Molecular Identification of Endophytic Fungi Associated with Cynometra ramiflora L. and Wrightia pubescens (R. Br.) Using the Internal Transcribed Spacer (ITS) Region of rDNA and Its Morphotypes
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Abstract
In this study, endophytic fungi from Philippine medicinal plants namely, Cynometra ramiflora and Wrightia pubescens were isolated and identified. The endophytic fungi were isolated using potato dextrose agar, and were identified by amplifying a fragment of their internal transcribed spacer (ITS) regions of rDNA using polymerase chain reaction. The identified endophytic fungi were Colletotrichum karsti, Diaporthe longicolla, Phomopsis columnaris, D. pseudomangiferae, C. acutatum, and D. inconspicua. All of the isolates belonged to phylum Ascomycota. Two isolates (C. karsti and C. acutatum) belonged to Glomerellaceae family and four isolates (D. longicolla, P. columnaris, D. pseudomangiferae, and D. inconspicua) were under the Diaporthaceae family. In addition, based on cultural morphology, the isolated endophytic fungi were described as irregular and circular morphotypes. This is the first report on the isolation of endophytic fungi associated with healthy leaves of C. ramiflora and W. pubescens.
Keywords: Cynometra ramiflora; Wrightia pubescens; endophytic fungi; Diaporthaceae; Glomerellaceae
*Corresponding author: Tel.: (+63) 9171031333
E-mail: jerwinundan@clsu.edu.ph
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References
Schulz, B. and Boyle, C., 2005. The endophytic continuum. Mycological Research, 109, 661-686.
Romero, A., Carrion, G. and Rico-Gray, V., 2001. Fungal latent pathogens and endophytes from leaves of Parthenium hysterophorus (Asteraceae). Fungal Diversity, 7, 81-87.
Strobel, G. and Daisy, B., 2003. Bioprospecting for microbial endophytes and their natural products. Microbiology Molecular Biology Reviews, 67, 491-502.
Arnold, A.E., Mejia, L. C., Kyllo, D., Rojas, E. I., Maynard, Z., Robbins N. and Herre, E.A., 2003. Fungal endophytes limit pathogen damage in a tropical tree. Proceedings of the National Academy of Science of the United Science of America, 100, 15649-15654.
Ratnaweera, P.B., De Silva, E.D., Williams, D.E. and Andersen, R.J., 2015. Antimicrobial activities of endophytic fungi obtained from the arid zone invasive plant Opuntia dillenii and the isolation of equisetin, from endophytic Fusarium sp.. BMC Complementary and Alternative Medicine, 15, 220, https//doi.org/10.1186/s12906-015-0722-4
Haryoto, M., Azizaha, T., Indrayudha, P., Suhendi, A. and Heng, K.H., 2014. Antioxidant, cytotoxic activities and chemical constituents of Cynometra ramiflora L.. International Journal of Indigenous Medicinal Plants, 47, 1578-1586.
Jittimanee, J., Panomket, P. and Wanrum, S., 2013. Inhibition of prostaglandin E2 by substances derived from Wrightia pubescens latex in LPS-activated RAW 264.7 mouse macrophages. Journal of Medical Technology and Physical Therapy, 25, 36-42.
Delos Reyes, M.M., Oyong, G.G., Ng, V.A.S., Shen, C. and Ragasa, C.Y., 2018. Cytotoxic compounds from Wrightia pubescens (R.Br.). Pharmacognosy, 10, 9-15.
Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V. Spouge, J.L., Andre Levesque, L., Chen, W. and Fungal Barcoding Consortium, 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proceedings of the National Academy of Science of the United Science of America, 109, 6241-6246.
Wang, X., Liu, C., Huang, L., Bengtsson-Palme, J. Chen, H., Zhang, J., Cai, D and Li, J., 2014. ITS1: a DNA barcode better than ITS2 in eukaryotes? Molecular Ecology Resources, 15, 573-586.
Hebert, P.D.N., Ratnasingham, S. and deWaard, J.R., 2003. Barcoding animal life: Cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society B: Biological Science, 270 (Suppl 1), S96-S99.
Mbareche, H., Veillette, M., Bilodeau, G. and Duchaine, C., 2020. Comparison of the performance of ITS1 and ITS2 as barcodes in amplicon-based sequencing of bioaerosols. Peerj, 8, e8523, https://doi.org/10.7717/peerj.8523
Gardes, M. and Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycets-application to the identification of mycorrhizae and rusts. Molecular Ecology, 2, 113-118.
White, T.J., Bruns, T., Lee, S. and Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White, eds. PCR Protocols: A Guide to Methods and Applications. San Diago: Academic Press, pp. 315-322.
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389-3402.
Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K., 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35, 1547-1549.
Tamura, K. and Nei, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 10, 512-526.
Stecher, G., Tamura, K. and Kumar, S., 2020. Molecular evolutionary genetics analysis (MEGA) for macOS. Molecular Biology and Evolution, 37(4), 1237-1239.
Taheri, H., Javan-Nikkhah, M., Elahinia, S.A., Khodaparast, S.A. and Golmohammadi. M., 2016. Species of Colletotrichum associated with citrus trees in Iran. Mycologia Iranica, 3, 1-14.
Yang, Q., Jiang, N. and Tian, CM., 2020. Three new Diaporthe species from Shaanxi Province, China. MycoKeys, 67, 1-18.
Farr, D.F., Lisa, A., Castlebury, A.M.Y., Rossman, Y. and Putnam, M.L., 2002. A new species of Phomopsis causing twig dieback of Vaccinium vitis-idaea (lingonberry). Mycological Research, 106, 745-752.
Peres, N.A., Timmer L.W., Adaskaveg, J.E. and Corell, J.C., 2005. Lifestyle of Colletotrichum acutatum. Plant Disease, 89, 784-796.
Crous, P.W., Wingfield, M.J., Burgess, T.I., Hardy, G., Gené, J., Guarro, J., Baseia, I.G., Garcia, D., Gusmão, L., Souza-Motta, C.M., Thangavel, R., Adamčík, S., Barili, A., Barnes, C.W., Bezerra, J., Bordallo, J.J., Cano-Lira, J.F., de Oliveira, R., Ercole, E., Hubka, V., … Groenewald, J.Z. 2018. Fungal planet description sheets: 716-784. Persoonia, 40, 240-393.
Lima, N.B., Batista, M.V.D.A., De Morais, Jr., M.A., Barbosa, M.A.G., Michereff, S.J., Hyde, K.D. and Câmara, M.P.S., 2013. Five Colletotrichum species are responsible for mango anthracnose in northeastern Brazil. Fungal Diversity, 61, 75-88.
Gangadevi, V. and Muthumary, J., 2008. Isolation of Colletotrichum gloeosporioides: a novel endophytic taxol-producing fungus from the leaves of a medicinal plant, Justicia gendarussa. Mycologia Balcanica, 5, 1-4.
Olson, T.R., Gabreil, A., Micijevic, A., Bradley, C.A., Wise, K.A., Mueller, D.S., Chilvers, M.I. and Mathew, F.M., 2015. Association of Diaporthe longicolla with black zone lines on mature soybean plants. Plant Health Progress, 16, 118-122.
Preeti, V., Ramesha, B.T., Singh, S., Ravikanth, G., Ganeshaiah, K.N., Suryanarayanan, T.S. and Shaanker, R.U., 2009. How promising are endophytic fungi as alternative sources of plant secondary metabolites? Current Science, 104, 178-182.
Serrato-Diaz, L.M., Rivera-Vargas, L.I. and French-Monar, R.D., 2014. First report of Diaporthe pseudomangiferae causing inflorescence rot, rachis canker and flower abortion of mango. Plant Disease, 98, 1004-1005.
Haryoto, H., Muhtadi, M., Indrayudha, P., Azizah, T. and Suhendi, A., 2013. Activity of sitotoksik ethanol extract plant growth (Cynometra ramiflora Linn) on heavy cell, T47D and WiDR. Journal Penelitian Saintek, 18, 21-28.
Freeman, S., Shabi, E. and Katan, T., 2000. Characterization of Colletotrichum acutatum causing anthracnose of anemone (Anemone coronaria L.). Applied and Environmental Microbiology, 66, 5267-5272.
Quirós, M.B., Peres, N.A. and Arauz, L.F., 2013. Presence of Colletotrichum acutatum and Colletotrichum gloeosporioides on leatherleaf fern, key lime, papaya, star fruit, and mango in Costa Rica and Florida (United States). Agronomía Costarricense, 37, 23-38.
Liu, H.X., Tan, H.B., Chen, Y.C., Li, S.N., Li, H.H. and Zhang, W.M., 2018. Secondary metabolites from the Colletotrichum gloeosporioides A12, an endophytic fungus derived from Aquilaria sinensis. Natural Product Research, 32, 2360-2365.
Zang, L.Y., Wei, W., Wang, T., Guo, Y., Tan, R.X. and Hui-Ming, G.E., 2012. Isochromophilones from an endophytic fungus Diaporthe sp. Natural Products and Bioprospecting, 2, 117-120.
Hemtasin, C., Kanokmedhakul, S., Kanokmedhakul, K., Hahnvajanawong, C., Soytong, K., Prabpai, S. and Kongsaeree, P., 2011. Cytotoxic pentacyclic and tetracyclic aromatic sesquiterpenes from Phomopsis archeri. Journal of Natural Products, 74, 609-13.