**3. Results**

244 Antimicrobial Agents

(5'GGTGGCGAAGGCGGA 3') and Sm5R (5' GAACTGAGACCGGCTTTTTGA 3') were used to amplify the 16S rDNA. Amplification conditions followed Arzanlou et al. (2008) for the fungi and Monciardini et al. (2002) for the actinomycete. Amplicons were sequenced using both PCR primers and DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBACE (Amersham Biosciences). Sequences were manually aligned using Mega v. 5 software (Kumar et al., 2004) by inserting gaps. The obtained sequences were aligned according to existing sequences at the data base NCBI though the BLASTn program. Phylogenetic analyses of the aligned sequence data were performed with PAUP

Endophytes were selected for the extraction of active metabolites by fermentation. After the growth in potato-dextrose-agar medium in Petri dishes for 7-14 days at 28ºC, fragments of the endophytes with a diameter of 10mm were removed and sowed in Erlenmeyers with 50mL and 100mL of the liquid medium Czapeck (Silva et al., 2004), MPE (Hamada et al., 1974) and malt extract broth (20 g l¹ malt extract, 1 g l1 peptone, 20 g l1 glucose), and were incubated at 28ºC at 120rpm. The 50mL cultures were incubated for 24 hours, while the ones with 100mL of medium were cultivated for 7 days. After the predetermined period the mycelium was separated of the metabolic medium by paper Whatman n°4 vacuum filtration and then stored. Either compounds from the culture and the ones retained on the cell structures were extracted with ethyl acetate p. a. (EtOAc; Merck). Solvent evaporation was carried out using a rotaevaporator at 45°C. The final extract was weighed and diluted in methanol, methanolic extracts (ME) at a concentration of 10 mg/mL (Corrado & Rodrigues, 2004). The fermentative liquid was lyophilized, weighed and also diluted in ultrapure sterilized water, aqueous extracts (AE) at

For the evaluation of the antimicrobial activity of the secondary metabolites obtained from the culture of the endophytes was used bioautographic TLC agar overlay assay (Corrado & Rodrigues, 2004). To evaluate the activity of the extracts obtained through the maceration of the endophytic cell mass, an adaptation of a manual patterned by Clinical and Laboratory Standards Institute (2003*a*) was used. The results were collected through the measurement of the growth inhibition halo formed around the well. The microorganisms used on the tests were: *Staphylococcus aureus* (ATCC 27213 and ATCC 25923), *Escherichia coli* (ATCC 25922), *Pseudomonas aeruginosa* (ATCC 27853), *Enterococcus faecalis* (ATCC 29212), *Klebsiella pneumonia* (ATCC 700603), *Micrococcus luteus* (ATCC 9314), methicillin-resistant *Staphylococcus aureus* (MRSA) and *Candida albicans* (ATCC 10231). Test organisms were grown overnight in a Müeller-Hinton broth (MH , Merck) at 37 °C and were diluted until reaching the concentration of 106 cells/mL. As positive control chloranphenicol 1mg/mL for bacterial strains and nystatin 100000UI/mL for *C. albicans* were used. Methanol was applied as solvent control and saline solution was used

(Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford, 1998).

**2.4 Endophytes extracts** 

concentration of 10 mg/mL.

as negative control.

**2.5 Antimicrobial activity of endophytes extracts** 
