CONDITIONS
Leite, C.A.1 Golçalves, R.C.R1.; Pretti, T.S1.; Marques, M.M1; Caporalin, J.B1.,
Berlinck, R.G.S
2. &Sponchiado, S. R. P
1.1
Instituto de Química de Araraquara, UNESP, R. Francisco Degni, s/n., Araraquara-SP,Brasil 2
Instituto de Química de São Carlos, USP, Av. Trabalhador São-Carlense, 400, São Carlos-SP, Brasil *carlaandrealeite@iq.unesp.br
Keywords: Antioxidant, Aspergillus terreus, secondary metabolites.
INTRODUCTION: In recent years there has been a growing interest in the discovery of the natural antioxidants due to the possibility of large-scale production at a cost lower than the chemical synthesis. Antioxidants may prevent damage caused by free radicals, which in excess are associated with various diseases such as Alzheimer and Parkinson. Fungi have been reported to be producers of secondary metabolites with broad range of biological activities. In early studies, we show that the melanin extracted from A. nidulans presents antioxidant activity for biological oxidants, as HOCl and H2O2 (GONÇALVES & POMBEIRO-SPONCHIADO, 2004). Studies in our laboratory also showed that the growth conditions affected the chemical profile of crude extracts obtained from 57 marine-derived fungal strains (VITA-MARQUES et al., 2008). In this work, we evaluated the influence of the culture medium and phase of fungal growth in the production of antioxidant metabolites by A. terreus.
MATERIALS AND METHODS: A. terreus (105conidia/mL) was cultivated in Sabouraud Dextrose Agar during 12 and 20 days or in artificial sea complete medium for 5 days, on orbital shaker (250rpm) at room temperature. Each culture broth was separated from mycelium by filtration under vacuum and submitted to three times partition with ethyl acetate and butanol, in sequence, and afterwards they were recovered by evaporation. The mycelia dried at 40ºC overnight was triturated and extracted for 30min with methanol under ultrasonically agitation. The resulting material was kept in the dark, for 5 days, a process repeated 3 times in order to complete the extraction of intracellular metabolites. The organic solvent was evaporated under airflow. All dried crude extracts were assayed for their antioxidant activity using ABTS (2,2’–azinobis-(3-ethyl- benzothiazoline-6-sulfonic) assay.
RESULTS AND DISCUSSION: With regard to Sabouraud’s medium, the intracellular (MeOH) and extracellular (EtOAc and BuOH) extracts, obtained after 12 days of growth, exhibited lower antioxidant activity than those cultivated for 20 days, when the fungus is in stationary phase. These results are in accordance with literature because they showed that production of active metabolites is associated to growth phase. To evaluate the effect of the composition of medium in the production of antioxidants, the fungus was cultivated in artificial sea complete medium by 5 days, which corresponds to the stationary phase. In this condition, all obtained extracts showed the lowest antioxidant activity, while the biomass produced was 2-fold greater compared with Sabouraud’s culture. This result can be explained by the fact that in the rich medium, as Marine Agar, the fungus uses the nutrients to produce biomass instead of active compounds, i.e., there is a inverse correlation between growth rate and production of secondary metabolites.
CONCLUSION: The results of this work showed that the variation of culture conditions may alter the production of active metabolites and the most promising antioxidant activity was obtained when A. terreus was cultivated on Sabouraud Agar for 20 days.
REFERENCES: GONÇALVES, R. C. R.; POMBEIRO-SPONCHIADO, S. R. Antioxidant Activity of the Melanin Pigment Extracted from Aspergillus nidulans. Biol. Pharm. Bull., v. 28, n. 6, p. 1129-1131, Nov. 2005.
VITA-MAQUES, A.M. et al. A multi-screening approach for marine-derived fungal metabolites and the isolation of cyclodepsipeptides from Beauveria felina. Quimica Nova., v.31, n.5, p.1099- 1103, 2008.
EVALUATION OF Burkholderia spp. CHARACTERISTICS ASSOCIATED TO PLANT GROWTH PROMOTION AND CONTROL OF DESEASE IN SUGARCANE
Luvizotto, D.M.1*, Dini-Andreote1, F., Dias, A.C.F1, Araújo, W.L..1,2, Pizzirani-Kleiner, AA1 1
MATERIAL AND METHODS: It was used 39 bacteria identified as Burkholderia spp., which ones 19 were isolated from rhizosphere and 20 from sugarcane root, as endophytic. It was determined, using specific methodologies, the potential of nitrogen fixation (NF) (Döbereiner et al. 1995), the production of plant growth hormone indole-acetic-acid (IAA) (Bric et al., 1991) and siderophores (SP) (Schwyn & Neilands, 1987), the inorganic phosphate solubilization activity (IFS) (Verma et al., 2001) and the antagonism potential against sugarcane pathogens: Fusarium
verticillioides (Paired Culture Method) and Xhantomonas albilineans (Agar Underlayer Method)
(Pugsley & Oudega, 1987, Gross & Vidaver, 1990).
RESULTS AND DISCUSSION: The obtained results show that, among the evaluated isolates, 87.2% were positive for NF, 100% were positive for production of IAA, 64.1% were positive for SP, 100% were positive for IFS, 97.4% of isolates were able to inhibit F. verticillioides and 74.4% to inhibit X. albilineans.
CONCLUSION: This study show the potential of Burkholderia bacteria genus in the production of compounds associated to plant growth promotion and pathogens inhibition, especially for sugarcane pathogens as F. verticillioides and X. albilineans. Future studies associated with specific methodologies for the practical usage of these isolates can be developed, aiming the production of bio-product, which ones show as a highly feasible in the current sustainable agriculture.
REFERENCES:
Bric, J.M., et al. Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl. Environ. Microbiol., 57: 535-538, 1991.
Coenye, T. & Vandamme, P. Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ. Microbiol., 5: 719-729, 2003.
Döbereiner, J., et al. Como isolar e identificar bactérias diazotróficas de plantas não- leguminosas. Brasília: Embrapa - SPI, 1995, 60 p.
Gross, D.C. & Vidaver, A.K. Methods in phytobacteriology, 1990, p. 245-249.
Pugsley, A.P. & Oudega, B. Methods for studying colicins and their plasmids. In: Hardy, K.G. (Ed.), Plasmids: a Practical Approach, Oxford: IRL Press, Oxford, p. 105-161, 1987.
Schwyn, B. & Neilands, J.B. Universal chemical assay for the detection and determination of siderophores. Analyt. Biochem., 160: 47-56, 1987.
Verma, S.C., et al. Evaluation of plant growth promoting and colonization ability of endophytic diazotrophs from deep water rice. J. Biotechnol., 91: 127-141, 2001.
Financial support: CNPQ, FAPESP.
Department of Genetics; ESALQ/USP, Piracicaba/SP * e-mail: luvizott@esalq.usp.br
Keywords: indole-acetic-acid, plant growth, siderophores.
INTRODUCTION: Sugarcane (Saccharum spp.) occupies a prominent position among the crops with economic importance, especially in Brazil, the largest producer in the world. Recent studies, about the endophytic bacterial community associated with this culture, have been showing that several bacteria genus with plant grow promotion and control of disease potentials can be found. In this context, we must consider the Burkholderia genus with approximately 30 species, where some of then had been describe as plant hormones producer, inorganic phosphate solubilizing, nitrogen fixation and antagonists of pathogens (Coenye & Vandamme, 2003). This study aimed to evaluate the characteristics of 39 sugarcane Burkholderia spp. isolates, trying to establish parameters to be used in future studies associated to the biotechnology usage of this isolates.