Antibacterial activity of endophytic fungi from the medicinal plant Uncaria tomentosa (Willd.) DC

This study was designed to determine the diversity and antibacterial activity of endophytic fungi isolated from Uncaria tomentosa. Leaf and stem were disinfected superficially and inoculated in PDA and SDA medium, with and without plant extract and incubated at 18 and 28°C for isolation of endophytic fungi. Endophytic fungi were inoculated in BD medium and the metabolites extracted with ethyl acetate. Endophytic fungi extracts were tested for antibacterial activity by the disk diffusion test. One hundred and seventy endophytic fungi were isolated and identified as Aspergillus, Asterosporium, Aureobasidium, Botrytis, Colletotrichum, Curvularia, Didymostilbe, Fusarium, Guignardia, Nigrospora, Penicillium, Pestalotiopsis, Phomopsis, and sterile mycelium. Staphylococcus aureus was the most resistant bacterium, with only two fungal extracts inhibiting its growth, while the most sensitive was Escherichia coli, with 23 extracts inhibiting its growth. Five extracts inhibited Enterococcus faecalis and four Klebsiella pneumoniae. No fungal extract was able to inhibit the four tested bacteria. Extracts from endophytic fungi isolated from U. tomentosa showed in vitro antibacterial activity against grampositive and gram-negative bacteria.


INTRODUCTION
Endophytes are microorganisms that colonize internal tissues of plants for at least part of their life cycle without causing disease symptoms in their hosts (Petrini, 1991).Fungal endophytes can inhabit host tissues in different organs, including leaves, stems, barks, roots, fruits, flowers, and seeds (Stone et al., 2004).In this symbiotic relationship, fungal endophytes receive protection and nutrients from the host, while the host plant receives protection against natural enemies, such as pathogens and herbivores (Azevedo et al., 2000), promoting plant growth (Hamayun et al., 2010) and increasing its resistance to abiotic stress factors (Khan et al., 2014).
Many medicinal plants are known to harbor endophytic fungi, which are producers of important bioactive secondary metabolites for the industry.Therefore, efforts have been made to characterize and identify endophytic fungi isolated from medicinal plants (Strobel et al., 2004).Uncaria tomentosa (Willd.)DC belongs to the *Corresponding author.E-mail: asfuryy@gmail.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Rubiaceae family, being a medicinal plant widely used by Amazon peoples.This species is used to treat infections, rheumatism, gastritis, cancer, asthma, cirrhosis, fever, and has a wide range of other medicinal applications (Keplinger et al., 1998;Dreifuss et al., 2013).Several chemical compounds, such as oxindole alkaloids and quinolinic acids, have anti-inflammatory (Akhtar et al., 2011), anticancer (Dietrich et al., 2014), and antimicrobial activity (Sá et al., 2014).However, there are no studies on the endophytic community of this plant.Thus, this study was designed to determine the diversity and antibacterial activity of endophytic fungi isolated from U. tomentosa.

Plant samples
Healthy and mature plant tissues were collected from three U. tomentosa trees in Rio Branco, Acre, Brazil (10°01′ S and 67°42′ W) in September 2015.Voucher specimens were deposited in the Herbarium of the Universidade Federal do Acre under the identification number 22.002.Leaves and stems were collected from each plant and brought directly to the laboratory, being processed within 24 h after collection (Azevedo et al., 2010).

Isolation of endophytic fungi
Each sample of plant material was washed with running water and surface sterilized with 70% ethanol for 1 min, followed by treatment with 2.5% active chlorine solution for 3 min, 70% ethanol for 30 s, and final rinsing in sterile water (Pereira et al., 1993).Prior to surface decontamination, the ends of stem fragments were sealed with paraffin to prevent the entry of germicidal agents into the plant tissue and thus inhibit the death of endophytic fungi.To assess whether the disinfection method was effective in the removal of fungi from the surface, 200 µL wash water were inoculated in the same culture media used for the isolation of endophytic fungi, and these plates were observed for emergent fungi (Azevedo et al., 2010).
After superficial disinfection, two plates of potato dextrose agar (PDA), Sabouraud dextrose agar (SDA), PDA+plant extract, and SDA+plant extract, supplemented with chloramphenicol (100 µg mL −1 ), each of them containing 10 fragments of plant material, were prepared for each of the two types of samples (leaf and stem) and maintained in the dark at 18 and 28°C.For producing the plant tissue extract, 100 g of fresh tissue were ground in 500 mL distilled water, filtered on filter paper, and 500 mL of an infusion of 200 g of potato were added to prepare PDA+extract medium or 500 mL distilled water for SDA+extract (Lima et al., 2011).
Fragments of mycelium emerging from plant fragments were transferred to new PDA plates without chloramphenicol to obtain pure cultures for identification (Azevedo et al., 2010).

Identification of endophytic fungi
Fungal cultures were maintained at ambient temperature (22 to 25°C) under natural photoperiod for 14 days and then visually examined regarding macroscopic (morphology, size, mycelial and agar color) and microscopic (presence of spores or other reproductive structures) characteristics (Barnett and Hunter, 1998).Isolates with similar morphological characteristics were grouped into morphospecies.Each morphospecies is represented by several isolates, being an isolate representative of each selected for microscopic identification and antibacterial activity (Azevedo et al., 2010).

Antibacterial test
A fungus from each morphospecies was inoculated in PDA medium and incubated at 28°C for 14 days, and ten 5 × 5 mm plugs were inoculated into 20 mL potato dextrose broth (PD) incubated at 28°C, without agitation, for 14 days.Moreover, 2 mL of medium containing fungal metabolites were extracted by a liquid-liquid partition with ethyl acetate and solubilized in 300 µL dimethylsulfoxide 99.9%(DMSO) (Azevedo et al., 2010).
Pathogenic bacteria were cultured at 3°C for 4 to 6 h and their turbidity adjusted to 0.5 McFarland scale.Bacteria were inoculated into Petri dishes containing Muller-Hinton (MH) medium, deposited on these paper discs, and then 20 µL of endophytic fungal extracts and incubated at 37°C for 24 h.The endophytic extract that did not allow bacterial growth around the disc was considered as having antibacterial activity and the inhibition halos produced were measured in millimeters (CLSI, 2003).Antibacterial tests were done in triplicate.

Statistical analysis of data
The infection index (FI) was calculated from the relationship between the number of fragments from which the endophytic fungi emerged and the total number of fragments used in the experiment (Azevedo et al., 2010).
The relative frequency of isolation (RF) was calculated as the number of isolates of a species divided by the total number of isolates, being expressed as a percentage.
For the diversity analysis of the endophytic community of U. tomentosa, the number of dominant species was calculated by using the Simpson and Shannon indices.The formula for calculating the Simpson diversity index is 1 − Σ (pi) 2 .Shannon-Wiener diversity (H') = −Σ pi ln pi, where pi is the proportion of species colonization frequency in a sample.Equivalence of Eveness (E) was calculated by using the following formula: E = H / ln S, where S is the number of species in the sample (Bezerra et al., 2015).

Isolation and identification of endophytic fungi
A total of 170 isolates belonging to 101 morphospecies, including isolates from sterile mycelium, were obtained from leaves and stems of U. tomentosa (Table 1).Isolation frequency of endophytic fungi was 89.6%, being higher in leaves (93.7%) than in stem (85.6%).
More endophytes were recovered from leaves (54.12%) than stems (45.88%) (Table 1).This difference may be related to the anatomical characteristics of U. tomentosa, which is a liana vine with more stems than leaves, facilitating the entry of microorganisms by  stomata and leaf grooves, as well as some fungi with hyphal growth on the leaf surface (Wagner and Lewis, 2000).Among the total isolated species, 28.23% were Hyphomycetes, 21.18% were Coelomycetes, and 50.59% were sterile mycelium.Among the endophytic species, Penicillium (8.82%), Nigrospora (7.65%), Colletotrichum (7.06%), and sterile mycelium (50.59%) predominated.As specialists and isolated only once, Asterosporium, Aureobasidium, Botrytis, and Didymostilbe were observed.These fungi indicated an intimate relationship with this plant, which suggests a genotypic interaction between fungus and plant, which may depend exclusively on the plant for its survival (Malcolm et al., 2013).
The highest recovery rate of endophytic fungi of U. tomentosa may also be related to the variation in the used nutritional and environmental conditions (Huang et al., 2007;Putra et al., 2015).
A different genus of endophytic fungi was isolated.Some of them are common in tropical regions and are often isolated in this type of study, being called generalists.However, other endophytic fungi are not very frequent and are known as specialists.Those with a preference for a particular culture medium, tissue, and/or temperature were classified as specialists of this isolation condition (Toju et al., 2013).
Among the culture media used, the highest fungal recovery occurred in PDA regardless of the type of tissue used, with 54 isolates (31.76%), followed by SDA, with 45 isolates (26.47%) (Figure 1).Some fungal genera showed to be specialists in relation to the culture medium.Fusarium was isolated only in PDA medium, Botrytis and Didymostilbe in PDA+extract, and Guignardia, Asterosporium, and Aureobasidium in SDA, showing the need to use different nutritional sources to increase the recovery rate and richness of endophytic fungi.Studies on endophytic fungal isolation usually use only PDA medium (Hilarino et al., 2011;Katoch et al., 2014;Bezerra et al., 2015).
Another factor of relevance in this study was the isolation temperature.Lower temperatures, such as 18°C, allow the development of fastidious fungi (Souza Leite et al., 2013).However, for U. tomentosa, a temperature of 28°C provided the highest number of endophytes with 90 isolates (52.94%).
Some fungal genera also presented specificity to the isolation temperature, being isolated only at 18 or 28°C.Asterosporium and Aureobasidium were isolated only at 18°C, while Guignardia, Aspergillus, Botrytis, and Didymostilbe only at 28°C.Studies on endophytic fungi generally use temperatures between 25 and 28°C (Premalatha and Kalra, 2013;Campos et al., 2015;Ferreira et al., 2015).Some fungi were not identified due to the absence of reproductive structures, called sterile mycelium.In U. tomentosa, 86 isolates could not be identified, representing 50.59%.
The diversity of the endophytic community isolated from different U. tomentosa tissues was compared by using α-diversity indices.Simpson diversity of endophytic  fungi was the same for both tissues.Both the Shannon-Wiener diversity and Eveness indices were higher in the stem.Species richness was also higher in the stem (Table 2).

Antibacterial activity
Among the 98 endophytic fungal extracts selected for testing against pathogenic strains, 23 were positive against at least one of the tested pathogenic bacteria.
Five extracts were active against E. faecalis, two against S. aureus, four against K. pneumoniae, and 23 against E. coli (Table 3).Extracts from Penicillium spp. 2 (2.378), Penicillium spp. 4 (2.4055), and Fusarium spp. 1 (2.3952) showed antibacterial activity against gram-positive and gramnegative bacteria (S. aureus, E. coli, and K. pneumoniae)  reproductive structures.Any fungal extracts presented antibacterial activity against the four bacteria tested.Penicillium spp. is the most studied bioprospecting fungus since penicillin was discovered and produces several defense metabolites with several biological activities such as antibacterial and antifungal agents (Supaphon et al., 2013).Endophytic Penicillium was observed with antibacterial activity in other studies (Jouda et al., 2004;Padhi and Tayung, 2015).
Nigrospora has not been a fungus commonly reported as a producer of antibiotics.However, in this study, six morphospecies presented this activity and Nigrospora spp. 1 (2.3831) presented strong activity against E. coli.
Antimicrobial activity is frequently detected among species of the genera Fusarium and Phomopsis (Radić and Štrukelj, 2012), as confirmed in this study.

Conclusion
This study demonstrated the diversity of endophytic fungi in the medicinal species U. tomentosa as the first report of endophytic studies for this plant.Crude extracts prepared from endophytic fungi isolated from leaves and stems demonstrated in vitro antibacterial activity against gram-positive and gram-negative bacteria.

Figure 1 .
Figure 1.Endophytic fungal communities isolated from Uncaria tomentosa according to plant tissue, culture medium, and temperature.

Figure 2 .
Figure 2. Antimicrobial activity (inhibition halo, in cm 2 ) against gram-positive and gram-negative bacteria presented by endophytic fungi isolated from Uncaria tomentosa.Each value is expressed as the average of three independent.

Table 1 .
Number and relative frequency percentages of endophytic fungi isolated from Uncaria tomentosa according to plant tissue, culture medium, and temperature.

Table 2 .
Diversity indices of endophytic fungi from Uncaria tomentosa according to plant tissue, culture medium, and temperature.

Table 3 .
Antibiosis results of extract from endophytic fungi isolated from of U. tomentosa, which presented some activity against pathogenic strains.