Antibacterial potential of endophytic fungi of the Amazon medicinal plant mulateiro Calycophyllum spruceanum (Benth.) Hook. f. ex K. Schum.
Abstract
Calycophyllum spruceanum, known as mulateiro, is an Amazonian medicinal plant traditionally used to treat skin infections, scars, and gastrointestinal and uterine conditions. Some of its therapeutic properties are attributed to bioactive compounds produced in association with endophytic fungi. This study aimed to evaluate the antibacterial potential of endophytic fungi isolated from C. spruceanum. Leaves and stems underwent surface sterilization and were cultured on four types of media: PDA, PDA supplemented with plant extract, SDA, and SDA supplemented with plant extract, incubated at 18 °C and 28 °C. The isolated fungi were classified into morphospecies using classical taxonomy. Crude extracts of fungal metabolites were tested for antibacterial activity against Gram-positive and Gram-negative bacteria through the disk diffusion assay. In total, 650 fungal isolates were obtained, 486 filamentous fungi and 164 yeasts, grouped into 248 morphospecies. The most frequently isolated genus was Phomopsis (30.9%), followed by Colletotrichum (16.7%) and Guignardia (5.1%). A higher number of fungi were recovered from leaves (60.3%) compared to stems (39.7%). Among the isolates, 23 demonstrated antibacterial activity against Escherichia coli, and one isolate exhibited inhibitory activity against Klebsiella pneumoniae, both Gram-negative bacteria of clinical relevance. This is the first report describing the endophytic fungal community associated with C. spruceanum, highlighting its potential as a source of novel antibacterial compounds, particularly effective against Gram-negative pathogens. These findings contribute to the bioprospecting of Amazonian biodiversity and open perspectives for the development of natural products with pharmaceutical applications.
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Adewale, A. M., Kheng, G. J., Ping, L. S., & Yien, T. A. S. (2015). Antimicrobial and enzymatic activities of endophytic bacteria isolated from Mentha spicata (MINT). Malaysian Journal of Microbiology, 11, 102-108. https://doi.org/10.21161/mjm.12014
Ahmed, S. A., Saleh, S. A., Mostafa, F. A., Abd El Aty, A. A., & Ammar, H. A. (2016). Characterization and valuable applications of xylanase from endophytic fungus Aspergillus terreus KP900973 isolated from Corchorus olitorius. Biocatalysis and Agricultural Biotechnology, 7, 134-144. https://doi.org/10.1016/j.bcab.2016.05.015
Arantes, T. D., Theodoro, R. C., Teixeira, M. D. M., Bosco, S. D. M. G., & Bagagli, E. (2016). Environmental mapping of Paracoccidioides spp. in Brazil reveals new clues into genetic diversity, biogeography and wild host association. PLoS Neglected Tropical Diseases, 10(5), 1-18. https://doi.org/10.1371/journal.pntd.0004606
Araújo, B. H. P., de Sousa, M. A. R., Nascimento, H. E. M., Zanuncio, A. J. V., Rodrigues, D. D. S., & Guedes, M. C. (2016). Physical properties of Calycophyllum spruceanum Benth. wood according on the diameter and position (base and top) in stem. Forest Sciences, 44(111), 759-768. https://doi.org/10.18671/scifor.v44n111.22
Baldin, T., Siegloch, A. M., & Marchiori, J. N. C. (2016). Compared anatomy of species of Calycophyllum DC. (Rubiaceae). Revista Árvore, 40(4), 759-768. https://doi.org/10.1590/0100-67622016000400020
Banhos, E. F. D., Souza, A. Q. L. D., Andrade, J. C. D., Souza, A. D. L. D., Koolen, H. H. F., & Albuquerque, P. M. (2014). Endophytic fungi from Myrcia guianensis at the Brazilian Amazon: Distribution and bioactivity. Brazilian Journal of Microbiology, 45(1), 153-161. https://doi.org/10.1590/S1517-83822014005000027
Barnett, H. L., & Hunter, B. B. (1999). Illustrated genera of imperfect fungi. APS.
Bezerra, J. D., Santos, M. G., Barbosa, R. N., Svedese, V. M., Lima, D. M., Fernandes, M. J. S., & Souza-Motta, C. M. (2013). Fungal endophytes from cactus Cereus jamacaru in Brazilian tropical dry forest: A first study. Symbiosis, 60, 53-63. https://doi.org/10.1007/s13199-013-0243-1
Buell, C. B., & Weston, W. H. (1947). Application of the mineral oil conservation method to maintaining collections of fungous cultures. American Journal of Botany, 34(9), 555-561. https://doi.org/10.2307/2437337
Cai, R., Chen, S., Liu, Z., Tan, C., Huang, X., & She, Z. (2017). A new alpha-pyrone from the mangrove endophytic fungus Phomopsis sp. HNY29-2B. Natural Product Research, 31(2), 124-130. https://doi.org/10.1080/14786419.2016.1214833
Castellani, A. (1963). The "water cultivation" of pathogenic fungi. American Journal of Tropical Medicine, 66(3), 283-284.
CLSI - Clinical and Laboratory Standards Institute. (2003). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard. http://www.sbac.org.br/pt/pdfs/biblioteca/clsi_OPASM7_A6.pdf
Costa, L. M. D., Santos, V. A. D., Ohana, D. T., Lima, E. S., Pereira, M. D. M., & Souza, T. P. D. (2011). Technological development of aqueous extracts from Calycophyllum spruceanum (Benth.) Hook. f. ex K. Schum., Rubiaceae, (mulateiro) using factorial design. Revista Brasileira de Farmacognosia, 21(1), 181-186. https://doi.org/10.1590/S0102-695X2011005000027
Ferreira, M. C., Vieira, M. D. L. A., Zani, C. L., de Almeida Alves, T. M., Junior, P. A. S., Murta, S. M., & Rosa, L. H. (2015). Molecular phylogeny, diversity, symbiosis and discover of bioactive compounds of endophytic fungi associated with the medicinal Amazonian plant Carapa guianensis Aublet (Meliaceae). Biochemical Systematics and Ecology, 59, 36-44. https://doi.org/10.1016/j.bse.2014.12.017
Figueiredo-Filho, D. A., FSEDV, F., Rodriguez, A. F. R., Vale, P. A. A., Do Egito, E. S. T., & Marcal, H. (2016). Mulateiro (Calycophyllum spruceanum) Stem Cell Extract: An evaluation of its anti-aging effect on human adult fibroblasts. International Journal of Scientific & Engineering Research, 7(1), 87-90.
Gouda, S., Das, G., Sen, S. K., Shin, H. S., & Patra, J. K. (2016). Endophytes: A treasure house of bioactive compounds of medicinal importance. Frontiers in Microbiology, 7, 1538. https://doi.org/10.3389/fmicb.2016.01538
Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95-98.
Hardoim, P. R., Van Overbeek, L. S., Berg, G., Pirttilä, A. M., Compant, S., Campisano, A., & Sessitsch, A. (2015). The hidden world within plants: Ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiology and Molecular Biology Reviews, 79(3), 293-320. https://doi.org/10.1128/MMBR.00050-14
Jin, Z., Li, D., Liu, T., Yu, F., Zhang, Z., Su, C., & Liu, Z. (2017). Cultural endophytic fungi associated with Dendrobium officinale: Identification, diversity estimation and their antimicrobial potential. Current Science, 112(8), 1690-1697. https://doi.org/10.18520/cs/v112/i08/1690-1697
Jouda, J. B., Tamokou, J. D. D., Mbazoa, C. D., Douala-Meli, C., Sarkar, P., Bag, P. K., & Wandji, J. (2016). Antibacterial and cytotoxic cytochalasins from the endophytic fungus Phomopsis sp. harbored in Garcinia kola (Heckel) nut. BMC Complementary and Alternative Medicine, 16(1), 462. https://doi.org/10.1186/s12906-016-1454-9
Jukes, T. H., & Cantor, C. R. (1969). Evolution of protein molecules. In H. R. Munro (Org.), Mammalian protein metabolism, 33, 21-132.
Kasaei, A., Mobini-Dehkordi, M., Mahjoubi, F., & Saffar, B. (2017). Isolation of taxol-producing endophytic fungi from Iranian yew through novel molecular approach and their effects on human breast cancer cell line. Current Microbiology, 74(6), 702-709. https://doi.org/10.1007/s00284-017-1231-0
Kathiravan, G., & Raman, V. S. (2010). In vitro taxol production by Pestalotiopsis breviseta - A first report. Fitoterapia, 81(6), 557-564. https://doi.org/10.1016/j.fitote.2010.01.021
Krishnapura, P. R., & Belur, P. D. (2016). Partial purification and characterization of L-asparaginase from an endophytic Talaromyces pinophilus isolated from the rhizomes of Curcuma amada. Journal of Molecular Catalysis B: Enzymatic, 124, 83-91. https://doi.org/10.1016/j.molcatb.2015.12.007
Lima, A. M. D., Salem, J. I., Souza, J. V. B. D., Cortez, A. C. A., Carvalho, C. M., Chaves, F. C. M., & Veiga Junior, V. F. D. (2011). Effects of culture filtrates of endophytic fungi obtained from Piper aduncum L. on the growth of Mycobacterium tuberculosis. Electronic Journal of Biotechnology, 14(4), 1-6. https://doi.org/10.2225/vol14-issue4-fulltext-11
Macias-Rubalcava, M., & Sanchez-Fernandez, R. E. (2017). Secondary metabolites of endophytic Xylaria species with potential applications in medicine and agriculture. World Journal of Microbiology and Biotechnology, 33(1), 1-22. https://doi.org/10.1007/s11274-016-2174-5
Mahmoud, F. M., Krimi, Z., Maciá-Vicente, J. G., Errahmani, M. B., & Lopez-Llorca, L. V. (2017). Endophytic fungi associated with roots of date palm (Phoenix dactylifera) in coastal dunes. Revista Iberoamericana de Micología, 34(2), 116-120. https://doi.org/10.1016/j.riam.2016.06.007
Malhadas, C., Malheiro, R., Pereira, J. A., de Pinho, P. G., & Baptista, P. (2017). Antimicrobial activity of endophytic fungi from olive tree leaves. World Journal of Microbiology and Biotechnology, 33(3), 1-12. https://doi.org/10.1007/s11274-017-2216-7
Monteiro, M. C. P., Alves, N. M., Queiroz, M. V. D., Pinho, D. B., Pereira, O. L., Souza, S. M. C. D., & Cardoso, P. G. (2017). Antimicrobial activity of endophytic fungi from coffee plants. Bioscience Journal, 33(2), 381-389. https://doi.org/10.14393/BJ-v33n2-34494
Nascimento, T. L., Oki, Y., Lima, D. M. M., Almeida-Cortez, J. S., Fernandes, G. W., & Souza-Motta, C. M. (2015). Biodiversity of endophytic fungi in different leaf ages of Calotropis procera and their antimicrobial activity. Fungal Ecology, 14, 79-86. https://doi.org/10.1016/j.funeco.2014.10.004
Pandi, M., Manikandan, R., & Muthumary, J. (2010). Anticancer activity of fungal taxol derived from Botryodiplodia theobromae Pat., an endophytic fungus, against 7, 12 dimethyl benz(a)anthracene (DMBA)-induced mammary gland carcinogenesis in Sprague dawley rats. Biomedicine & Pharmacotherapy, 64(1), 48-53. https://doi.org/10.1016/j.biopha.2009.03.001
Passarini, M. R., Santos, C., Lima, N., Berlinck, R. G. S., & Sette, L. D. (2013). Filamentous fungi from the Atlantic marine sponge Dragmacidon reticulatum. Archives of Microbiology, 195(2), 99-111. https://doi.org/10.1007/s00203-012-0854-6
Polonio, J. C., Almeida, T. T., Garcia, A., Mariucci, G. E. G., Azevedo, J. L., Rhoden, S. A., & Pamphile, J. A. (2015). Biotechnological prospecting of foliar endophytic fungi of guaco (Mikania glomerata Spreng.) with antibacterial and antagonistic activity against phytopathogens. Genetics and Molecular Research, 14(3), 7297-7309. https://doi.org/10.4238/2015.July.3.5
Pylro, V. S., Roesch, L. F. W., Ortega, J. M., do Amaral, A. M., Tótola, M. R., & Hirsch, P. R. (2014). Brazilian Microbiome Project: Revealing the unexplored microbial diversity-challenges and prospects. Microbial Ecology, 67(1), 237-241. https://doi.org/10.1007/s00248-013-0302-4
Rao, H. C. Y., & Satish, S. (2015). Genomic and chromatographic approach for the discovery of polyketide antimicrobial metabolites from an endophytic Phomopsis liquidambaris CBR-18. Frontiers in Life Science, 8(3), 200-207. https://doi.org/10.1080/21553769.2015.1033768
Record, S. J., & Hess, R. W. (1943). Timbers of the new world. Yale University Press.
Revilla, J. (2001). Plantas da Amazônia: Oportunidades econômicas e sustentáveis. Sebrae/INPA.
Rizzini, C. T. (1978). Árvores e madeiras úteis no Brasil: Manual de dendrologia brasileira (2a ed.). Editora Blücher.
Santos, A., Ribeiro-Oliveira, J., & Carvalho, C. (2016). Sobre a botânica, a etnofarmacologia e a química de Calycophyllum spruceanum (Benth.) Hook. f. ex K. Schum. Revista Brasileira de Plantas Medicinais, 18(1), 383-389. https://doi.org/10.1590/1983-084X/15_152
Sharma, V. K., Kumar, J., Singh, D. K., Mishra, A., Verma, S. K., Gond, S. K., & Kharwar, R. N. (2017). Induction of cryptic and bioactive metabolites through natural dietary components in an endophytic fungus Colletotrichum gloeosporioides (Penz.) Sacc. Frontiers in Microbiology, 8, 1-9. https://doi.org/10.3389/fmicb.2017.01126
Siebers-Wolff, S., Arfmann, H. A., Abraham, W. R., & Kieslich, K. (1993). Microbiological hydroxylation and n-oxidation of cinchona alkaloids. Biocatalysis, 8(1), 47-58. https://doi.org/10.3109/10242429309030955
Silva, G. H., Teles, H. L., Zanardi, L. M., Young, M. C. M., Eberlin, M. N., Hadad, R., & Araújo, Â. R. (2006). Cadinane sesquiterpenoids of Phomopsis cassiae, an endophytic fungus associated with Cassia spectabilis (Leguminosae). Phytochemistry, 67(18), 1964-1969. https://doi.org/10.1016/j.phytochem.2006.06.004
Somjaipeng, S., Medina, A., & Magan, N. (2016). Environmental stress and elicitors enhance taxol production by endophytic strains of Paraconiothyrium variabile and Epicoccum nigrum. Enzyme and Microbial Technology, 90, 69-75. https://doi.org/10.1016/j.enzmictec.2016.05.002
Szilagyi-Zecchin, V. J., Adamoski, D., Gomes, R. R., Hungria, M., Ikeda, A. C., Kava-Cordeiro, V., & Galli-Terasawa, L. V. (2016). Composition of endophytic fungal community associated with leaves of Maize cultivated in South Brazilian field. Acta Microbiologica et Immunologica Hungarica, 63(4), 449-466. https://doi.org/10.1556/030.63.2016.020
Toghueo, R. M. K., Zabalgogeazcoa, I., de Aldana, B. V., & Boyom, F. F. (2017). Enzymatic activity of endophytic fungi from the medicinal plants Terminalia catappa, Terminalia mantaly and Cananga odorata. South African Journal of Botany, 109, 146-153. https://doi.org/10.1016/j.sajb.2016.12.021
Ugarte-Guerra, L. J., & Domínguez-Torrejón, G. (2010). Índice de Sitio (IS) de Calycophyllum spruceanum Benth. en relación con la altura dominante del rodal en ensayos de plantación en la Cuenca del Aguaytía, Ucayali, Perú. Ecología Aplicada, 9(2), 101-111.
Vargas, F. S., Almeida, P., de Boleti, A. P. A., Pereira, M. M., de Souza, T. P., de Vasconcellos, M. C., & Lima, E. S. (2016). Antioxidant activity and peroxidase inhibition of Amazonian plants extracts traditionally used as anti-inflammatory. BMC Complementary and Alternative Medicine, 16(1), 1-8. https://doi.org/10.1186/s12906-016-1061-9
Vieira, M. L., Johann, S., Hughes, F. M., Rosa, C. A., & Rosa, L. H. (2014). The diversity and antimicrobial activity of endophytic fungi associated with medicinal plant Baccharis trimera (Asteraceae) from the Brazilian savannah. Canadian Journal of Microbiology, 60(12), 847-856. https://doi.org/10.1139/cjm-2014-0449
Wang, Y., & Guo, L. D. (2007). Comparative study of endophytic fungi in needles, bark, and xylem of Pinus tabulaeformis. Canadian Journal of Botany, 85(9), 911-917. https://doi.org/10.1139/B07-084
White, T. J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White (Eds.), PCR protocols: A guide to methods and applications (pp. 315-322). Academic Press.
Zuleta, L. M. C., Cavalheiro, A. J., Silva, D. H. S., Furlan, M., Young, M. C. M, Albuquerque, S., & da Silva Bolzani, V. (2003). Seco-iridoids from Calycophyllum spruceanum (Rubiaceae). Phytochemistry, 64(2), 549-553. https://doi.org/10.1016/S0031-9422(03)00153-5
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