• Agus Eko Prasetyo Pusat Penelitian Kelapa Sawit
  • Kikin Hamzah Mutaqin Departemen Proteksi Tanaman, Fakultas Pertanian, Institut Pertanian Bogor
  • Giyanto Departemen Proteksi Tanaman, Fakultas Pertanian, Institut Pertanian Bogor




coconut wilt disease, phytoplasmas, nested PCR, sequencing


Characterization of phytoplasmas associated with coconut wilt disease in Derawan Island using in silico RFLP. Coconutwilt disease has been reported in Derawan Island that resulted in eradication up to 10% of the total cultivated palms. Theobjective of this study was to detect and characterize phytoplasmas associated with coconut wilt disease in Derawan islandusing nested PCR technique and in silico RFLP based on 16S rRNA gene sequences. Detection of phytoplasmas was performedusing nested PCR method, cloning of nPCR products, sequencing, and analysis of sequencing results using in silico RFLP.The results revealed that phytoplasmas could not be detected by PCR using P1/P7 primer pairs however it could be amplifiedby nested PCR using R16F2n/R16R2 primer pairs resulting amplicon at about 1.25 kb. In silico RFLP analysis indicated thatphytoplasmas associated with coconut wilt disease in Derawan Island belong to 16SrII (witches broom phytoplasma). PCRproduct of the nPCR need to be sequenced because the R16F2n/R16R2 primer will also amplify the other bacterial species, mainly from Gram positive bacteria.


Anonim. 2007. Prospek dan Arah Pengembangan Agribisnis Kelapa. Ed ke-2. Jakarta: Departemen Pertanian.

Bai XD, Zhang JH, Ewing A, Miller SA, Radek AJ, Shevchenko DV, Tsukerman K, Walunas T, Lapidus A, Campbell JW, & Hogenhout SA. 2006. Living with genome instability, the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. J. Bacteriol. 188(10): 3682–3696.

Deng S & Hiruki C. 1991. Amplification of 16S rRNA genes from culturable and non culturable mollicutes. J. Microbiol. Methods 14(1): 53–61.

Dickinson M, Tuffen M, & Hodgetts J. 2013. The phytoplasma: an introduction. In: Dickinson M & Hodgetts J (Eds.) Phytoplasma Methods and Protocols. pp. 1–14. Humana Press, New York.

Gundersen DE & Lee IM. 1996. Ultrasensitive detection of phytoplasmas by nested PCR assays using two universal primer pairs. Phytopathol. Mediterr. 35(3): 144–151.

Harrison NA, Myrie W, Jones P, Carpio ML, Castilo M, Doyle MM, & Oropeza C. 2002. 16S rRNA interoperon sequence heterogeneity distinguishes strain populations of palm lethal yellowing phytoplasma in the Carribean region. Ann. Appl. Biol. 141(2): 183–193.

Leon R, Santamaria JM, Alpizar L, Escamilla JA, & Oropeza C. 1996. Physiological and biochemical changes in shoots of coconut palms affected by lethal yellowing. New Phytol. 134(2): 227–234.

Musetti R. 2010. Biochemical changes in plant infected by phytoplasma. In: Weintraub PG & Jones P (Eds.). Phytoplasmas: Genomes, Plant Hosts and Vectors. pp. 132–146. CAB International, Wallingford.

Myrie WA, Paulraj L, Dollet M, Wray D, & Been BO. 2006. First report of lethal yellowing disease of coconut palms caused by phytoplasma on Nevis Island. Plant Dis. 90(6): 834.

Nejat N, Sijam K, Abdullah SNA, Vadamalai G, & Dickinson M. 2009. Molecular characterization of a phytoplasma associated with coconut yellow decline (CYD) in Malaysia. Am. J. Appl. Sci. 6(7): 1331–1340.

Oropeza C, Cordova I, Narvaez M, & Harrison N. 2002. Palm Trunk Sampling for DNA Extraction and Phytoplasma Detection. University of Florida, Florida.

Sambrook J & Russel DW. 2001. Molecular Cloning, A Laboratory Manual. Ed ke-3. Cold Sping Harbor Laboratory Press, New York.

Tymon AM, Jones P, & Harrison NA. 1998. Phylogenetic relationships of coconut phytoplasmas and the development of specific oligonucleotide PCR primers. Ann. Appl. Biol. 132(3): 437–452.

Warokka JS, Jones P, & Dickson MJ. 2006. Detection of phytoplasma associated with kalimantan wilt disease of coconut by the polymerase chain reaction. J. Littri 12(4): 154–160.

Wei W, Davis RE, Lee IM, & Zhao Y. 2007. Computer-simulated RFLP analysis of 16S rRNA genes, identification of ten new phytoplasma groups. Int. J. Syst. Evol. Microbiol. 57: 1855–1867.

Weintraub PG & Wilson MR. 2010. Control of phytoplasma disease and vectors. In: Weintraub PG & Jones P (Eds.). Phytoplasmas: Genomes, Plant Hosts and Vectors. pp. 233–266. CAB International, Wallingford.

Zhang YP, Uyemoto JK, & Kirkpatrick BC. 1998. A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay. J. Virol. Methods 71(1): 45–50.



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