Antimicrobial Activity of Crude Extracts of Endophytic Fungi Isolated from Medicinal Plant Sapindus saponaria L.

ABSTRACT


INTRODUCTION
Endophytic microorganisms are fungi and bacteria that colonize inter or intracellular spaces of plant tissues during at least one phase of their life cycle (Azevedo et al., 2000;Kaneko et al., 2010). Mostly, they maintain a stable relationship with the host plants, since they live asymptomatically (Pamphile and Azevedo 2002), without causing apparent damage to them (Kaneko et al., 2010).
. . . substances of biotechnological interest, including:s econdary metabolites with pharmaceutical application (Schulz et al., 1999;Schulz and Boyle, 2005;Strobel, 2003;Strobel, 2006), being used in the production of antimicrobials that inhibit the development of pathogens (Chareprasert et al., 2006;Phongpaichit et al., 2006;Strobel et al., 1999;Weber et al., 2007). Also, they can protect the hosts against several biotic and abiotic factors, such as the attack of insects, pathogens and herbivores (Arnold et al., 2003;Firáková et al., 2007;Mejía et al., 2008).The in vitro inhibition of pathogens by antagonists is considered an indication of antibiosis caused by antagonistic substances possibly produced in the culture media (Rocha et al., 2009).
The genus Phomopsis is commonly found as endophyte in tropical medicinal plants, such as reported by Bernardi-Wenzel et al. (2010), Garcia et al. (2012 and Rhoden et al. (2012a). This genus is a rich source of biologically active secondary metabolites with antimicrobial activity against several pathogens including Mycobacterium tuberculosis, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Micrococcus luteus and Candida albicans (Jayanthi et al., 2011;Rukachaisirikul et al., 2008) and it is also potential for protecting plants from fungal diseases (Brayford, 1990).
Tilley and Walker (2002), isolated Cochliobolus intermedius from diseased crabgrass (Digitaria sp.) and evaluted its potential as a microbial herbicide for control of large crabgrass (Digitaria sanguinalis), observing that these fungus could be a potential microbial herbicide for control of large crabgrass in crops such as soybean, cotton, and peanut.
The aim of the present work was to obtain crude extracts of fungal endophytes (G2-20 Cochliobolus intermedius, G1-74 non-identified -NI, G22-97 Phomopsis sp. and G23-100 NI) isolated from S. saponaria and investigate their biotechnological potential against five pathogenic bacteria.

Obtainment of crude extracts
The crude extracts were obtained following the methodology described by Li et al. (2005) modified. The endophytic fungi were incubated in culture medium, at 25º C for 10 days in Potato Dextrose (PD), which prepared according to Smith and Onions (1983) modified by Pamphile et al. (2004). The fermented medium obtained was centrifuged at 3.600 rpm for 10 minutes. The supernatant was transferred to a separatory funnel to which was added the same volume of crude ethyl acetate. The funnel was strongly agitated and then the separation of the phases occurred by polarity differences. This process was repeated twice. Ethyl acetate solution containing the fungal metabolite was 98% concentrated in a Büchi R-3000 Rotavapor at 40º C and the material obtained from the evaporation was suspended with 1ml of absolute methanol and storaged at 4º C until its use.
The fungal mycelium obtained in the PD incubation was maintained for 48 hours in methanol. After this period, the methanol was centrifuged and the supernatant was collected and 98% concentrated a Büchi R-3000 Rotavapor at 40º C. The material obtained from the evaporation was suspended with 1 ml of absolute methanol and also stored at 4º C.

Assessment of antimicrobial activity
The antimicrobial activity was tested by qualitative biological analysis in triplicate, using the disk diffusion technique (cup plate). The microorganisms used in this test were the human pathogenic bacteria Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Salmonella typhi (ATCC 19430), Micrococcus luteus (ATCC 9341) and Enterococcus hirae (ATCC 1227).
To evaluate the antibacterial activity, bacteria were grown in liquid LB (Luria Bertani) medium (Sambrook and Russel, 2001) for 24 h, adjusted at a concentration of 10 6 cells/ml and spread (100 μl) on Petri dishes containing solid LB medium. In each dish were placed, equidistantly, four disks of sterile filter paper Whatman n o . 4 (6 mm) inoculated with 10 μl of extracts. As negative controls, paper disks were inoculated with autoclaved distilled water and absolute methanol. As positive control, Tetracycline (50 µg.ml -1 in absolute ethanol) was used. The dishes were incubated at 37° C in B.O.D for 24 h. The antibacterial activity was evaluated by the formation of inhibition halos, according to Souza et al. (2004).
All the experiments were carried out using a completely randomized design (CRD), with 3 repetitions. In order to test the efficiency of the metabolite extracts, statistical analyses through WinBUGS (Spiegelhalter et al., 1994) software were employed, which is followed by the Bayesian analysis, admitting normal distribution due to the growth inhibition halo data.

RESULTS AND DISCUSSION
The increasing use of chemical products in order to implant and maintain healthy crops and high productivity has been caused negative effects for the biotic complex of nature, affecting animals, humans and plants (Mochi et al., 2005).
Discovery of new and potential drugs molecules can be focused on the production of bioactive compounds by plants, microbial and marine organisms. Endophytic microorganisms isolated from plants constitute a source of search for novel secondary metabolites (Firákóva et al., 2007), since a single endophyte may be able to produce a variety of bioactive metabolites (Ramasamy et al., 2010).
The antibacterial tests with the fungal isolates G2-20 (Cochliobolus intermedius), G22-97 (Phomopsis sp.), G1-74 and G23-100 (non-identified endophytes) showed that when the extracts were obtained by the incubation of mycelium with methanol, it was verified no difference in comparison to the negative controls. Considering the endophytic extracts obtained from the fermented medium extracted with ethyl acetate, the results were statistically significant in comparison to the negative controls for at least one of the five human pathogenic bacterium tested (E. coli, S. aureus, S. typhi, M. luteus and E. hirae) (Figure-1 and Table-1). However, the only one extract that showed antibacterial activity against all pathogenic bacteria was the endophytic strain G2-20, molecularly characterized as C. intermedius. For S. aureus, extract from G2-20 (C. intermedius) showed the highest antimicrobial activity. Those produced by G1-74 (NI) and G23-100 (NI) also showed inhibitory activity, whereas the extract produced by G22-97 strain (Phomopsis sp.) was statistically no significant in relation to negative control. For E. coli, only one crude extract that showed antimicrobial activity was the produced by G2-20 (C. intermedius) strain. For M. luteus, all extracts tested showed positive results. Among them, the most efficient was also G2-20 (C. intermedius). Against S. typhi, highest indexes of antimicrobial activity were produced by the isolates G2-20 (C. intermedius) and G1-74 (NI), followed by G23-100 (NI). The same was observed for E. hirae.
Many studies are evaluating metabolic activity from endophytic fungi isolated, mostly, of plants with properties medicinal. Pileggi et al. (2002), studied antimicrobial action in endophyte fungi isolated form medicinal plant Simphytum officinale, popularly known as confrei. These isolated inhibit growing of pathogenic bacteria S. aureus.
According to Borges (2008), an important factor to be considered is the fungal growth condition, since the fungus requires excellent conditions for an optimal development, expressing thus its enzymatic potential in the production of metabolites. In his study, this author demonstrated that, for the same fungus, the metabolites production varied depending on the conditions of cultivation.
In our study, the most efficient method for the obtainment of fungal secondary metabolites was the extraction with ethyl acetate, where results statistically significant in relation to negative control were obtained for at least one bacterium tested. The obtainment by incubation of fungal mycelium in methanol did not present positive results for none of the isolates. Rhoden et al. (2012b), using the same methodology and tested against same bacterial observed that endophytic fungi Cordyceps memorabilis inhibit growing against E. coli, M. luteus and E. hirae, Phomopsis longicolla inhibit S. typhy and E. hirae, and two another fungi not identified inhibit growing of M. luteus and E. hirae.
Similarly, Bernardi-Wenzel (2008) and Rhoden et al. (2012b) also observed minor efficiency of endophytic secondary metabolites extracted with methanol regarding antimicrobial tests. Bernardi-Wenzel (2008) researched the antimicrobial activity of fungal secondary metabolites produced by endophytes from Luehea divaricata against the human pathogenic bacteria Escherichia coli and Staphylococcus aureus. None extracts showed antagonistic activity against S.aureus, while some extracts inhibited the E. coli growth. Phongpaichit et al. (2006) isolated fungal endophytes from five medicinal Garcinia plants and verified that the metabolites produced by 70 isolates and extracted with ethyl acetate showed antimicrobial activity by agar diffusion test against at least a pathogen microorganism tested: Staphylococcus aureus, C.albicans, Cryptococcus neoformans and Microsporum gypseu. These authors identified the genera Aspergillus, Botryosphaeria, Eutypella, Fusarium, Guignardia,Penicillium, Phomopsis and Xylaria as the ones which showed the greatest results for the inhibition of microbial growth. Among these endophytes, three strains showed most active for production of secondary metabolites were Phomopsis sp., Botryo sp, haeria sp. and a nonidentified fungi.
In a subsequent study employing endophytes from the same plant genus, Phongpaichit et al. (2007) verified the biological activity of sixty-five fungal metabolites extracted with ethyl acetate. Their results were promising, which 80% of the extracts showed some bioactivity, such as: cytotoxic, antimycobacterial, antimalarial, antiviral, antioxidant and anticancer. Souza et al. (2004), tested the antimicrobial activity of endophytes from Amazonian toxic plants Palicourea longiflora and Strychnos cogens. From total of 79 fungal isolates whose metabolites were tested, 19 inhibited at least one of the pathogenic microorganisms tested: Bacillus sp., B.subtilis, S.aureus, E. coli, Candida albicans, Trichoderma sp., and Aspergillus flavus. Gomes-Figueiredo (2007) observed that the metabolic extracts produced by endophytes of Pestalotiopsis genus, isolated from medicinal plant Maytenus ilicifolia, have antimicrobial activity against a variety of human pathogens and some isolates showed be potential for the biological control of the phytopathogenic fungus Guignardia citricarpa. Similarly, some authors reported activity of metabolites from endophytic fungi in the inhibition of different pathogenic bacteria and fungi, showing that some plant properties can be present in their endophytes.
Using Chinese medicinal plants Li et al.(2005) isolated 130 endophytic fungi and tested the antitumour and antifungal activities from their extracts. As result, 9.2% of them presented antitumour activity and 30% had antifungal activity, what indicates that some fungal compounds can be associated with the host plants.
Similarly to the present study, Teles et al. (2006) also extracted secundary metabolites from Periconia atropurpurea, an endophyte from Xylopia aromatica, using ethyl acetate. Besides identifying these compounds, the authors tested their biological activity, proving their cytotoxic and antifungal potential.
Hoffman et al. (2008), working with a Phoma strain, endophytically isolated from Saurauias caberrinae, verified that it produced phomodione, a substance with inhibitory action against phatogenic bacterium Staphylococcus aureus. Hormazabal and Piontelli (2009) showed that, among the endophytes isolated from Chilean native gymnosperms, the metabolite produced by Curvularia protubera had the best effect on Bacillus subtilis, M. luteus and S. aureus, with growth inhibition zones of 12, 9 and 16 mm, respectively. None of the metabolites tested by these authors showed effect on E. coli, on the contrary to the result obtained by our research group, where one metabolite tested was effective against this bacterium. Sutjaritvorakul et al. (2011) used the paper disk susceptibility test to evaluate the antimicrobial activity of metabolites produced by fungal endophytes against five reference human pathogenic microorganisms (S. aureus, B. subtilis, Pseudomonas aeruginosa, E. coli and C. albicans). The fungal metabolites inhibited two or more pathogens and the growth of Gram positive bacteria were more inhibited than those Gram negative, with inhibition halos up to 20.1 mm, which was produced by extract of Pestalotiopsis sp. DO2 against B. subtilis.
The results reported in this present study demonstrate the potential of endophytic fungi from S. saponaria for the biological control of human pathogens. Tests with the crude extracts produced by the endophytic isolates showed promising results for growth inhibition of human pathogenic bacteria. Therefore, it indicates that these endophytes can be important sources of bioactive substances of biotechnological interest.

CONCLUSION
Crude extracts from endophytes of S. saponaria showed in this study a greater antimicrobial activity against some human pathogenic bacteria. So studies on safety and efficacy should be performed for these fungi for use as pharmaceutical drugs.

ACKNOWLEDGMENTS
To the (CAPES) for the scholarship. To the CNPq for financial support.