DIVERSITY AND ANTIMALARIAL ACTIVITY OF ENDOPHYTIC FUNGI ASSOCIATED WITH Myrciaria dubia FROM THE CANTÃO STATE PARK, TOCANTINS - BRAZIL
Palabras clave:
Camu – camu., Fungal microbiota, Plasmodium falciparum, HepG2Resumen
O camu-camu (Myrciaria dubia) é uma planta Amazônica de grande potencial econômico, devido à alta concentração de vitamina C em seus frutos, sendo a polpa utilizada na indústria farmacológica, de cosméticos e de alimentos. Os fungos endofíticos são micro-organismos que colonizam os tecidos internos das plantas e, associados aos vegetais, representam uma fonte inexplorada de novos produtos naturais bioativos. Neste contexto, este trabalho teve como objetivo identificar a diversidade de fungos endofíticos isolados de amostras de folhas e caules de M. dubia e analisar o potencial antimalárico dos extratos fúngicos contra o protozoário Plasmodium falciparum em ensaio imunoenzimático anti-HRPII (proteína rica em histidina II). Os ensaios de citotoxicidade dos extratos foram realizados contra linhagem celular de hepatoma humano (HepG2), sendo determinado pelo método colorimétrico MTT (3-(4,5-dimetiltiazol-2yl)-2,5-difenil brometo de tetrazolina). Um total de 308 isolados fúngicos foram obtidos e identificados como pertencentes a 22 táxons por meio de métodos moleculares. Os extratos dos fungos endofíticos Diaporthe sp. 2 (IC50= 2,05), Diaporthe miriciae (IC50= 2,31), Diaporthe sp. 4 (IC50= 9,28), e da família Xylariaceae (IC50= 39,00), foram ativos contra o P. falciparum. A identificação da microbiota fúngica associada a vegetais contribui com o conhecimento taxonômico, colaborando para a bioprospecção dos endofíticos que podem ser utilizados principalmente na agricultura e indústria farmacêutica.
Citas
2. Tan RX, Zou WX. Endophytes: a rich source of functional metabolites. Nat Prod Rep. 2001; 18(4): 448-59.
3. Campos FF, Rosa LH, Cota BB, Caligiorne RB, Rabello AL, Alves TM, et al. Leishmanicidal metabolites from Cochliobolus sp., an endophytic fungus isolated from Piptadenia adiantoides (Fabaceae). PLoS Negl Trop Dis. 2008; 2(12): e348.
4. Hidayat H, Karsten K, Siegfried D, Kathrin M, Barbara S. Bioactive chemical constituents of a sterile endophytic fungus from Meliotus dentatus. Rec Nat Prod. 2009; 3(2): 114-7.
5. Gunatilaka AAL. Natural products from plant associated microorganisms: distribution, structural diversity, bioactivity, and implications of their occurrence. J Nat Prod. 2012; 69(3): 509-26.
6. Peixoto-Neto PAS, Azevedo JL, Araújo WL. Microrganismos endofíticos. Biotecnologia C&D. 2002; 29: 62-76.
7. Hyde KD, Soytong K. The fungal endophyte dilemma. Fungal Divers. 2008; 33: 163-73.
8. Arnold AE. Understanding the diversity of foliar endophytic fungi: progress, challenges, and frontiers. Fungal Biol Rev. 2007; 21(2-3): 51-66.
9. Rakotoniriana EF, Munaut F, Decock C, Randriamampionona D, Andriambololoniaina M, Rakotomalala T, et al. Endophytic fungi from leaves of Centella asiatica: occurrence and potential interactions within leaves. Antonie Van Leeuwenhoek. 2008; 93(1-2): 27-36.
10. Stone JK, Polishook JD, White Jr. JF. Endophytic Fungus. In: Mueller JM, Bills GF, Foster MS. Biodiversity of fungi: inventory and monitoring methods. Boston: Elsevier Academic Press; 2004. p. 241-70.
11. Arnold AE, Maynard Z, Gilbert GS, Coley PD, Kursar TA. Are tropical fungal endophytes hyperdiverse? Ecol Lett. 2007; 3(4): 267-74.
12. Akello J, Dubois T, Gold SC, Nakavuma J, Paparu P. Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.). J Invertebr Pathol. 2007; 96(1): 34-42.
13. Oses R, Valenzuela S, Freer J, Sanfuentes E, Rodriguez J. Fungal endophytes in xylem of healthy chilean trees and their possible role in early wood decay. Fungal Divers. 2008; 33: 77- 86.
14. Bae H, Sicher RC, Kim MS, Kim SH, Strem, MD, Melnick RL. The beneficial endophyte Trichoderma hamatum isolate DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao. J Exp Bot. 2009; 60(11): 3279-95.
15. Bayat F, Mirlohi A, Khodambashi M. Effects of endophytic fungi on some drought tolerance mechanisms of tall fescue in a hydroponics culture. Russ J Plant Physiol. 2009; 56(4): 510-6.
16. Soliman SSM, Trobacher CP, Tsao R, Greenwood JS, Raizada MN. A fungal endophyte induces transcription of genes encoding a redundant fungicide pathway in its host plant. BMC Plant Biol. 2013; 13(93): 1-10.
17. Weber RWS, Kappe R, Paululat T, Mosker E, Anke H. Anti-candida metabolites from endophytic fungi. Phytochemistry. 2007; 68(6): 886-92.
18. Justi KC, Visentainer JV, Souza NE, Matsushita M. Nutritional composition and vitamin C stability in stored camu-camu (Myrciaria dubia) pulp. Arch Latinoam Nutr. 2000; 50(4): 405-8.
19. Taylor L. Herbal Secrets of the Rainforest. 1st ed. Califórnia: Prima Lifestyles; 1998. 360p.
20. Yuyama K, Aguiar JPL, Yuyama LKO. Camu-camu: um fruto fantástico como fonte de vitamina C. Acta Amazon. 2002; 32(1): 169-74.
21. Azevedo-Meleiro CH, Rodriguez-Amaya DB. Confirmation of the Identity of the carotenoids of tropical fruits by HPLC-DAD and HPLC-MS. J Food Compost Anal. 2004; 17(3-4): 385-96.
22. Fisk OS, Middaugh AL, Rhee YS, Brunt AR. Few favorable associations between fruit and vegetable intake and biomarkers for chronic disease risk in American adults. Nutr Res. 2011; 31(8): 616-24.
23. Collado J, Platas G, Peláez F. Fungal endophytes in leaves, twigs and bark of Quercus ilex from Central Spain. Nova Hedwigia. 1996; 63(3): 347-60.
24. Araújo WL, Lima ADS, Azevedo JL, Marcon J, Sobral JK, Lacava PT. Manual: isolamento de microrganismos endofíticos. 1a ed. Piracicaba: CALQ; 2002. 86 p.
25. Hammer O, Harper DAT, Ryan PD. PAST: Paleontological Statistics software package for education and data analysis. Palaeontol Electronica. 2001; 4: 4-9.
26. Colwell RK. EstimateS: Statistical estimation of species richness and shared species from samples. Version 9; 2013. [updated 2013 June 14; cited 2016 May 29]. Available from: http://viceroy.eeb.uconn.edu/EstimateS.
27. Rosa LH, Vaz ABM, Caligiorne RB, Campolina S, Rosa CA. Endophytic fungi associated with the Antarctic Grass Deschampsia antarctica Desv. (Poaceae). Polar Biol. 2009; 32(2): 161-7.
28. White TJ, Bruns TD, Lee SB, Taylor JW. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR protocols: a guide to methods and applications. New York: Academic Press; 1990. p. 315-22.
29. Glass NL, Donaldson GC. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol. 1995; 61(4): 1323-30.
30. Godinho VM, Furbino L, Santiago IF, Pellizzari FM, Yokoya NS, Pupo D, et al. Diversity and bioprospecting of fungal communities associated with endemic and cold-adapted macroalgae in Antarctica. ISME J. 2013; 7(7): 1434-51.
31. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997; 25(17): 3389-402.
32. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28(10): 2731-9.
33. Kirk P, Cannon PF, Minter DW, Stalpers JA. Ainsworth and Bisby's Dictionary of the Fungi. 10th ed. Wallingford: CAB International; 2008. 770p.
34. Rosa LH, Queiroz SC, Moraes RM, Wang X, Techen N, Pan Z, et al. Coniochaeta ligniaria: antifungal activity of the cryptic endophytic fungus associated with autotrophic tissue cultures of the medicinal plant Smallanthus sonchifolius (Asteraceae). Symbiosis. 2013; 60(3): 133-42.
35. Trager W, Jensen EJ. Human malaria parasites in contínuous culture. Science. 1976; 193(4254): 673-5.
36. Lambros C, Vanderberg J. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979; 65(3): 418-20.
37. Penna-Coutinho J, Cortopassi WA, Oliveira AA, França TCC, Krettli AU Antimalarial activity of potential inhibitors of Plasmodium falciparum lactate dehydrogenase enzyme selected by docking studies. PloS One. 2011; 6(7): e21237.
38. Noedl H, Wongsrichanalai C, Miller RS, Myint KSA, Looareesuwan S, Sukthana Y, et al. Plasmodium falciparum: effect of anti-malarial drugs on the production and secretion characteristics of histidine-rich protein II. Exp Parasitol. 2002; 102 (3-4): 157-63.
39. Reynertson AK, Basile MJ, Kennelly EJ. Antioxidant potential of seven Myrtaceous fruits. Ethnobot Res App. 2005; 3: 25-35.
40. Calvo-Calle J, Moreno A, Eling W, Nardin E. In vitro development of infectious liver stages of P. yoelii and P. berghei malaria in human cell lines. Exp Parasitol. 1994; 79(3): 362-73.
41. Madureira M, Martins AP, Gomes M, Paiva J, Proença da Cunha A, Rosário V. Antimalarial activity of medicinal plants used in traditional medicine in S. Tomé and Príncipe Islands. J Ethnopharmacol. 2002; 8(1): 23-9.
42. Bézivin C, Tomasi S, Deveat FL, Boustie J. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines. Phytomedicine. 2003; 10(6-7): 499-503.
43. Santamaría J, Bayman P. Fungal epiphytes and endophytes of coffee leaves (Coffea arabica). Microb Ecol. 2005; 50(1): 1-8.
44. Ravijara NS, Maria GL, Sridhar KR. Antimicrobial evaluation of endophytic fungi inhabiting medicinal plants of the Western ghats of India. Eng Life Sci. 2006; 6(5): 515-20.
45. Gazis R, Chaverri P. Diversity of fungal endophytes in leaves and stems of wild rubber trees (Hevea brasiliensis) in Peru. Fungal Ecol. 2010; 3(3): 240-54.
46. González V, Tello ML. The endophytic mycota associated with Vitis vinifera in central Spain. Fungal Divers. 2011; 47(1): 29-42.
47. Higginbotham SJ, Arnold AE, Ibañez A, Spadafora C, Coley PD, Kursar TA. Bioactivity of fungal endophytes as a function of endophyte taxonomy and the taxonomy and distribution of their host plants. PLoS One. 2013; 8: e73192.
48. Brand A, Gow NA. Mechanisms of hipha orientation of fungi. Curr Opin Microbiol. 2009; 12(4): 350-7.
49. Rubini MR, Silva-Ribeiro RT, Pomella AWV, Maki CS, Araújo WL, Santos DR, et al. Diversity of endophytic fungal community of cacao (Theobroma cacao L.) and biological control of Crinipellis perniciosa, causal agent of Witches’ Broom Disease. Int J Biol Sci. 2005; 1(1): 24-33.
50. Magalhães WCS, Missagia RV, Costa FAF, Costa MCM. Diversidade de fungos endofíticos em candeia Eremanthus erythropappus (DC.) MacLeish. Cerne. 2008; 14(3): 267-73.
51. Siqueira VM, Conti R, Araújo JM, Souza-Motta CM. Endophytic fungi from the medicinal plant Lippia sidoides Cham. and their antimicrobial activity. Symbiosis. 2011; 53(2): 89-95.
52. Rodrigues KF. The foliar fungal endophytes of the Amazonian palm Euterpe oleracea. Mycologia. 1994; 86(3): 376-85.
53. Arnold AE, Mejia LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, et al. Fungal endophytes limit pathogen damage in a tropical tree. PNAS. 2003; 100(26): 15649-54.
54. Pinto WS, Perim MC, Borges JC, Pimenta RS, Rosa LH, Silva JFM, et al. Diversity and antimicrobial activities of endophytic fungi isolated from Myrcia sellowiana in Tocantins, Brazil. Acta Hortic. 2011; 905(905): 283-6.
55. Banhos EFD, Souza AQLD, Andrade JCD, Souza ADLD, Koolen HHF, Albuquerque PM. Endophytic fungi from Myrcia guianensis at the Brazilian Amazon: distribution and bioactivity. Braz J Microbiol. 2014; 45(1): 153-62.
56. Taylor JE, Hyde KD, Jones EBG. Endophytic fungi associated with the temperate palm, Trachycarpus fortunei, within and outside its natural geographic range. New Phytol. 1999; 142(2): 335-46.
57. Murali TS, Suryanarayanan TS, Venkatesan G. Fungal endophytes communities in two tropical forest of southern India: diversity and host affiliation. Mycol. Prog. 2007; 6(3): 191-99.
58. Kumaresan V, Suryanarayanan TS. Occurrence and distribuition of endophytic fungi in a mangrove community. Mycol Res. 2001; 105(11): 1388-91.
59. Vieira PDS, Silva FG, Silva WMT, Cavalcanti PA, Lima D. Primeiro registro de fungos endofíticos em folhas de Ixora coccinea L. em Pernambuco, Brasil. Rev Bras Biocienc. 2012; 10(1): 1-4.
60. Carvalho CR, Gonçalves VN, Pereira CB, Johann S, Galliza IV, Alves TMA, et al. The diversity, antimicrobial and anticancer activity of endophytic fungi associated with the medicinal plant Stryphnodendron adstringens (Mart.) Coville (Fabaceae) from the Brazilian savannah. Symbiosis. 2012; 57(2): 95-107.
61. Pandey AK, Reddy MS, Suryanarayanan TS. ITS-RFLP and ITS sequence analysis of a foliar endophytic Phyllosticta from different tropical trees. Mycol Res. 2003; 107(4): 439-44.
62. Murali TS, Suryanarayanan TS, Geeta R. Endophytic Phomopsis species: host range and implications for diversity estimates. Can J Microbiol. 2006; 52(7): 673-80.
63. Sieber TN. Endophytic fungi in forest trees: are they mutualists? Fungal Biol Rev. 2007; 21(2-3): 75-89.
64. Rosa LH, Gonçalves VN, Caligiorne RB, Alves TMA, Rabello A, Sales PA. Leishmanicidal, trypanocidal, and cytotoxic activities of endophytic fungi associated with bioactive plants in Brazil. Braz. J. Microbiol. 2010; 41(2): 114-22.
65. Suryanarayanan TS, Thirunavukkarasu N, Govindarajulu MB, Sasse F, Jansen R, Murali TS. Fungal endophytes and bioprospecting. Fungal Biol Rev. 2009; 23(1-2): 9-19.
66. Bernardi-Wenzel J, Garcia A, Filho CJ, Prioli, AJ, Pamphile JA. Evaluation of foliar fungal endophyte diversity and colonization of medicinal plant Luehea divaricata (Martius et Zuccarini). Biol Res. 2010; 43(4): 375-84.
67. Corrado M, Rodigues K. Evaluation of fungal extracts produced by endophytic strains of Phomopsis sp. J Basic Microbiol. 2004; 44(2): 157-60.
68. Correia VCS, Lima NO, Oliveira FAS, Santos APA, Teles, CBG, Oliveira Júnior WP, et al. Evaluation of the antiplasmodial and leishmanicidal potential of the Myrciaria dubia (Myrtaceae) extract. Rev Soc Bras Med Trop. 2016; 49(5): 586-92.
69. Gonçalves FJT, Freire FCO, Lima JS. Fungos endofíticos e seu potencial como produtores de compostos bioativos. Essentia. 2013; 15(1): 71-92.
70. Radiastuti N, Rahayu G, Okane I, Hidayat I, Achmadi SS. Alkaloid profile of endophytic Diaporthe spp. from Cinchona calisaya. Jurnal Penelitian Teh dan Kina. 2015; 18(1): 81-93.
71. Ash GJ, Stodart B, Sakuanrungsirikul S, Anschaw E, Crump N, Hailstones D, et al. Genetic characterization of a novel Phomopsis sp., a putative biocontrol agent for Carthamus lanatus. Mycologia. 2010; 102(1): 54-61.
72. Lin X, Huang Y, Fang M, Wang J, Zheng Z, Su W. Cytotoxic and antimicrobial metabolites from marine lignicolous fungi, Diaporthe sp. FEMS Microbiol Lett. 2005; 251(1): 53-8.
73. Sebastianes FL, Cabedo N, El Aouad N, Valente AMMP, Lacava PT, Azevedo JL, et al. 3-Hydroxypropionic acid as an antibacterial agent from endophytic fungi Diaporthe phaseolorum. Curr Microbiol. 2012; 65(5): 622-32.
74. Isaka M, Jaturapat A, Rukseree K, Danwisetkanjana K, Tanticharoen M, Thebtaranonth Y. Phomoxanthones A and B, novel xanthone dimers from the endophytic fungus Phomopsis species. J Nat Prod. 2001; 64(8): 1015-8.
75. Dai J, Krohn K, Floerke U, Gehle D, Aust HJ, Draeger S, et al. Novel highly substituted biraryl ethers, phomopsines D-G, isolated from endophytic fungus Phomopsis sp. from Adenocarpus foliolosus. European J Org Chem. 2005; 2005(23): 5100-5.
76. Elsaesser B, Krohn K, Floerke U, Root N, Aust HJ, Draeger S. X ray structure determination absolute configuration and biological activity of phomoxanthone. European J Org Chem. 2005; 2005(21): 4563-70.
77. Kumaran RS, Hur B. Screening of species of the endophytic fungus Phomopsis for the production of the anticancer drug taxol. Biotechnol Appl Biochem. 2009; 54(1): 21-30.
78. Silva GH, Teles HL, Trevisan HC, Bolzani VS, Young MCM., Pfenning LH, et al. New bioactive metabolites produced by Phomopsis cassiae, an endophytic fungus in Cassia spectabilis. J Braz Chem Soc. 2005; 16(6b): 1463-6.
79. Wu SH, Chen YW, Shao SC, Wang L, Li Z, Yang L, et al. Ten-membered lactones from Phomopsis sp., an endophytic fungus of Azadirachta indica. J Nat Prod. 2008; 71(4): 731-4.
80. Zhang C, Ondeyka JG, Herath KB, Guan Z, Collado J, Platas G, Pelaez F, et al. Tenellones A and B from a Diaporthe sp.: two highly substituted benzophenone inhibitors of parasite cGMP-dependent protein kinase activity. J Nat Prod. 2005; 68(4): 611-3.
81. Calcul L, Waterman C, Ma WS, Lebar MD, Harter C, Mutka T, et al. Screening mangrove endophytic fungi for antimalarial natural products. Mar Drugs. 2013; 11(12): 5036-50.
82. Ferreira MC, Vieira MLA, Zani CL, Alves TMA, Sales Junior PA, Murta SMF, et al. Characterization of bioactive compounds produced by endophytic fungi associated with Vellozia gigantea, an endemic Brazilian plant. Planta Med. 2014; 80(10): PC25.
83. Kornsakulkarn J, Somyong W, Supothina S, Boonyuen N, Thongpanchang C Bioactive oxygen-bridged cyclooctadienes from endophytic fungus Phomopsis sp. BCC 45011. Tetrahedron. 2015; 71(48): 9112-6.
84. Hemtasin C, Kanokmedhakul S, Kanokmedhakul K, Hahnvajanawong C, Soytong K, Prabpai S, et al. Cytotoxic Pentacyclic and tetracyclic aromatic sesquiterpenes from Phomopsis archeri. J Nat Prod. 2011; 74(4): 609-13.
85. Martínez-Luis S, Cherigo L, Higginbotham S, Arnold E, Spadafora C, Ibañez A, et al. Screening and evaluation of antiparasitic and in vitro anticancer activities of Panamanian endophytic fungi. Int Microbiol. 2001; 14(2): 95-102.
86. Tansuwan S, Pornpakakul S, Roengsumran S, Petsom A, Muangsin N, Sihanonta P, et al. Antimalarial benzoquinones from an endophytic fungus, Xylaria sp. J Nat Prod. 2007; 70(10): 1620-3.
87. Correia VCS, Pereira RKO, Lima NO, Coelho MB, Oliveira Júnior WP, Pimenta RS, et al. Avaliação da atividade antagonista in vitro de fungos endofíticos associados ao camu-camu (Myrciaria dubia). JBFS. 2015; 2(4): 201-7.
88. Strobel G. Muscodor albus and its biological promise. J Ind Microbiol Biotechnol. 2006; 33(7): 514-22.
89. Suwannarach N, Kumla J, Bussaban B, Lumyong S. Biocontrol of Rhizoctonia solani AG-2, the causal agent of damping-off by Muscodor cinnamomi CMU-Cib 461. World J Microbiol Biotechnol. 2012; 28(11): 3171-7.
90. Siri-udom S, Suwannarach N, Lumyong S. Existence of Muscodor vitigenus, M. equiseti and M. heveae sp. nov. in leaves of the rubber tree (Hevea brasiliensis Müll. Arg.), and their biocontrol potential. Ann Microbiol. 2015; 66(1): 437-48.
91. Fill TP, Silva BF, Rodrigues-Fo E. Biosynthesis of phenylpropanoid amides by an endophytic Penicillium brasilianum found in root bark of Melia azedarach. J Microbiol Biotechnol. 2010; 20(3): 622-9.
92. Wang FW, Hou ZM, Wang CR, Li P, Shi DH, et al. Bioactive metabolites from Penicillium sp., an endophytic fungus residing in Hopea hainanensis. World J Microbiol Biotechnol. 2008; 24(10): 2143-7.
93. Arnold AE. Endophytic fungi: hidden components of tropical community ecology. In: Carson WP, Schnitzer SA. Tropical Forest Community Ecology. 1st ed West Sussex: Wiley-Blackwell; 2008. p. 254-71.
94. Ferreira MC, Vieira MLA, Zani CL, Alves TMA, Sales Junior PA, Murta SMF, et al. Molecular phylogeny, diversity, symbiosis and discover of bioactive compounds of endophytic fungi associated with the medicinal Amazonian plant Carapa guianensis Aublet (Meliaceae). Biochem Syst Ecol. 2015; 59: 36-44.
95. Azevedo JL, Maccheori WJ, Araújo WL, Pereira JO. Microrganismos endofíticos e seu papel em plantas tropicais. In: Serafini LA, Barros NM, Azevedo JL, editores Biotecnologia: avanços na agricultura e na agroindústria. 1a ed. Caxias do Sul: EDUCS; 2002. p. 235-68.
96. Firáková S, Sturdíková M, Múcková M. Bioactive secondary metabolites produced by microorganisms associated with plants. Biologia. 2007; 62(3): 251-7.
97. Brown JR. Ancient horizontal gene transfer. Nat Rev Genet. 2003; 4(2): 121-32.