Prospecção de metabólitos especiais com potencial antimicrobiano produzidos por fungos endofíticos associados à gramínea Axonopus Leptostachyus (Flüggé) Hitchc (Poaceae)
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Data
2020-03-31
Autores
Neponuceno, Ricardo Aparecido Rodrigues
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Universidade Federal de Mato Grosso
Resumo
Endophytic fungi contribute considerably to the production of bioactive metabolites of
great economic value and with several applications, such as antibiotics and
agrochemistry. In this context, the present work aimed to perform the isolation,
purification and structural elucidation of secondary metabolites produced by endophytic
fungi associated with Axonopus leptostachyus, aiming at the search for substances with
potential antimicrobial action. In this way, 64 endophytes isolated from A.
leptostachyus, were submitted to assays to evaluate antimicrobial activity, against 5
strains of bacteria and 5 of human pathogenic candida, semi-quantified and classified
into groups of high (AIA), medium (MAI) and low (BAI) inhibitory activity.
Additionally, six selected endophytes were also evaluated for the detection of possible
antagonists of the phytopathogen Corynespora cassicola. The results of the bioassays
added to the multivariate analysis allowed the selection of the endophytic fungi with the
best potential for obtaining the bioactive metabolites, with Microsphaeropsis arundinis
(P21) being the only one that showed excellent activity in relation to all tests. Against
pathogenic bacteria and candida, the fungi Talaromyces varreculosus (P24),
Gongronella butleri (P48), Penicillium javanicum (P70) and the unidentified endophyte
(P4) stood out with the best antimicrobial activities. The tests also identified that
Cladosporum flabelliforme (P5) and Neocosmospora strita (P28) showed excellent
activities against pathogenic candida and phytopathogen, while Fusarium succisae
(P59) and Fusarium oxysporum (P61) stood out only with activity for C. cassicola.
After the bioassays, the endophytes with the best potentials were submitted to
cultivation in PDA (Potato Dextrose Agar) and minimal medium, in a reduced and
enlarged scale. In the PDA medium, the endophytes were grown in 100 mL of medium
(5 Petri dishes) under incubation at 37 °C for 15 to 23 days. Pieces with PDA and
mycelia were randomly cut and macerated for 24 h with 150 mL of ethyl acetate
(EtOAc). The EtOAc phase was vacuum filtered, concentrated in a rotary evaporator
and dried in an air jet, obtaining the crude extracts EtOAc. In the minimal medium, the
fungi grew in duplicate of 200 mL under incubation and orbital shaking for 20 days.
Then, the micellar suspension was filtered, partitioned with EtOAc, and dried to obtain
the crude extracts. In sequence, the extracts from the screening and growth stages on a
larger scale were solubilized and analyzed using HPLC-PDA (High-Performance Liquid
Chromatography – Photodiode Array) and 1H NMR (Proton Nuclear Magnetic
Resonance). After analysis, the P5, P21 and P28 endophytes were chosen for the
chromatographic fractionation of their extracts, aiming at the isolation of the
compounds responsible for the detected activities. The isolated substances were
analyzed using 1H and 13C NMR spectroscopy, gCOSY, gHMBC and gHSQC for the
identification or elucidation of their structures. The fractionation of the EtOAc extract
of C. flabelliforme (P5) resulted in the isolation of a compound in Tr 26 min. (FR1.16-
17.fim.3), not yet identified. The study of M. arundinis (P21) allowed the identification
of a benzolactonic dimer (1), an unprecedented molecular structure for the genus
Microsphaeropsis. For the fungus N. striata (P28), substances extracted from the PDA
medium were isolated, such as the monorden (radicicol), a lactonic derivative of
resorcylic acid (2), and the compounds in fractions FR4.fim.4, FR6.1, FR6.3, FR6.5 and
FR1.precipitate, not yet identified, as well as a substance extracted from the minimal
medium, under study for structural identification (3).
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NEPONUCENO, Ricardo Aparecido Rodrigues. Prospecção de metabólitos especiais com potencial antimicrobiano produzidos por fungos endofíticos associados à gramínea Axonopus Leptostachyus (Flüggé) Hitchc (Poaceae). 2020. 189 f. Dissertação (Mestrado em Gestão e Tecnologia Ambiental) - Universidade Federal de Mato Grosso, Instituto de Ciências Agrárias e Tecnológicas, Rondonópolis, 2020.