* Corresponding Author
Perancangan Primer Spesifik Subspesies Berbasis Gen Endoglukanase untuk Deteksi Ralstonia syzygii subsp. syzygii
https://doi.org/10.22146/jpti.32217 Bambang Trianom(1), Triwidodo Arwiyanto(2), Tri Joko(3*)
(1) Balai Karantina Pertanian Bandar Lampung Jln. Soekarno-Hatta km. 7, Bandar Lampung, Lampung 35241
(2) Departemen Hama dan Penyakit Tumbuhan, Fakultas Pertanian, Universitas Gadjah Mada Jln. Flora No. 1, Bulaksumur, Yogyakarta 55281
(3) Departemen Hama dan Penyakit Tumbuhan, Fakultas Pertanian, Universitas Gadjah Mada Jln. Flora No. 1, Bulaksumur, Yogyakarta 55281
(*) Corresponding Author
Abstract
Ralstonia syzygii subsp. syzygii that belong to Ralstonia solanacearum species complex is the cause of Sumateran disease of clove. The disease was reported to cause widespread devastation on clove plantings in Indonesia. One of the control strategies is to reduce the spread of the disease through early detection on clove seedlings. The study aimed to design the specific primers based on endoglucanase (egl) gene of R. syzygii subsp. syzygii as a tool for early diagnosis. The analyses were conducted on development of specific primers design using egl sequences retrieved from GenBank, Polymerase Chain Reaction (PCR), primers sensitivity and specificity test. The pair of primers UGMRss-F (5’-GCTCACCATCGC CAAGGACAGCG-3’) and UGMRss-R (5’-TTCGATCGAACGCCTGGTTGAGC-3’) could amplify R. syzygii subsp. syzygii at ~378 base pairs with 0.8 ng/µl minimum concentration of DNA. The primers was specific to R. syzygii subsp. syzygii but not to other bacterial species even in the same phylotype.
Intisari
Ralstonia syzygii subsp. syzygii merupakan bakteri yang termasuk dalam kelompok Ralstonia solanacearum species complex yang menyebabkan penyakit Sumatera pada tanaman cengkih. Penyakit ini menyebabkan kerugian yang sangat besar dan sampai saat ini belum ditemukan cara pengendalian yang efektif. Salah satu upaya pencegahan penyakit adalah melalui deteksi dini dan mencegah penyebaran penyakit melalui peredaran bibit dari areal yang endemis. Penelitian ini bertujuan untuk merancang primer spesifik berbasis gen endoglukanase (egl) sebagai upaya deteksi dini penyakit Sumatera. Analisis yang dilakukan meliputi desain primer spesifik dengan menggunakan data sekuens gen egl dari GenBank, Polymerase Chain Reaction (PCR), uji kepekaan primer dan uji kekhususan primer. Desain primer yang berhasil dirancang terdiri dari UGMRss-F (5’- GCTCACCATCGCCAAGGACAGCG-3’) dan UGMRss-R (5’-TTC GATCGAACGCCTGGTTGAGC-3’) dengan amplikon ~378 pasang basa. Pada konsentrasi DNA 0,8 ng/µl, secara peka R. syzygii subsp. syzygii masih dapat teramplifikasi dengan baik. Primer ini juga hanya dapat mendeteksi R. syzygii subsp. syzygii dan tidak untuk bakteri lain bahkan pada filotipe yang sama.
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Arwiyanto, T. 2013. Ralstonia solanacearum, Biologi Penyakit yang Ditimbulkan dan Pengelolaannya. Gadjah Mada University Press,Yogyakarta. 99 hlm.
Bennet, C.P.A., P. Hunt, & A. Asman. 1985. Association of a Xylem-Limited Bacterium with Sumatra Disease of Cloves in Indonesia. Plant Pathology 34: 487–494.
Chuan, L.Y., Y.H. Cheng, & C.H. Yang. 2013. Specific Primer Design for the Polymerase Chain Reaction. Biotechnology Letter 35: 1541–1549.
Danaatmadja, Y., S. Subandiyah, T. Joko, & C.U. Sari. 2009. Isolasi dan Karakterisasi Ralstonia syzygii. Jurnal Perlindungan Tanaman Indonesia 15:7–12.
Dieffenbach, C.W., T.M. Lowe, & G.S. Dveksler. 1993. General Concepts for PCR Primer Design. Genome Research 3: S30-S37.
Dwimartina, F., T. Arwiyanto, & T. Joko. 2017. Potential of Endophytic and Rhizobacteria as an Effective Biocontrol for Ralstonia syzygii subsp. syzygii. Asian Journal of Plant Pathology 11: 191–198.
Eden-Green, S.J., R. Balfas, & T. Sutarjo. 1992. Characteristics of the Transmission of Sumatra Disease of Cloves by Tube-Building Cercopoids, Hindola spp. Plant Pathology 41: 702–712.
Fegan, M. & P. Prior. 2005. How Complex is The ‘‘Ralstonia solanacearum Species Complex’’, p. 449–461. In C. Allen, A.C. Hayward & P. Prior (eds.), Bacterial Wilt Disease and the Ralstonia solanacearum Species Complex. American Phyto-pathological Society, St Paul.
Frank, J.A, C.L. Reich, S. Sharma, J.S. Weisbaum, B.A. Wilson, & G.J. Olsen. 2008. Critical Evaluation of Two Primers Commonly Use for Amplification of Bacterial 16S rRNA Genes. Applied and Enviromental Microbiology 74: 2461–2470.
Handiyanti, M., T. Joko, & S. Subandiyah. 2018. Deteksi Molekuler Burkholderia glumae, Penyebab Penyakit Hawar Malai Padi. Jurnal Perlindungan Tanaman Indonesia 22: 98−107
Ismiyatuningsih, T. Joko, & S. Hartono. 2016. Survey and Detection of Pectobacterium atrosepticum in Major Potato-Growing Areas in Central Java Province, Indonesia. Ilmu Pertanian 1: 1−6.
Joko, T., S. Subandiyah, & S. Somowiyarjo. 2000. The Role of Extracellular Protein on the Pathogenicity of Xanthomonas campestris pv. citri. Jurnal Perlindungan Tanaman Indonesia 6: 32−38.
Joko, T., H. Hirata, & S. Tsuyumu. 2007a. Sugar Transporter (MfsX) of Major Facilitator Superfamily is Required for Flagella-Mediated Pathogenesis in Dickeya dadantii 3937. Journal of General Plant Pathology 73: 266−273.
Joko, T., H. Hirata, & S. Tsuyumu. 2007b. A Sugar Transporter (MfsX) is Also Required by Dickeya dadantii 3937 for in Planta Fitness. Journal of General Plant Pathology 73: 274−280.
Joko, T., D. Kiswanti, S. Subandiyah, & Hanudin. 2011a. Occurence of Bacterial Soft Rot of Phalaenopsis Orchids in Yogyakarta and West Java, Indonesia, p. 255–265. In Y. Koentjoro (ed.), Proceeding of International Seminar on“Natural Resources, Climate Change, and Food Security in Developing Countries”, June 27−28, 2011. Surabaya, Indonesia.
Joko, T., N. Kusumandari, & S. Hartono. 2011b. Optimasi Metode PCR untuk Deteksi Pectobacterium carotovorum, Penyebab Penyakit Busuk Lunak anggrek. Jurnal Perlindungan Tanaman Indonesia 17: 54−59.
Joko, T., M.P. Koentjoro, S. Somowiyarjo, M.S. Rohman, A. Liana, & N. Ogawa. 2012. Response of Rhizobacterial Communities in Watermelon to Infection with Cucumber Green Mottle Mosaic Virus as Revealed by Cultivation-Dependent RISA. Archives of Phytopathology and Plant Protection 45: 1810−1818.
Joko, T., A. Subandi, N. Kusumandari, A. Wibowo, & A. Priyatmojo. 2014. Activities of Plant Cell Wall-Degrading Enzymes by Bacterial Soft Rot of Orchid. Archives of Phytopathology and Plant Protection 47: 1239−1250.
Joko, T., M. Umehara, T. Murata, H. Etoh, K. Izumori, & S. Tsuyumu. 2018. Hyperinduction of Pectate Lyase in Dickeya chrysanthemi EC16 by Plant-Derived Sugars. Journal of Plant Interactions 13: 141−150.
Liu, H., S. Zhang, M.A. Schell, & T.P. Denny. 2005. Pyramiding Unmarked Deletions in Ralstonia solanacearum Shows that Secreted Proteins in Addition to Plant Cell-Wall-Degrading Enzymes Contribute to Virulence. Molecular Plant Microbe Interactions 18: 1296−1305.
Mahfut, T. Joko, & B.S. Daryono. 2016. Molecular Characterization of Odontoglossum Ring Spot Virus (ORSV) in Java and Bali, Indonesia. Asian Journal of Plant Pathology 10: 9−14.
Nurjanah, N., T. Joko, & S. Subandiyah. 2017. Characterization of Pantoea ananatis Isolated from Garlic and Shallot. Jurnal Perlindungan Tanaman Indonesia 21: 120−126.
Poretsky, R., L.M. Rodriguez-R, C. Luo, D. Tsementzi, & K.T. Konstantinidis. 2014. Strengths and Limitations of 16S rRNA Gene Amplicon Sequencing in Revealing Temporal Microbial Community Dynamics. PLoS ONE 9: e93827.
Roberts, D.P., T.P. Denny, & M.A. Schell. 1988. Cloning of the egl Gene of Pseudomonas solanacearum and Analysis of Its Role in Phytopathogenicity. Journal of Bacteriology 170: 1445−1451.
Schaad, NW, J.B. Jones, & W. Chun. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. American Phytopathological Society, St Paul. 373 p.
Safni, I., I. Cleenwerck, P.D. Vos, M. Fegan, L. Sly, & U. Kappler. 2014. Polyphasic Taxonomic Revision of the Ralstonia solanacearum Species Complex: Proposal to Emend the Descriptions of Ralstonia solanacearum and Ralstonia syzygii and Reclassify Current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum Phylotype IV Strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., Banana Blood Disease Bacterium Strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum Phylotype I and III Strains as Ralstonia pseudosolanacearum sp. nov. International Journal of Systematic and Evolutionary Microbiology 64: 3087–3103.
Safni, I., S. Subandiyah, & M. Fegan. 2018. Ecology, Epidemiology and Disease Management of Ralstonia syzygii in Indonesia. Frontiers in Microbiology 9: 1–11.
Suharti, T., T. Joko, & T. Arwiyanto. 2017. Deteksi Bakteri Patogen Terbawa Benih Akor (Acacia auriculiformis A. Cunn. ex Benth.). Jurnal Hama dan Penyakit Tumbuhan Tropika 17: 19–36.
Trianom, B., T. Arwiyanto, & T. Joko. Morphological and Molecular Characteri- zation of Sumatra Disease of Clove in Central Java, Indonesia. Tropical Life Sciences Research (In Press).
Van Pelt-Verkuil, E., A. Van Belkum, & J.P. Hays. 2008. Principals and Technical Aspects of PCR Amplification. Springer, Netherland. 30 p.
Widyaningsih, S., S.N.H. Utami, T. Joko, & S. Subandiyah. 2017. Development of Disease and Growth on Six Scion/Root Stock Combinations of Citrus Seedlings under Huanglongbing Pressure. Journal of Agricultural Science 9: 229−238.
Windari, U., T. Joko, & S. Subandiyah. 2015. Deteksi Penyakit Bacterial Fruit Blotch pada Melon Menggunakan ELISA. Jurnal Perlindungan Tanaman Indonesia 19: 1−5.
Yamamoto, S. & S.Harayama. 1995. PCR Amplification and Direct Sequencing of gyrB Genes with Universal Primers and Their Application to the Detection and Taxonomic Analysis of Pseudomonas putida Strains. Applied and Environmental Microbiology 61: 1104−1109.
Yuwono, T. 2006. Teori dan Aplikasi Polymerase Chain Reaction. Penerbit Andi, Yogyakarta. 246 hlm.
DOI: https://doi.org/10.22146/jpti.32217
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