Molecular Identification of Mycorrhizal Fungi Species in the Roots of Azadirachta excelsa Trees in South Bengkulu Regency, Bengkulu Province
Abstract
Azadirachta excelsa trees are cultivated in many regions of South Bengkulu Regency. This species is known to form associations with mycorrhizal fungi, which assist the trees in absorbing nutrients and water while protecting their roots from pathogenic attacks. The present study aimed to identify the types of mycorrhizal fungi associated with the roots of A. excelsa trees through biomolecular analysis in three villages of South Bengkulu Regency, Bengkulu Province. Root samples were collected from A. excelsa stands located in Pagar Dewa Village, Manna Subdistrict; Tanjung Eran Village, Pino Subdistrict; and Batu Ampar Village, Kedurang Subdistrict, South Bengkulu Regency. Root DNA extraction was conducted using a Geneaid Fungal Kit, followed by Polymerase Chain Reaction (PCR) with forward ITS5.8S and reverse ITS4 primers. Electrophoresis was performed using a 1.5% agarose gel in TAE 1X buffer at 100V for 32 minutes, and sequencing was carried out at 1st BASE PT Genetic Science Indonesia. The resulting sequences were then compared with those available in the GenBank database, and a phylogenetic tree was reconstructed using MEGA XI. Sequencing results revealed three species of mycorrhizal fungi associated with A. excelsa roots from samples collected in Pagar Dewa. These included a species from the Agaricaceae family (Delicatula integrella), belonging to the order Agaricales, class Agaricomycetes, and division Basidiomycota. Two additional species—Basidiomycota sp. (MT645155.1), Uncultured fungus (MF568791.1), Uncultured fungus (MT672409.1), and Uncultured fungus (MH636718.1)—were classified as unclassified Basidiomycota or could not be definitively categorised in the GenBank database.
Keywords:
Azadirachta excelsa, Basidiomycota, Biomolecular, Mycorrhiza, Sequencing
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References
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Kitur, A., Stedje, B., Wabuyele, E., & Løken, S. B. (2025). Exploring the phylogenomics of Dipcadi (Asparagaceae, Scilloideae) and assessing the utility of ITS as a DNA barcode. Plant Systematics and Evolution, 311(3), 17. https://doi.org/10.1007/s00606-025-01947-0
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Padmalatha, K., & Prasad, M. N. V. (2006). Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India. African Journal of Biotechnology, 5(3), 230-234. https://doi.org/10.5897/AJB05.188
Parwito, P., & Anwar, G. (2024). Literature Review Study of Google Scholar Indexed Articles on Mycorrhiza in Plant Disease Control. Journal of Tropical Mycorrhiza, 3(1), 29-38. https://doi.org/10.58222/jtm.v3i1.76
Peng, S., Guo, T., & Liu, G. (2013). The effects of arbuscular mycorrhizal hyphal networks on soil aggregations of purple soil in southwest China. Soil Biology and Biochemistry, 57, 411-417. https://doi.org/10.1016/j.soilbio.2012.10.026
Phillips, R. P., Brzostek, E., & Midgley, M. G. (2013). The mycorrhizal‐associated nutrient economy: a new framework for predicting carbon–nutrient couplings in temperate forests. New Phytologist, 199(1), 41-51. https://doi.org/10.1111/nph.12221
Premono, T., & Lestari, S. (2013). Analisis Finansial Agroforestri Kayu Bawang) Dan Kebutuhan Lahan Minimum Dysoxilum Mollissimum Blume Di Provinsi Bengkulu [Financial Analysis of Dysoxillum mollisimum Blume) and Minimum Land Requirements in Bengkulu Province]. Journal of Social and Economic Forestry Research. 10(4): 211-223. https://doi.org/10.20886/jsek.2013.10.4.211-223
Redecker, D., Kodner, R., & Graham, L. E. (2000). Glomalean Fungi from the Ordovician. Science, 289(5486), 1920-1921. https://doi.org/10.1126/science.289.5486.1920
Riviere, T., Diedhiou, A. G., Diabate, M., Senthilarasu, G., Natarajan, K., Verbeken, A., ... & Ba, A. M. (2007). Genetic diversity of ectomycorrhizal Basidiomycetes from African and Indian tropical rain forests. Mycorrhiza, 17(5), 415-428. https://doi.org/10.1007/s00572-007-0117-6
Sambrook, J., & Russell, D. W. (1989). Molecular cloning: A laboratory manual (2nd ed., Vol. 3). Cold Spring Harbor Laboratory Press.
Sharma, S., & Kumar, S. (2021). Fast and accurate bootstrap confidence limits on genome-scale phylogenies using little bootstraps. Nature Computational Science, 1(9), 573-577. https://doi.org/10.1038/s43588-021-00129-5
Siahaan, H., Suhendang, E., Rusolono, T., & Sumadi, A. (2011). Growth of Dysoxylum molissimum Bl. in various planting patterns and stand densities. Journal of Plantation Forest Research, 8(4), 225–237.
Lim, G. S., & Bottrell, D. G. (1994). Neem pesticides in rice: Potential and limitations. International Rice Research Institute. http://books.irri.org/9712200477_content.pdf
Steidinger, B. S., Crowther, T. W., Liang, J., Van Nuland, M. E., Werner, G. D., Reich, P. B., ... & Peay, K. G. (2019). Climatic controls of decomposition drive the global biogeography of forest-tree symbioses. Nature, 569(7756), 404-408. https://doi.org/10.1038/s41586-019-1128-0
Taylor, D. L., Walters, W. A., Lennon, N. J., Bochicchio, J., Krohn, A., Caporaso, J. G., & Pennanen, T. (2016). Accurate estimation of fungal diversity and abundance through improved lineage-specific primers optimized for Illumina amplicon sequencing. Applied and Environmental Microbiology, 82(24), 7217-7226. https://doi.org/10.1128/aem.02576-16
Tedersoo, L., Bahram, M., Cajthaml, T., Põlme, S., Hiiesalu, I., Anslan, S., ... & Abarenkov, K. (2016). Tree diversity and species identity effects on soil fungi, protists and animals are context dependent. The ISME journal, 10(2), 346-362. https://doi.org/10.1038/ismej.2015.116
Thompson, J. R., Marcelino, L. A., & Polz, M. F. (2002). Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Research, 30(9), 2083-2088. https://doi.org/10.1093/nar/30.9.2083
Ulfa, M., Faridah, E., Sumardi, S., Lee, S. S., Mansor, P., ie Roux, C., & Galiana, A. (2019). Identifikasi molekuler jenis-jenis jamur mikoriza ekto yang berasosiasi dengan dipterokarpa di hutan hujan tropika sekunder [Molecular Identification of Ectomycorrhizal Fungi Species Associated with Dipterocarpa in Secondary Tropical Rain Forest]. Jurnal Wana Tropika, 9(1). https://jurnal.instiperjogja.ac.id/index.php/JWT/article/view/158
van Der Heijden, M. G., Martin, F. M., Selosse, M. A., & Sanders, I. R. (2015). Mycorrhizal ecology and evolution: the past, the present, and the future. New Phytologist, 205(4), 1406-1423. https://doi.org/10.1111/nph.13288
Anwar, G., Lilleskov, E. A., & Chimner, R. A. (2020). Arbuscular mycorrhizal inoculation has similar benefits to fertilization for Thuja occidentalis L. seedling nutrition and growth on peat soil over a range of pH: implications for restoration. New Forests, 51(2), 297-311. https://doi.org/10.1007/s11056-019-09732-x
Chubo, J. K. A. (2009). Mycorrhizal spore suppression and phosphorus effects on root morphology, growth, and nutrient content of seedlings of Azadirachta excelsa [Master’s thesis, Universiti Sains Malaysia]. Universiti Sains Malaysia.
Dahlberg, A. (2001). Community ecology of ectomycorrhizal fungi: an advancing interdisciplinary field. New Phytologist, 150(3), 555-562. https://doi.org/10.1046/j.1469-8137.2001.00142.x
Delavaux, C. S., LaManna, J. A., Myers, J. A., Phillips, R. P., Aguilar, S., Allen, D., ... & Averill, C. (2023). Mycorrhizal feedbacks influence global forest structure and diversity. Communications biology, 6(1), 1066. https://doi.org/10.1038/s42003-023-05410-z
Delavaux, C. S., Smith‐Ramesh, L. M., & Kuebbing, S. E. (2017). Beyond nutrients: a meta‐analysis of the diverse effects of arbuscular mycorrhizal fungi on plants and soils. Ecology, 98(8), 2111-2119. https://doi.org/10.1002/ecy.1892
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39(4), 783-791. https://doi.org/10.2307/2408678
Hillis, D. M., & Bull, J. J. (1993). An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology, 42(2), 182-192. https://doi.org/10.1093/sysbio/42.2.182
Huat, O. K., Awang, K., Hashim, A., & Majid, N. M. (2002). Effects of fertilizers and vesicular–arbuscular mycorrhizas on the growth and photosynthesis of Azadirachta excelsa (Jack) Jacobs seedlings. Forest Ecology and Management, 158(1-3), 51-58. https://doi.org/10.1016/S0378-1127(00)00668-X
Kanokmedhakul, S., Kanokmedhakul, K., Prajuabsuk, T., Panichajakul, S., Panyamee, P., Prabpai, S., & Kongsaeree, P. (2005). Azadirachtin Derivatives from Seed Kernels of Azadirachta e xcelsa. Journal of Natural Products, 68(7), 1047-1050. https://doi.org/10.1021/np050064t
Khairani, M. (2022). Meta-analysis of the diversity of ectomycorrhizal fungi in Indonesia. JP25 Journal. STKIP Asy-Syafi'iyah International Medan.
Kitur, A., Stedje, B., Wabuyele, E., & Løken, S. B. (2025). Exploring the phylogenomics of Dipcadi (Asparagaceae, Scilloideae) and assessing the utility of ITS as a DNA barcode. Plant Systematics and Evolution, 311(3), 17. https://doi.org/10.1007/s00606-025-01947-0
Kõljalg, U., Larsson, K. H., Abarenkov, K., Nilsson, R. H., Alexander, I. J., Eberhardt, U., ... & Ursing, B. M. (2005). UNITE: a database providing web‐based methods for the molecular identification of ectomycorrhizal fungi. New phytologist, 166(3), 1063-1068. https://doi.org/10.1111/j.1469-8137.2005.01376.x
Lilleskov, E. A., Fahey, T. J., Horton, T. R., & Lovett, G. M. (2002). Belowground ectomycorrhizal fungal community change over a nitrogen deposition gradient in Alaska. Ecology, 83(1), 104-115. https://doi.org/10.1890/0012-9658(2002)083[0104:BEFCCO]2.0.CO;2
Mutakim, D., Nugroho, J., Wanggai, J., Rahmadaniarti, & Mahmud, A. (2022). Fungi mikoriza arbuskula (fma) berasosiasi dengan tiga jenis tegakan pohon asal papua. [Arbuscular mycorrhizal fungi (AMF) associated with three types of tree stands native to Papua]. Papuasia Forestry Journal, 8(2), 297–308. https://doi.org/10.46703/jurnalpapuasia.Vol8.Iss2.363
Padmalatha, K., & Prasad, M. N. V. (2006). Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India. African Journal of Biotechnology, 5(3), 230-234. https://doi.org/10.5897/AJB05.188
Parwito, P., & Anwar, G. (2024). Literature Review Study of Google Scholar Indexed Articles on Mycorrhiza in Plant Disease Control. Journal of Tropical Mycorrhiza, 3(1), 29-38. https://doi.org/10.58222/jtm.v3i1.76
Peng, S., Guo, T., & Liu, G. (2013). The effects of arbuscular mycorrhizal hyphal networks on soil aggregations of purple soil in southwest China. Soil Biology and Biochemistry, 57, 411-417. https://doi.org/10.1016/j.soilbio.2012.10.026
Phillips, R. P., Brzostek, E., & Midgley, M. G. (2013). The mycorrhizal‐associated nutrient economy: a new framework for predicting carbon–nutrient couplings in temperate forests. New Phytologist, 199(1), 41-51. https://doi.org/10.1111/nph.12221
Premono, T., & Lestari, S. (2013). Analisis Finansial Agroforestri Kayu Bawang) Dan Kebutuhan Lahan Minimum Dysoxilum Mollissimum Blume Di Provinsi Bengkulu [Financial Analysis of Dysoxillum mollisimum Blume) and Minimum Land Requirements in Bengkulu Province]. Journal of Social and Economic Forestry Research. 10(4): 211-223. https://doi.org/10.20886/jsek.2013.10.4.211-223
Redecker, D., Kodner, R., & Graham, L. E. (2000). Glomalean Fungi from the Ordovician. Science, 289(5486), 1920-1921. https://doi.org/10.1126/science.289.5486.1920
Riviere, T., Diedhiou, A. G., Diabate, M., Senthilarasu, G., Natarajan, K., Verbeken, A., ... & Ba, A. M. (2007). Genetic diversity of ectomycorrhizal Basidiomycetes from African and Indian tropical rain forests. Mycorrhiza, 17(5), 415-428. https://doi.org/10.1007/s00572-007-0117-6
Sambrook, J., & Russell, D. W. (1989). Molecular cloning: A laboratory manual (2nd ed., Vol. 3). Cold Spring Harbor Laboratory Press.
Sharma, S., & Kumar, S. (2021). Fast and accurate bootstrap confidence limits on genome-scale phylogenies using little bootstraps. Nature Computational Science, 1(9), 573-577. https://doi.org/10.1038/s43588-021-00129-5
Siahaan, H., Suhendang, E., Rusolono, T., & Sumadi, A. (2011). Growth of Dysoxylum molissimum Bl. in various planting patterns and stand densities. Journal of Plantation Forest Research, 8(4), 225–237.
Lim, G. S., & Bottrell, D. G. (1994). Neem pesticides in rice: Potential and limitations. International Rice Research Institute. http://books.irri.org/9712200477_content.pdf
Steidinger, B. S., Crowther, T. W., Liang, J., Van Nuland, M. E., Werner, G. D., Reich, P. B., ... & Peay, K. G. (2019). Climatic controls of decomposition drive the global biogeography of forest-tree symbioses. Nature, 569(7756), 404-408. https://doi.org/10.1038/s41586-019-1128-0
Taylor, D. L., Walters, W. A., Lennon, N. J., Bochicchio, J., Krohn, A., Caporaso, J. G., & Pennanen, T. (2016). Accurate estimation of fungal diversity and abundance through improved lineage-specific primers optimized for Illumina amplicon sequencing. Applied and Environmental Microbiology, 82(24), 7217-7226. https://doi.org/10.1128/aem.02576-16
Tedersoo, L., Bahram, M., Cajthaml, T., Põlme, S., Hiiesalu, I., Anslan, S., ... & Abarenkov, K. (2016). Tree diversity and species identity effects on soil fungi, protists and animals are context dependent. The ISME journal, 10(2), 346-362. https://doi.org/10.1038/ismej.2015.116
Thompson, J. R., Marcelino, L. A., & Polz, M. F. (2002). Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Research, 30(9), 2083-2088. https://doi.org/10.1093/nar/30.9.2083
Ulfa, M., Faridah, E., Sumardi, S., Lee, S. S., Mansor, P., ie Roux, C., & Galiana, A. (2019). Identifikasi molekuler jenis-jenis jamur mikoriza ekto yang berasosiasi dengan dipterokarpa di hutan hujan tropika sekunder [Molecular Identification of Ectomycorrhizal Fungi Species Associated with Dipterocarpa in Secondary Tropical Rain Forest]. Jurnal Wana Tropika, 9(1). https://jurnal.instiperjogja.ac.id/index.php/JWT/article/view/158
van Der Heijden, M. G., Martin, F. M., Selosse, M. A., & Sanders, I. R. (2015). Mycorrhizal ecology and evolution: the past, the present, and the future. New Phytologist, 205(4), 1406-1423. https://doi.org/10.1111/nph.13288
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Anwar, G., Sipriyadi, S. and Manik, M. (2025) “Molecular Identification of Mycorrhizal Fungi Species in the Roots of Azadirachta excelsa Trees in South Bengkulu Regency, Bengkulu Province”, International Journal of Advancement in Life Sciences Research, 8(4), pp. 11-21. doi: https://doi.org/10.31632/ijalsr.2025.v08i04.002.
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