Istiana Prihatini, Farah Aulya Faradilla, Suranto Suranto


In vivo control activities test of BCA candidate is required to evaluate its effectiveness. This test involved identification of the fungal species that inhabited the experimental plant after inoculation of BCA and pathogen. Identification of inhabitant fungi in the BCA control activity test can be conducted by culturing the fungi on artificial media. Fungal species can be identified based on morphological characters or genetic characters of fungal culture. This study was conducted to determine the potential of PCR ITS-RFLP molecular markers in the initial selection process of fungal isolates before identification based on DNA sequences and to select the enzyme that produce polymorphic PCR ITS RFLP pattern. Three enzymes (DraI, EcoRI and HinfI) were successfully separated 8 fungal cultures used in this study into three groups based on the pattern of PCR ITS DNA, while five other enzymes (BamHI, BclI HaeII, HpaI, and HindIII) were failed to cut the DNA ITS fragments, except for one isolate.


fungal identification; isolates selection; restriction enzyme

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Agustini, L., Wahyuno, D., Indrayadi, H., & Glen, M. (2014). In vitro interaction between Phlebiopsis sp. and Ganoderma philippii isolates. Forest Pathology, 44(6), 472–476. https://doi.org/10.1111/efp.12143

Buana, R. F. N., Wahyudi, A. T., & Toruan-Mathius, N. (2014). Control Activity of Potential Antifungal-Producing Burkholderia sp. in Suppressing Ganoderma boninense Growth in Oil Palm. Asian Journal of Agricultural Research, 8, 259–268.

Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., & Sayers, E. W. (2016). GenBank. Nucleic Acids Research, 44(D1), D67–D72. https://doi.org/10.1093/nar/gkv1276

Datta, S., Choudhary, R. G., Shamim, M., & Dhar, V. (2011). Polymorphism in the internal transcribed spacer (ITS) region of the ribosomal DNA among different Fusarium species. Archives of Phytopathology and Plant Protection, 44(6), 558–566. https://doi.org/10.1080/03235400903187402

Dean, T. R., Kohan, M., Betancourt, D., & Menetrez, M. Y. (2005). A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi. Molecular Biotechnology, 31(1), 21–27. https://doi.org/10.1385/MB:31:1:021

Diguta, C. F., Proca, I. G., Jurcoane, S., & Matei, F. (2018). Molecular characterization by PCR-RFLP of indigenous fungal isolates from hypersaline stream water in Romania. Folia Microbiologica, in press, 1–13.

Diguta, C. F., Vincent, B., Guilloux-Benatier, M., Alexandre, H., & Rousseaux, S. (2011). PCR ITS-RFLP: A useful method for identifying filamentous fungi isolates on grapes. Food Microbiology, 28, 1145e1154. https://doi.org/10.1016/j.fm.2011.03.006

Duttweiler, K. B., Sun, G. Y., Batzer, J. C., Harrington, T. C., & Gleason, M. L. (2008). An RFLP-Based Technique for Identifying Fungi in the Sooty Blotch and Flyspeck Complex on Apple. Plant Disease, 92(5), 794–799. https://doi.org/10.1094/PDIS-92-5-0794

Elad, Y., Baker, S. C., & Faull, J. L. (2004). Multi trophic relationships – interaction of a biocontrol agent and a pathogen with the indigenous micro-flora on bean leaves Multi trophic relationships – interaction of a biocontrol agent and a pathogen with the indigenous micro-flora on bean leaves. Management of Plant Diseases and Arthropod Pests by BCAs, 27(January), 151–154.

Endang, A., & Farikhah, H. N. (2010). Infestation of Xystrocera festiva in Paraserianthes falcataria plantation in East Java, Indonesia. Journal of Tropical Forest Science, 22(4), 397–402. https://doi.org/10.1007/BF01586686

Fliegerová, K., Paîoutová, S., Mrázek, J., & Kopeân, J. (2002). Special properties of polycentric anaerobic fungus Anaeromyces mucronatus, 441–444.

Gardes, M., & Bruns, T. D. (1993). ITS primers with enhanced specificity for basidiomycetes, application to the identification of mycorrihiza and rusts. Molecular Ecology, 2, 113–118. https://doi.org/Doi 10.1111/J.1365-294x.1993.Tb00005.X

Glen, M., Bougher, N. L., Francis, A. A., Nigg, S. Q., Lee, S. S., Irianto, R. S. B., … Mohammed, C. L. (2009). Ganoderma and Amauroderma species associated with root-rot disease of Acacia mangium plantation trees in Indonesia and Malaysia. Australasian Plant Pathology, 38(4), 345–356. https://doi.org/10.1071/AP09008

Harsono, W. N., Rakhmawati, A., & Prihatini, I. (2016). Isolasi dan identifikasi kapang endofit dari pohon sengon provenan kepulauan Solomon berdasarkan morfologi dan molekuler (analisa rDNA ITS (Internal Transcribed Spacer). Jurnal Biologi, 5(6), 15–27.

Hong, S. G., Liu, D., & Pryor, B. M. (2005). Restriction mapping of the IGS region in Alternaria spp. reveals variable and conserved domains. Mycological Research, 109(1), 87–95. https://doi.org/10.1017/S0953756204001388

Kowalski, T., & Drozynska, K. (2011). Mycobiota in needles and shoots of Pinus nigra following Infection by Dothistroma septosporum. Phyton-Annales Rei Botanicae, 51(2), 277–287.

Lawrence, D. P., Holland, L. A., Nouri, M. T., Travadon, R., Abramians, A., Michailides, T. J., & Trouillas, F. P. (2018). Molecular phylogeny of Cytospora species associated with canker diseases of fruit and nut crops in California, with the descriptions of ten new species and one new combination. IMA Fungus, 9(2), 333–370. https://doi.org/10.5598/imafungus.2018.09.02.07

Mohammed, H. A. (2013). A PCR ITS-RFLP method for identifying fungal contamination of date palm (Phoenix dactylifera L.) tissue cultures. African Journal of Biotechnology, 12(32), 5054–5059. https://doi.org/10.5897/AJB2013.12407

Nilsson, R. H., Glöckner, F. O., Saar, I., Tedersoo, L., Kõljalg, U., Abarenkov, K., … Picard, K. (2018). The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic Acids Research, 47(D1), D259–D264. https://doi.org/10.1093/nar/gky1022

Nusaibah, S. A., Latiffah, Z., & Hassaan, A. R. (2011). ITS-PCR-RFLP analysis of Ganoderma sp. infecting industrial crops. Pertanika Journal of Tropical Agricultural Science, 34(1), 83–91.

O’Donnell, K., Sutton, D. A., Rinaldi, M. G., Sarver, B. A. J., Balajee, S. A., Schroers, H. J., … Geiser, D. M. (2010). Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. Journal of Clinical Microbiology, 48(10), 3708–3718.

Old, K. M., Lee, S. S., Sharma, J. K., & Zi, Q. Y. (2000). A Manual of Diseases of Tropical Acacias in Australia, South-East Asia and India Design and production: Vlad Mosmondor. Jakarta: Center for International Forestry Research.

Palapala, V. A., Aimi, T., Inatomi, S., & Morinaga, T. (2002). ITS-PCR-RFLP Method for Distinguishing Commercial Cultivars of Edible Mushroom, Flammulina velutipes. Journal of Food Science, 67(7), 2486–2490. https://doi.org/10.1111/j.1365-2621.2002.tb08763.x

Pei, N., Chen, B., & Kress, W. J. (2017). Advances of Community-Level Plant DNA Barcoding in China. Frontiers in Plant Science, 8, 225. https://doi.org/10.3389/fpls.2017.00225

Prihatini, I., Glen, M., Wardlaw, T. J., & Mohammed, C. L. (2015). Lophodermium pinastri and an unknown species of Teratosphaeriaceae are associated with needle cast in a Pinus radiata selection trial. Forest Pathology, 45(4). https://doi.org/10.1111/efp.12169

Prihatini, I., Glen, M., Wardlaw, T. J., & Mohammed, C. L. (2016). Diversity and identification of fungi associated with needles of Pinus radiata in Tasmania. Southern Forests, 78(1), 19–34. Retrieved from https://www.scopus.com/inward/record.uri?eid=2-s2.0-84959239010&partnerID=40&md5=39d3a9910727e63a477ab2baaf4808f5

Prihatini, I., Glen, M., Wardlaw, T., & Mohammed, C. (2014). Multigene phylogenetic study of Cyclaneusma species. Forest Pathology, 44(2).

Prihatini, I., Rimbawanto, A., Puspitasari, D., & Fauzi, D. (2018). PENGUJIAN PENANDA JENIS SPESIFIK PADA JAMUR YANG BERPOTENSI SEBAGAI AGENS PENGENDALI HAYATI PENYAKIT BUSUK AKAR PADA AKASIA. Jurnal Pemuliaan Tanaman Hutan; Vol 12, No 1 (2018): Jurnal Pemuliaan Tanaman Hutan. https://doi.org/10.20886/jpth.2018.12.1.1-12

Puspitasari, D., Wibowo, A., Rahayu, S., Prihatini, I., & Rimbawanto, A. (2016). Karakter morfologi isolat Phlebiopsis sp . 1 jamur pengendali hayati yang potensial untuk Ganoderma philippii. Jurnal Pemuliaan Tanaman, 10(1), 51–62.

Putri, K. P., & Bramasto, Y. (2017). Pengendalian cendawan Uromycladium tepperianum pada bibit sengon (Falcataria moluccana ( Miq .) Barneby & J . W . Grimes ). Jurnal Perbenihan Tanaman Hutan, 5(1), 13–22.

Raeder, U., & Broda, P. (1985). Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology, 1(1), 17–20.

Shamim, M., Kumar, P., Kumar, R. R., Kumar, M., Kumar, R. R., & Singh, K. N. (2017). Assessing Fungal Biodiversity Using Molecular Markers. In B. P. Singh & V. K. Gupta (Eds.), Molecular Markers in Mycology: Diagnostics and Marker Developments (pp. 305–333). Cham: Springer International Publishing. https://doi.org/10.1007/978-3-319-34106-4_15

Srivastava, S., Gupta, P. S., Lal, S., & Sinha, O. K. (2017). Rapid identification of endophytic fungi of sugarcane (Saccharum spp. hybrid) using PCR-RFLP of rDNA. Journal of Environmental Biology, 38(1), 21–26. https://doi.org/10.3399/bjgp09X453576

Szemiako, K., Śledzińska, A., & Krawczyk, B. (2017). A new assay based on terminal restriction fragment length polymorphism of homocitrate synthase gene fragments for Candida species identification. Journal of Applied Genetics, 58(3), 409–414. https://doi.org/10.1007/s13353-017-0394-5

Tigini, V., Prigione, V., Di Toro, S., Fava, F., & Varese, G. C. (2009). Isolation and characterisation of polychlorinated biphenyl (PCB) degrading fungi from a historically contaminated soil. Microbial Cell Factories, 8(1), 5. https://doi.org/10.1186/1475-2859-8-5

Viaud, M., Pasquier, A., & Brygoo, Y. (2000). Diversity of soil fungi studied by PCR-RFLP of ITS. Mycological Research, 104(9), 1027–1032. https://doi.org/10.1017/S0953756200002835

White, T. J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: A guide to methods and applications. Academic Press, Inc.

Xiang, L., Gong, S., Yang, L., Hao, J., Xue, M., Zeng, F., … Yu, D. (2016). Biocontrol potential of endophytic fungi in medicinal plants from Wuhan Botanical Garden in China. Biological Control, 94, 47–55. https://doi.org/10.1016/j.biocontrol.2015.12.002

Yeh, F. C., Yang, R.-C., Boyle, T. B. J., Ye, Z.-H., & Mao, J. X. (1999). POPGENE, the user-friendly shareware for population genetic analysis. Edmonton: Molecular Biology and Biotechnology Centre, University of Alberta, Canada. Retrieved from http://www.ualberta.ca/-fyeh/fyeh

Yuskianti, V., Glen, M., Puspitasari, D., Francis, A., Rimbawanto, A., Gafur, A., … Mohammed, C. L. (2014). Species-specific PCR for rapid identification of Ganoderma philippii and Ganoderma mastoporum from Acacia mangium and Eucalyptus pellita plantations in Indonesia. Forest Pathology, 44(6), 477–485. https://doi.org/10.1111/efp.12144

DOI: https://doi.org/10.20886/jpth.2019.13.1.33-43


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