoplasmas but also other bacterial species that mainly belong to Gram Positive group. Phytoplasmas that belong to 16SrII are also detected in some leafhoppers and C. filliformis.