**2.3 Endophytes identification**

An analysis based on a polyphasic approach integrating taxonomic information, morphological traits and the sequencing of the ITS1–5.8S–ITS2 of the rDNA or 16S was used, as described by Gomes-Figueiredo et al. (2007). Isolates were initially identified based on their microscopic and macroscopic characteristics including their morphology and characteristics when grown on the following culture media: PDA, oatmeal agar (OA) (20 g l-1 oat, 20 g l-1 glucose, 15 g l-1 agar), malt extract agar (MEA), and complete medium (CM) (Pontecorvo et al., 1953). Isolates were incubated for 7 days at 22 or 280C and a 12 h light: 12 h dark photoperiod. The experimental design was completely randomized with 3 replicates. Colonies were analyzed with respect to their average diameter (cm), the aspect of their borders, the aspect and coloration of the mycelium, sporulation, mycelium characteristics, the production of acervuli, the coloration of the reverse of the Petri dish, the viscosity and coloration of the medium, and the size and coloration of the conidia. A total of 20 conidia from each culture medium were observed under light microscopy (x 1000 magnification) after being grown for 7, 14, and 21 days. Conidia were assessed with respect to their width and length and the length of the apical appendages. The coloration of the median cells was also recorded. For actinomycetes identification, characteristics of colonies were used, after growth in AC medium. The isolates Gram-stained were observed under light microscopy (x 1000 magnification).

The fungi isolates were randomly selected as morphotypes according to Arnold et al. (2000), and the endophytes that presented at least one of the extracts with antimicrobial activity were submitted to identification using ITS sequences of the rDNA. DNA extraction followed method described by Raeder & Broda (1985), modified by Glienke-Blanco et al. (2002). For the fungi, the primers V9G (De Hoog et al., 2003) and ITS4 (White et al., 1990) were used to amplify the ITS1-5.8S-ITS2 of the nuclear ribosomal RNA, in the following reaction mixture (50 µl): 0,2 mM of each dNTP, 1X Tris/HCl, 1.5 mM MgCl2, 1.5 U Taq polymerase, 0.06 µM each primer and 50ng of DNA; the PCR was processed in a Mastercycler Gradient (Eppendorf®) with the following program: 94 °C for 2 min at the start followed by 35 cycles of 94 °C for 30 s, 55 °C for 1 min and 72 °C for 1 min and a final extension of 72 °C for 3 min. For the actinomycete the primers Sm6F

Antimicrobial Activity of Endophytes from Brazilian Medicinal Plants 245

The dried leaves were put in contact with petroleum ether for six days, having the solvent freshened when saturated. After removing the filtrated with petroleum ether the leaves were exposed to methanol for nine days having the solvent freshened when necessary (Harbone, 1998). The concentrated crude methanolic extract was partitioned using the following gradient elution: petroleum ether, petroleum ether: dichloromethane 1:1, dichloromethane, dichloromethane: ethyl acetate 1:1, ethyl acetate, ethyl acetate: methanol 1:1 and methanol.

For this evaluation an adaptation from the macrodilution method (Clinical and Laboratory Standards Institute 2003*b*) has been used. In a test tube with Müller-Hinton broth already with the extract/fraction in a known concentration, the microorganism to be combated *(Staphylococcus aureus, Pseudomonas aeruginosa or Candida albicans)* was inoculated. To isolate the solvent influence (dimethyl sulfoxide) at the activity of the extract/fraction used, controls having different solvent concentration were prepared. The test tubes were incubated and after that the turbidity standard was analyzed. For an exact analysis of the results an aliquot of 100μL of each test tube was sowed in a Petri dish with Müller-Hinton agar and incubated at 35ºC for 24 hours for posterior growth analysis by colonies counting. For the yeast the

The bioactive extracts obtained from peppertree endophytes were compared with the compounds present in the crude methanolic extract of the leaves and fractions active by thin-layer chromatography. The revealing substances used were: Dragendorff reactive, potassium hydroxide, sulfuric vanillin, ferric chloride - 1.5%, anisaldehyde and ninhydrin

One hundred thirty-one endophytes were isolated from peppertree leaves. Nine endophytes active metabolites producers were identified. These, 2 were identified as *Alternaria* sp., 3 as *Phomopsis* sp., 1 as *Penicillium roseopurpureum*, 1 as a basidiomycete, and 1 as *Streptomyces* sp. One hundred ninety-one endophytes were isolated from leaf fragments of *M. ilicifolia*, belonging to 6 genera of fungi: *Alternaria*, *Phyllosticta*, *Xylaria, Phomopsis, Pestalotiopsis* and *Colletotrichum.* Eighteen actinomycetes were isolated from 4000 samples *Vochysia divergens,* with isolation rates of 0.47%, of these 61.1% (11) were isolated from petiole and 38.9% (07) leaves. Three taxa were identified: *Microbispora* sp. (10 isolates), *Micromonospora* sp. (2

Antibacterial activity of the methanolic extracts from endophytes of *S. terebinthifolius* was evaluated (Table 2). From the twenty isolates selected to fermentation, three released

**2.7 Antimicrobial activity of peppertree crude methanolic extract and fractions** 

incubation period was 48 hours at 35ºC. The test was carried out in duplicate.

**2.8 Chemical comparison** 

**3. Results** 

(Ordóñez et al., 2006; Rodrigues et al., 2009).

**3.1 Isolation and identification of endophytes** 

isolates) and *Streptomyces sampsonii* (2 isolates).

**3.2 Antimicrobial activity** 

**2.6 Peppertree crude methanolic extract and fractions** 

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

#### **2.4 Endophytes extracts**

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 concentration of 10 mg/mL.

#### **2.5 Antimicrobial activity of endophytes extracts**

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 as negative control.
