Diversidade e bioprospecção de fungos endofíticos associados à plantas bioativas do Brasil e Estados Unidos

Abstract

Endophytic fungi are microorganisms which live inside plant tissues or organs, without causing them any harmful symptoms. These microorganisms may be found in several species of plants and represent a promising source of bioactive metabolites for pharmacological and agricultural area. The medicinal plants have been used throughout human history in the world. Specifically in Brazil, the Copaifera genus are frequently used due its anti-inflammatory effects and wound healing of their oleoresin. In the United States, Echinacea purpurea is commonly used because it may stimulate the immune system. However, despite its ethnobotany use, few studies have been conducted in order to know their endophytic fungi communities. The aim of this study were to characterize the diversity of the community of endophytic fungi associated with Copaifera langsdorffii, C. pubiflora, Equinacea purpurea and evaluate it as a source of bioactive metabolites as well. Nine hundred-one endophytic fungal isolates were obtained in association with the medicinal plant C. langsdorffi (308 isolates), C. pubiflora (360 isolates) and E. purpurea (233 isolates). Cladosporium, Colletotrichum, Diaporthe, Fusarium, Guignardia/Phyllosticta and Pestalotiopsis genera were shared among the three host plants. Diaporthe sp. 6, Xylariaceae sp. 1, Diaporthales sp. 1 e Diaporthales sp. 2 were the dominant species associated wih C. langsdorffi; Diaporthe sp. 5, Guignardia sp. 2, Diaporthales sp., Diaporthe sp. 8 e Guignardia sp. 1 with C. pubiflora; and Alternaria alternata, Colletotrichum dematium and Stagonosporopsis sp. 2 with E. purpurea. The fungal community associated with C. langsdorffii showed the highest diversity (Fisher α = 16.6), followed by E. purpurea (Fisher α = 14.96) and C. pubiflora (Fisher α = 7.23). The fungal community of C. lansdorffii showed the highest species richness (Margalef = 8.41), followed by E. purpurea (Margalef = 7,52) and C. pubiflora (Margalef = 4,57). The Simpson index (dominance) showed close values for the three communities (C. langsdorffi = 0.94, C. pubiflora = 0.90, E. purpurea = 0.84). Only 11 (1.6%) extracts from Copaifera species showed ≥70% inhibition against at least one target microorganism, and those extracts were selected for the minimal inhibitory concentrations determination (MIC). The dichloromethane extracts of Alternaria daucifolii UFMGCB 7491, Diaporthe sp. 6 UFMGCB 7696 and 7720, Diaporthe sp. 7 UFMGCB 7927 and Diaporthe sp. 8 UFMGCB 7646, 7653, 7701, 7719, 7770, 7772 were submitted to 1H NMR spectrometry. The extracts of A. daucifolii UFMGCB 7491 and Diaporthe sp. 7 UFMGCB 7927 were predominately composed of fatty acids. The linoleic,palmitic and elaidic acids were the predominant in the A. daucifolii UFMGCB 7491 extract. The oleic, linoleic, stearidonic and palmitic acids in the Diaporthe sp. 7 UFMGCB 7927 extract. The Diaporthe sp. 8 UFMGCB 7719 extract was fractionated and two pure compounds were identified, cytochalasin H and cytochalasin J. Those compounds showed antifungal activity against Phomopsis obscurans e P. viticola. Eight extracts from endophytic fungi associated to E. purpurea were selected for chemical analysis. The 1H NMR analysis indicated that the extracts of Colletotrichum sp. 4 EPU8FB, Fusarium sp. EPU9CB, Nigrospora oryzae EPU13CA, Colletotrichum sp. 4 EPU30CA, Fusarium sporotrichioides EPU36CA and Sordariomycetes sp. 2 EPU40CE were predominately composed of fatty acids. Further analysis of all of the extracts confirmed the presence of a mixture of 16 fatty acids. Using a bioassay-guided purification approach (-)-5-methylmellein and (-) (3R)-8-Hydroxy-6-methoxy-3,5-dimethyl-3,4- dihydroisocoumarin were obtained from Biscognauxia mediterranea EPU38CA. The (-)-5- methylmellein showed antifungal activity against P. obscurans and P. viticola. The (-) (3R)-8- Hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin was not able to inhibited the C. gloeosporioides growth, however the substance showed activity against all the others microorganism tested in at least one concentration and time of incubation. The antifungal effects observed in extracts and fractions selected, as well as the cytochalasin H, cytochalasin J, (-)-5- methylmellein and (-) (3R)-8-hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin production give evidence that the symbiotic relationship between endophytic fungi and their host plants may be able to produce substances against pathogenic fungi and herbivores animals. The results obtained in this study showed that medicinal plants can represent a reservoir of endophytic fungi promising for bioprospecting.