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Endophytic fungi from Myrcia guianensis at the Brazilian Amazon: Distribution and bioactivity

Endophytic fungi from Myrcia guianensis at the Brazilian Amazon: Distribution and bioactivity Beneficial interactions between plants and microorganisms have been investigated under different ecological, physiological, biochemical, and genetic aspects. However, the systematic exploration of biomolecules with potential for biotechnological products from this interaction still is relatively scarce. Therefore, this study aimed the evaluation of the diversity and antimicrobial activity of the endophytic fungi obtained from roots, stems and leafs of Myrcia guianensis (Myrtaceae) from the Brazilian Amazon. 156 endophytic fungi were isolated and above 80% were identified by morpho- logical examination as belonging to the genera Pestalotiopsis, Phomopsis, Aspergillus, Xylaria, Nectria, Penicillium and Fusarium. Fermented broth of those fungi were assayed for antimicrobial activity and four inhibited the growth of Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Penicillium avellaneum. As the strain named MgRe2.2.3B (Nectria haematococca) had shown the most promising results against those pathogenic strains, its fermented broth was fractioned and only its two low polar fractions demonstrated to be active. Both fractions exhibited a minimum -1 bactericidal concentration of 50 g.mL against S. aureus and a minimum fungicidal concentration -1 of 100 g.mL against P. avellaneum. These results demonstrate the diversity of fungal genera in M. guianensis and the potential of these endophytic fungi for the production of new antibiotics. Key words: secondary metabolites, fungus/plant interaction, antibiosis, Amazonian endophytic fungi. asymptomatically in the apoplastic spaces or within the Introduction plant cells, at least during a significant part of their life cy- In the last two decades, the increasing number of sci cle (Petrini, 1991). A more recent definition considers entific studies involving endophytic fungi has allowed the endophytes to be any cultured or uncultured organism that development of their concept. One of the most accepted concepts is that endophytic fungi are those that live inhabits the interior of plant tissues and organs without Send correspondence to E.F. Banhos. Biology Program of the Federal University of Western Pará, Av. Marechal Rondon s/n, 68040-070, Santarém, PA, Brazil. E-mail: sandrobanhos@yahoo.com.br. 154 Banhos et al. causing damage to their host. These can be the type I, which understanding of endophyte communities and their poten- includes those microorganisms that do not produce external tial as new antibiotic sources. structures in the plant, or type II, those that produce exter- nal structures in the plant (Miller et.al, 2010). Materials and Methods The great interest in endophytic microorganisms co- mes from the perception that they occupy the same ecologi- Plant collection and isolation of the endophytic fungi cal niches of plant pathogens and therefore have great Three specimens of M. guianensis were collected in potential for use in biological control. The strict relation- April 2009, at 35 m distant from each other, in an Amazo- ship between endophytes and their hosts makes them natu- nian savanna area, located in the São Pedro community, ral candidates for use as agents of disease and insect control which belongs to the city of Santarém (Pará State), in the (Hallmann et al., 1997; Miller et.al, 2010). Over the last following coordinates: latitude 02°32’08.9” S and longi- years, other evidences have justified this interest emphasiz- tude 54°54’23.9” W, at an elevation of 19 meters relative to ing that endophytic fungi can be ecologically important to sea level. Their identifications were realized at INPA (Na- their host, sometimes giving them support, and other times tional Institute of Amazon Research) Herbarium, and a being the protagonists in fundamental processes of plant voucher specimen was deposited under the registration survival (Artursson et al., 2006; Yue et al., 2000). number 181913. The diversity of endophytic fungi that colonizes dif- Samples of roots, stems and leaves of the specimens ferent parts of the plants has promoted a wide variety of were washed with autoclaved distilled water, and stored in questions about the potential of these microorganisms as a sterile plastic bags at 6 °C. For the isolation process, the consequence of the various types of environments, such as samples were washed with detergent under tap water, frag- desert, tropical and arctic, in which these host species are mented into 10x12 cm pieces, and subjected to a sequence present (Rosa et al., 2009; Wali et al., 2008). The biotech- of submersions in different solutions in the following order nological potential of endophytic fungi is emphasized by and time: (i) for the leaves, 70% alcohol for 1 min; sodium the amount of scientific investigations in this area, showing hypochlorite 3% for 2.5 min, 70% alcohol for 30 seconds, that these microorganisms can produce a very large number and sterile distilled water for 2 min; (ii) for the roots and of compounds, many of which have biological activities of stems, 70% alcohol for 1 min; sodium hypochlorite 4% for interest, such as phytohormones (Vidal and Jaber 2009), 3 min, 70% alcohol for 30 seconds, and sterile distilled wa- antimalarial, antiviral (Isaka et al., 2007; Lehtonen et al., ter for 2 min (Souza et al., 2004). In this specific situation, 2006; Suryanarayanan et al., 2009), antioxidant, antileish- the water was plated and incubated at 26 °C as a control of manial (Cubilla-Rios et al., 2008; Khidir et al., 2010; sterilization procedure. Since the roots and stems fragments Schulz and Boyle, 2005), cytotoxic (Bashyal et al., 2005; were divided into cortex and bark for subsequent inocula- Davis et al., 2008), and antimicrobial (Aly et al., 2008; tion, the endophytes were isolated from five parts of the Schulz et al., 2002; Zhou et al., 2008) compounds. plants: leaves (L), stem cortex (S), stem bark (Sb), root cor- Thus, this type of research contributes to the elucida- tex (R) and root bark (Rb). tion of new fungal compounds that have potential applica- The plant material was cut into pieces of approxi- tions in the pharmaceutical industry. Furthermore, due to mately 5x5 mm and inoculated in Petri dishes (2 plates with the increase in poor soil and most adverse environment, 9 fragments for each tissue) containing PDA medium Amazonian plants can host fungi that may also contribute -1 added with chloramphenicol 50 g.mL . A total of 270 to the development of agribusiness sectors, through the dis- fragments were inoculated (45 fragments from each part of covery of new compounds for development of cultivars and the plant of the three specimens, in duplicate) and incubated to combat of pests, such as bacteria and pathogenic fungi. at 18 °C for 8 days, following by 4 days, at 26 °C. Accord- To this day, only a small number of studies on this ing to the cultivable endophytes that had been raised, they theme, focusing on species belonging to the Myrtaceae were transferred to test tubes containing inclined PDA me- family, was published, although this family has numerous dium. In order to identify the morphological characteristics species throughout Brazil, many of which are present in the of each isolate, they were inoculated individually into Petri Brazilian Amazon (Landrum and Kawasaki, 1997). The dishes and analyzed. choice of the host plant species is important for the isolation of endophytes of interest from the standpoint of the anti- The isolates were stored in duplicate into mineral oil microbial activity. In this sense, the family Myrtaceae is for the anamorphic fungi (Castellani, 1939); into 2 mL noted for producing a variety of compounds, some with microtubes containing 20% glycerol; and in Petri dishes proven antimicrobial activity (Cruz, 1982). Hence, the aim containing PDA medium, in duplicate, for sterile mycelia of this study was to isolate and evaluate the antimicrobial and Ascomycetes (telemorphic fungi). All isolated endo- activity of the endophytic fungi obtained from the roots, phytic fungi were deposited into the Collection of the Labo- stems and leafs of Myrcia guianensis, a common plant spe- ratory of the School of Health Sciences, State University of cies of the Brazilian Amazon, in order to contribute to the Amazonas (ESA/UEA). Endophytic fungi from Myrcia guianensis 155 Diversity analysis ing results was fractionated in an open column with normal phase silica (70-230 mesh) in a gradient mode. The mobile For the analyze of the diversity, the isolates were phases were: dichloromethane:ethyl acetate 1:1 (FA1), grouped according to their macro and micromorphological ethyl acetate 100% (FA2); ethyl acetate:acetone 1:1 (FA3), characteristics. They were identified by their macroscopic acetone 100% (FA4), 100% methanol (FA5) and metha- vegetative characteristics, which were color, texture, to- nol:water 8:2 (FA6). pography, diffuse pigmentation, color, and topography of the back of the colony, and well as by their microscopic re- Preliminary characterization of fungal metabolites productive structures, using the microculture technique and Chemical prospecting in ME and FBE extracts were comparing the obtained results with taxonomic keys (Bar- performed to observe the presence of four groups of com- nett and Hunter, 1972; Hanlin, 1996). pounds already found as having antimicrobial activity, The colonization rates (CR) of the fungi isolated were which can be present in the secondary metabolism of endo- achieved by the ratio between the total number of isolates phytic fungi (Yu et al., 2010). Phenols and tannis (reaction and the number of fragments within the sample, and the rel- with FeCl ), alkaloids (Hager, Mayer and Dragendorff re- ative frequency (RF) was based on the ratio of the total agents) and quinones (reaction with NH OH) were evalu- number of isolates of a group and the total number of iso- ated according to the method proposed by Matos (2009). lates. The microhabitats occupied by the isolated fungi (Azevedo, 1999) were also evaluated. For the statistical Qualitative analysis of the antimicrobial activity analysis of colonization rates, the Tukey test with p  0.05 The evaluation of the antimicrobial activity of the was used. crude fermentation broth was performed by the agar diffu- sion method (Souza et al., 2004). The fungal metabolites Fungal metabolites production were tested against standard strains obtained from the Col- For the fungal metabolic production, 20% of the iso- lection of Amazon Bacteria (CBAM) and from the Collec- lates from each morphological group were selected. These tion of Amazon Fungi (CFAM) of the Oswaldo Cruz Foun- strains were inoculated in triplicate into 50 mL Erlenmeyer dation, Manaus, Amazonas (FIOCRUZ-AM). The strains flasks containing 10 mL of potato dextrose liquid medium used were Staphylococcus aureus (CBAM-026), Bacillus (PD) added with 0.2% of yeast extract under sterile condi- cereus (CBAM-289), Enterococcus faecalis (CBAM-309) tions (Souza et al., 2004). The flasks were incubated into a Pseudomonas aeuriginosa (CBAM-024) and the fungal shaker during 8 to 11 days, according to the growth of each strains of Candida albicans (CFAM-1132) and Penicillium group, at 26 °C and 120 rpm. As negative control, flasks avellaneum. The fungus P. avellaneum is an species which containing only culture media were incubated under the has been used by many researchers as a target of anticancer same conditions. After the cultivation period, the crude fer- and antifungal compounds (Floss et al., 2004; Hanka and mentation broth was separated from the mycelium by vac- Barnett 1974; Kittakoop et al., 2009). uum filtration and sterilized by filtration through a 22 m Cell suspensions were prepared using the same media Millipore membrane. The crude fermentation broth was the microorganisms were inoculated in: Müller-Hinton for stored at 4 °C. bacteria and Sabouraud for the fungi strains. For the prepa- The fungal strain that had presented the most promis- ration of the inoculum suspension, a concentration of 10 ing results in the antimicrobial activity tests was grown for cfu for bacteria, and 10 cfu for fungi (fungal spore suspen- a preparative scale, in order to obtain a sufficient amount of sion) were standardized. An amount of 100 L from the cell the crude fermentation broth that would ensure the assess- suspension was applied and spread using a Drigalski spat- ment of the bioactive compound. At this stage, 75 Erlen- ula through a Petri dish (20x150 mm) containing specific meyer flasks (capacity 250 mL) containing 100 mL of PD solid medium: brain heart infusion (BHI) for bacteria and liquid medium were used. The methodology to obtain the Sabouraud for fungi. After that, 26 wells with 5 mm in di- metabolic medium was the same described above. ameter were placed on the culture medium of each plate and filled with 100 L of the crude fermentation broth. All Fungal metabolites extraction and fractioning plates were incubated under aerobic conditions for 24 and 72 h, at 28 °C for fungi and 37 °C for bacteria. After that, The mycelium was extracted three times with etha- the inhibition zones were measured. As positive control, nol, and after filtration the ethanolic mixture was concen- antibiotics were used: amoxicillin for bacteria, and keto- trated under vacuum, resulting in the mycelial extract -1 conazole for fungi, both at 2.0 mg.mL . As the negative (ME). The crude fermentation broth was partitioned with control, only the crude fermentation broth was used. ethyl acetate, and the organic extract was concentrated un- der reduced pressure, resulting in the fermentation broth Antimicrobial activities of extracts and fractions extract (FBE). The dried extracts (ME and FBE) were weighed and stored at 4 °C. After the assessment of anti- Extracts (ME and FBE) and fractions (FA1-FA6) microbial activity, the extract that showed the most promis- were tested against the same pathogenic strains as previ- 156 Banhos et al. ously described. On the case of positive results, new assays plants, the larger numbers of isolates were obtained from were done in order to determine the Minimum Inhibitory stem barks, 50 (32.1%) and leaves, 47 (30.1%). Root corti- Concentration (MIC), in accordance with the predetermi- ces provided the lowest number, with five fungi, totaling nation of the National Committee for Clinical Laboratory 3.2% of the isolates (Table 1). Standards (NCCLS), and using the broth microdilution Endophytic fungi diversity method, according to Cos and coworkers (2006). For these tests, the extracts and fractions were dissolved to All isolated fungi were assembled in 14 morphogroups, -1 2.0 mg.mL in DMSO:water (1:9), and 100 L of the solu- seven of known and seven of unknown genera. The genera tion were applied in ELISA plates in the serial dilution identified and their relative frequencies were: Pestalotiopsis mode. In each well, 20 L of the pathogenic organism sus- (33.3%), Phomopsis (25.0%), Aspergillus (11.5%), Xylaria pensions were inoculated. All tests were performed in trip- (5.1%), Penicillium (2.5%), Nectria (1.9%), Fusarium (0.6%) licate. The plates were incubated at 32 °C and after 24 and and Guignardia (0.6%). The relative frequencies of unidenti- 48 h the inhibition zone was measured in mm. fied groups were: Unidentified group 5 (5.1%), Unidentified Minimum Bactericidal Concentration (MBC) and group 6 (6.4%), Unidentified group 7 (2.5%), Unidentified Minimum Fungicidal Concentration (MFC) tests were also group 8 (1.9%), Unidentified group 10 (1.9%) and Unidenti- performed. Aliquots of the positive results observed for fied group 11 (1.2%) (Table 2). MIC tests were inoculated into Petri dishes containing ap- For strain MgRe2 3.3 that showed the best results in propriated culture media (Cos et al., 2006). The plates were the antimicrobial assays it was obtained DNA sequences, incubated at 37 °C for 24-48 h. The MBC or MFC was con- with 539 base pairs, which were compared with the NCBI sidered the lowest concentration of the fraction in which database. The pairwise comparisons revealed trustfully was observed no cell growth on the surface of the inocu- identity with 99% of Nectria haematococca. This result lated culture medium. was confronted with the morphological analyzes, and con- firmed using specialized bibliography (Grafenhan et al., Identification of the most promising strain 2011; Hanlin, 1996). The identification of the most promising strain was Antimicrobial activity confirmed by sequencing of the fungus ITS-1 and ITS-2 rDNA and compared with sequences from the GenBank. From the crude fermentation broth of the 46 endo- The strain was stirred for 6 days (120 rpm) on potato- phytic fungi selected for the tests against the pathogenic dextrose (PD) medium at room temperature. From the my- strains, three were positive against S. aureus, and E. celium separated by filtration, the genomic DNA was ex- faecalis, one against C. albicans and and two against P. tracted by the CTAB method (White et al., 1990) as avellaneum. None of the isolated fungi was active against adaptation of Souza and coworkers (2008). P. aeruginosa B. cereus (Table 3). 42 did not show any ac- tivity against the pathogens tested. Results Nectria haematococca: Extracts, fractions, MIC and MBC Isolation of endophytic fungi From the 270 inoculated fragments of M. guianensis, Antibiosis sensitivity tests performed with the myce- 156 endophytic fungi were isolated. Of these, 53 were iso- lia - ME (0.76 g) - and with the fermentation broth extracts - lated from the specimen 1, 54 from the specimen 2 and 49 FBE (3.58 g) - of this strain confirmed the presence of from the specimen 3 (Table 1). Considering the parts of the bioactive substances with inhibitory effect against S. Table 1 - Total of fungi isolated and colonization rates in each tissue of Myrcia guianensis. Plant parts Specimen 1 Specimen 2 Specimen 3 Averages n° of isolates CR n° of isolates CR n° of isolates CR n° of isolates CR L 15 0.166 19 0.211 13 0.144 15.66 0.173 R _ _ 3 0.033 2 0.022 1.66 0.018 Rb 10 0.111 9 0.100 11 0.122 10.00 0.111 S 11 0.122 7 0.077 6 0.066 8.00 0.088 Sb 17 0.188 16 0.177 17 0.188 16.66 0.184 Total 53 0.196 54 0.200 49 0.181 52.00 0.192 For the isolation it was used a total of 90 fragments from each specimen of M. guianensis. L – Leaves; R – Root Cortex; Rb – Root Bark; S – Stem Cortex; Sb – Stem Bark; CR – colonization rate. Averages with a same letter indicate no significant difference according to the Tukey test (p < 0.05). Endophytic fungi from Myrcia guianensis 157 Table 2 - Morphological groups and relative frequencies of endophytic fungi isolated from Myrcia guianensis. Morphogroup Specimen 1 Specimen 2 Specimen 3 Total RF (%) LR S T LR S T LR S T Pestalotiopsis 64 11 21 1258 869 23 52 33.3 Phomopsis 367 16 546 15 2248 39 25.0 Aspergillus 2125 2136 –167 18 11.5 Xylaria 1–12 1–34 11–28 5.1 Unknown 6 1–23 1–12 2–138 5.1 Unknown 7 –––– 4–48 1–12 10 6.4 Unknown 8 –––– 1124 ––––4 2.5 Unknown 9 1––1 1–12 ––––3 1.9 Penicillium ––11 ––22 ––114 2.5 Unknown 10 2––2 –1–1 ––––3 1.9 Unknown 11 –––– –1–1 –1–12 1.2 Nectria ––11 ––11 ––113 1.9 Fusarium –1–1 –––– ––––1 0.6 Guignardia –––– –––– 1––11 0.6 Total 16 12 24 53 16 10 28 54 15 11 24 49 156 53.3 L – leaves; R – root; S – stem; T – total of each specimen. Table 3 - Antibiosis results of the crude fermentation broth from endophytic fungi isolated from of M. guianensis which presented some activity against the pathogenic strains. Endophytic fungi Genera Tested microorganisms Strains Pa Sa Ef Bc Ca Pv MgF2.1.2 Pestalotiopsis –++ ++– – – MgF1.2.1 Phomopsis –++ ++– – – MgR2.1.1 Unknown 10 –––– ++ ++ MgRe2.2.3B Nectria – ++ + + – – +++ Negative control amoxicilin ketoconazole –––––– +++ ++ +++ +++ NT NT NT NT NT NT + + + + + + Pa - Pseudomonas auriginosa;Sa- Staphylococcus aureus;Ef - Enterococcus faecalis;Bc- Bacillus cereus;Ca- Candida albicans;Pv - Penicillium avellaneum; NT – Not tested. + Inhibition zone between 10 and 15 mm; + + Inhibition zone between 15 and 30 mm;+++ Inhibition zone between 30 and 45 mm. aureus, E. faecalis and P. avellaneum only in the FBE. Discussion Negative results were observed for ME (Table 4). The frac- Isolation of endophytic fungi tions FA1 (0.35 g), FA2 (0.52 g), FA3 (0.11 g), FA4 (0.09 g), FA5 (1.32 g) and FA6 (0.19 g) obtained from the The results showed that M. guianensis is a good chromatographic fractioning of FBE were tested against source of endophytic fungi, since only one type of culture the same pathogenic strains. FA1 and FA2 showed positive medium was used for the isolation process, and this unique result against the tested strains (Table 4). method allowed the acquirement of a considerable number of endophytes (totaling 57.7% of the inoculated frag- ments). No microorganism had appeared from the last Preliminary characterization of fungal metabolites washing water, so the surface disinfection method was con- Qualitative analysis of the compounds present in the sidered efficient. ME and FBE showed the presence of phenols in both extracts. The three specimens of M. guianensis presented simi- The presence of tannins was found only in the ME, and the lar relative distribution of isolated fungi among all parts of presence of quinones was observed only in the FBE. It was not the plant. While someone could expect the higher numbers observed the presence of alkaloids in any of the extracts. of isolates in the leaves and stem barks (Table 1), a smaller 158 Banhos et al. amount of fungi in the roots was somewhat unexpected, since as this tissue is in contact with the ground, which is a source of numerous microorganisms. However, several works have presented the leaves and stems as main parts where endophytes are found (Gazis and Chaverri, 2010; Suryanarayanan et al., 2009), which may be related to how the endophyte penetrates the plant (by its aerial interac- tions), and also to the favorable conditions of certain tissues for the fungus protection (Ahlholm, 2002; Brand and Gow, 2009). On the other hand, the similarity among the results for M. guianensis specimens suggests that the isolation pro- cess is expressing the real number of cultivable endophytic fungi present in the healthy tissues. Besides, almost every isolated genus presented a relative frequency greater than 1, except Guignardia, known as an orange’s pathogen (Spó- sito et al., 2011) and Fusarium, the agent that causes the addlement of the roots (Yu et al., 2004), both cases recog- nized as a systemic colonization (Table 2). In a study performed by Pinto (2011), in the isolation of endophytic fungi from leaves and stems of M. sellowiana, a lower frequency of isolates (12.7%) was ob- served when compared to the findings presented here (53.3%), as the treatment leaves in the disinfection step was the same surface, and the medium used for isolation also (BDA), the discrepancy in results may be related to season- ality of these microorganisms in the host, considering that the collection of plant material for the isolation occurred at different times in the year 2009, during the rainy season and the other during the dry season (Pinto, 2011). The statistical analyses of the averages of the fungi isolated in each tissue showed that there is no significant difference (p  0.05) between the number of isolates from the stem barks (Sb) and from the leaves (L). The same was observed for the number of isolates from the root barks (Rb) and from the stem cortices (S). On the other hand, there is a notable difference between the averages of colo- nization rate (CR) in the roots cortices (0.018), and in the stem barks (0.184). Clearly, for the conditions applied, the amount of cultivable isolates in the stem barks is ten times greater than in the root cortices, showing the importance of the plant fragmentation for the fungal isolation. Since many fungal species are not cultivable, in order to analyze the ef- ficiency of the endophyte isolation, it is necessary to re- member that these numbers just express the frequency of cultivable endophytes under determined isolation condi- tions, such as superficial disinfection, culture medium and growth temperature. Endophytic fungal diversity It was found a greater diversity of genera in the speci- men 2, than in 1 and 3. In contrast, specimens 1 and 3 pre- sented more isolates of the genus Pestalotiopsis (Table 2). Some of the most frequent isolates of M. guianensis,as Pestalotiopsis and Phomopsis are known as endophytes of other plant species from tropical climate, and some have Table 4 - Antibiosis activities of the extracts and fractions from the fermentation broth of the fungus Nectria haematococca cultivated in preparative scale. Tested strains Extracts Fractions Control FA1 FA2 ME FBE FA1 FA2 FA3-6 CON AMO KET MIC MBC MIC MBC -1 -1 -1 -1 (g.mL ) (g.mL ) (g.mL ) (g.mL ) S. aureus – –– NT 23.3  1.1 12.3  0.5 12.3  0.5 28.1  0.28  25  50  25  50 E. faecalis – –– NT – – 24.3  2.0 11.3  0.5 11.6  0.5 46.0  1.00  50  50 P. avellaneum – –– NT 22.3  2.0 23.3  1.1 21.3  0.5 43.3  0.6  12.5  100  12.5  100 ME – Ethanolic extract from the mycelium; FBE – ethyl acetate extract from the fermentation broth; FA1 – fraction from FBE obtained in dichloromethane:ethyl acetate (1:1); FA2 – fraction from FBE obtained in ethyl acetate 100%; FA3 – fraction from FBE obtained in ethyl acetate: acetone (1:1); FA4 – fraction from FBE obtained in acetone 100%; FA5 – fraction from FBE obtained in methanol 100%; FA6 – fraction -1 -1 from FBE obtained in methanol:water (8:2); AMO – Amoxicilin 2.0 mg.mL ; KET – ketoconazole 2.0 mg.mL ; CON – negative control DMSO: water (1:9); MIC – minimum inhibitory concentration; MBC – minimum bactericidal concentration; NT – not tested. Endophytic fungi from Myrcia guianensis 159 demonstrated significant potential for the production of Antimicrobial activity useful biotechnological compounds, such as isopestacin Even though only some of the isolated endophytes isolated from P. microspora. This compound showed anti- have been used in the analyses of the antimicrobial activity, fungal activity against Pythium ultimum, an oomycete that which reduced the possibility of finding strains that pro- causes the addlement of the roots in plants of agricultural duce bioactive compounds, and considering that they have importance (Strobel et al., 2002). been cultivated in only one culture medium, which cannot be suitable for gene expression that led to the production of Another example is the lactones extracted from the some bioactive metabolites, it was gratifying that the strain crude fermentation broth of Phomopsis sp., an endophytic MgRe2.2.3B, belonging to the genus Nectria, had shown fungus from Azadirachta indica. A recent work it was de- activity against S. aureus, E. faecalis and P. avellaneum,a scribed the antifungal action of these lactones against the signal of its great potential as a source of antibacterial and important plant pathogen, Ophiostoma minus, with a mini- -1 antifungal compounds. It should be pointed out that the re- mum inhibitory concentration (MIC) of 31.25 g.mL (Wu sult of the antibiosis test of the crude fermented broth of this et al., 2008). fungus against P. avellaneum was similar to the positive Regarding the diversity of endophytes, it is impor- control ketoconazole. This fact supported the selection of tant to note that the identified groups at the genus level this strain for subsequent assays, as well as for its molecular were also found in other studies concerning tropical identification. plants from the Amazon region (Hanada et al., 2010; Extracts, fractions, MIC and MBC Souza et al., 2008). This is the case of the genera Pestalotiopsis, Phomopsis, Aspergillus, Penicillium and The test results indicate that the active compounds are Xylaria, which demonstrates the versatility of these among the substances present in the fractions 1 and 2. A endophytic microorganisms in colonize diverse host spe- -1 MIC of 25 g.mL was obtained for FA1 and FA2 against cies. In this sense, it is important to consider that fre- -1 S. aureus and of 12 g.mL against P. avellaneum. Against quencies under 1% indicate genera not common in the -1 E. faecalis the obtained MIC was 50 g.mL for fraction 1 plant’s tissues. In some cases, those indicate that these -1 and 100 g.mL for fraction 2 (Table 4). The minimum fungi may not be a natural endophyte, but an epiphyte or bactericidal concentration (MBC) results were found at even a plant pathogen trying to colonize the plant tissue -1 50 g.mL for both FA1 and FA2 against S. aureus. The transitorily. In this work, it was possible to observe the fractions did not present bactericidal capacity against E. presence of two plant’s pathogen genera, Fusarium and faecalis in any of the tested concentrations. FA1 and FA2 Guignardia (Spósito et al., 2011; Yu, et al., 2004), both showed fungicidal ability against P. avellaneum, but this found at a frequency of 0.6%, indicating its momentarily -1 capacity is only observed at a dose of 100 g.mL .Al- colonization. though the most promising results for the antimicrobial ac- Plants of Myrcia genus appear as a promising source tivity were obtained against the P. avellaneum strain, of endophytic microorganisms, which produce metabolites different results were observed during the fungicidal tests. with proven antimicrobial activity. Pinto (2011) evaluated These findings demonstrate the importance of such tests, the antimicrobial activity of extracts from endophytic fungi since this evaluation confirms or denies the effective action isolated from M. sellowiana, and found 62 extracts active of the investigated compound against pathogenic microor- against S. aureus, 20 against E. coli and 11 against both ganisms. pathogens (Pinto, 2011). In addition to that, the author found 35 endophytic fungi strains that produced metabo- Preliminary characterization of fungal metabolites lites which are active against the pathogenic fungus The production of phenols and quinones by endo- Colletotrichum gloeosporioides. These findings justify the phytic fungi and antimicrobial activity of these compounds investigation among this vegetal genus as a source of anti- are fairly known. In a study carried out by Li and coworkers microbial producing endophytic fungi and corroborate the (2008), two new antimicrobial substances (pestalachloride results presented in this work. A and B) were isolated, which are phenolic compounds produced by the endophytic fungus Pestalotiopsis adusta In this work it was also possible to verify the isolation (Li et al., 2008). In another work, it was observed the pro- of a fungi from the genus Nectria, a known producer of pig- duction of quinones by Ampelomyces sp., an endophytic ments such as anthraquinone and naphthoquinone (Barbier fungus isolated from Urospermum picroides, with signifi- et al., 1988; Barbier, et al., 1990). The genus Nectria had cant activity against the pathogenic bacteria S. aureus, S. not been previously described as endophytic fungi from epidermidis and E. faecalis (Aly et al., 2008). Amazon hosts, or as a producer of antimicrobial com- pounds. Hence, the results obtained here demonstrate the Antimicrobial activity of compounds produced by limited knowledge of fungal species that comprise the Am- Nectria haematococca has not yet been described, but it is azon ecosystem. already known that this genus is a producer of a variety of 160 Banhos et al. Bashyal BP, Wijeratne EMK, Faeth SH, Gunatilaka AAL (2005) quinones, a class of compounds that could be identified in Globosumones A-C, Cytotoxic Orsellinic Acid Esters from the N. haematococca extract using a qualitative assay. Such the Sonoran Desert Endophytic Fungus Chaetomium information expands the possibilities of research about this globosum. J Nat Prod 68:724-728. genus and its secondary metabolites, in this case resulting Brand A, Gow NA (2009) Mechanisms of hipha orientation of in a biotechnological perspective toward obtaining new an- fungi. Curr Opin Microbiol 12:350-357. tibiotics. Castellani A (1939) Viability of mold culture of fungi in distilled water. J Trop Med Hyg 42:225. Final Remarks Cos P, Vlietinck AJ, Berghe DV, Maes L (2006) Anti-infective The Amazonian plant M. guianensis proved to be a potential of natural products: How to develop a stronger in valuable source of endophytic fungi, both in terms of mi- vitro `proof-of-concept’. J Ethnopharmacol 106:290-302. Cruz GL (1982) Dictionary of useful plants in Brazil. Civilização croorganisms number as in diversity, being detected at least Brasileira, Rio de Janeiro, Brazil. 14 distinct morphogroups. It was possible to identify some Cubilla-Rios L, Della-Togna G, Coley PD, Kursar TA, Gerwick known genera already described as producers of com- WH (2008) Antileishmanial Constituents of the Panamanian pounds with biotechnological interest. A fungus from the Endophytic Fungus Edenia sp. J Nat Prod 71:2011-2014. genus Nectria, which was not previously identified in the Davis RA, Longden J, Avery VM, Healy PC (2008) The isolation, Amazon region, was also described in this work. The re- structure determination and cytotoxicity of the new fungal sults of the antimicrobial tests demonstrate the production metabolite, trichodermamide C. Bioorg Med Chem of bioactive compounds from the isolated microorganisms, 18:2836-2839. both antifungal and antibacterial in some endophytic Floss HG, Cassady JM, Chan KK, Leistner E (2004) Recent De- strains. The bioguided fractioning of an active extract from velopments in the Maytansinoid Antitumor Agents. Chem Pharm Bull 52:1-26. the fungus Nectria haematococca yielded positive results Gazis R, Chaverri P (2010) Diversity of fungal endophytes in in the fractions of the crude fermentation broth which pres- leaves and stems of wild rubber trees (Hevea brasiliensis)in ent quinones and phenols as chemical constituents. Peru. Fungal Ecol 3:240-254. 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Tetrahedron 63:6855-6860. arbuscular micorrhizal fungi and bacteria and their potential Khidir HH, Eudy DM, Porras-Alfaro A, Herrera J, Natvig DO, for stimulating plant growth. Environ Microbiol 8:1-10. Sinsabaugh RL (2010) A general suite of fungal endophytes Azevedo JL (1999) Microrganismos endofíticos. In: Melo, I.S.; dominate the roots of two dominant grasses in a semiarid Azevedo, J.L. (eds). Ecologia Microbiana. EMBRAPA- CNPMA, Jaguariuma, Brazil, 117-137. grassland. J Arid Environ 74:35-42. Barbier M, Parisot D, Devys M (1988) Fusarubinoic acid, a new Kittakoop P, Chomcheon P, Wiyakrutta S, Sriubolmas N, naphthoquinone from the fungus Nectria haematococca. 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Endophytic fungi from Myrcia guianensis at the Brazilian Amazon: Distribution and bioactivity

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Pubmed Central
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Copyright © 2014, Sociedade Brasileira de Microbiologia
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1517-8382
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1678-4405
DOI
10.1590/S1517-83822014005000027
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Abstract

Beneficial interactions between plants and microorganisms have been investigated under different ecological, physiological, biochemical, and genetic aspects. However, the systematic exploration of biomolecules with potential for biotechnological products from this interaction still is relatively scarce. Therefore, this study aimed the evaluation of the diversity and antimicrobial activity of the endophytic fungi obtained from roots, stems and leafs of Myrcia guianensis (Myrtaceae) from the Brazilian Amazon. 156 endophytic fungi were isolated and above 80% were identified by morpho- logical examination as belonging to the genera Pestalotiopsis, Phomopsis, Aspergillus, Xylaria, Nectria, Penicillium and Fusarium. Fermented broth of those fungi were assayed for antimicrobial activity and four inhibited the growth of Staphylococcus aureus, Enterococcus faecalis, Candida albicans and Penicillium avellaneum. As the strain named MgRe2.2.3B (Nectria haematococca) had shown the most promising results against those pathogenic strains, its fermented broth was fractioned and only its two low polar fractions demonstrated to be active. Both fractions exhibited a minimum -1 bactericidal concentration of 50 g.mL against S. aureus and a minimum fungicidal concentration -1 of 100 g.mL against P. avellaneum. These results demonstrate the diversity of fungal genera in M. guianensis and the potential of these endophytic fungi for the production of new antibiotics. Key words: secondary metabolites, fungus/plant interaction, antibiosis, Amazonian endophytic fungi. asymptomatically in the apoplastic spaces or within the Introduction plant cells, at least during a significant part of their life cy- In the last two decades, the increasing number of sci cle (Petrini, 1991). A more recent definition considers entific studies involving endophytic fungi has allowed the endophytes to be any cultured or uncultured organism that development of their concept. One of the most accepted concepts is that endophytic fungi are those that live inhabits the interior of plant tissues and organs without Send correspondence to E.F. Banhos. Biology Program of the Federal University of Western Pará, Av. Marechal Rondon s/n, 68040-070, Santarém, PA, Brazil. E-mail: sandrobanhos@yahoo.com.br. 154 Banhos et al. causing damage to their host. These can be the type I, which understanding of endophyte communities and their poten- includes those microorganisms that do not produce external tial as new antibiotic sources. structures in the plant, or type II, those that produce exter- nal structures in the plant (Miller et.al, 2010). Materials and Methods The great interest in endophytic microorganisms co- mes from the perception that they occupy the same ecologi- Plant collection and isolation of the endophytic fungi cal niches of plant pathogens and therefore have great Three specimens of M. guianensis were collected in potential for use in biological control. The strict relation- April 2009, at 35 m distant from each other, in an Amazo- ship between endophytes and their hosts makes them natu- nian savanna area, located in the São Pedro community, ral candidates for use as agents of disease and insect control which belongs to the city of Santarém (Pará State), in the (Hallmann et al., 1997; Miller et.al, 2010). Over the last following coordinates: latitude 02°32’08.9” S and longi- years, other evidences have justified this interest emphasiz- tude 54°54’23.9” W, at an elevation of 19 meters relative to ing that endophytic fungi can be ecologically important to sea level. Their identifications were realized at INPA (Na- their host, sometimes giving them support, and other times tional Institute of Amazon Research) Herbarium, and a being the protagonists in fundamental processes of plant voucher specimen was deposited under the registration survival (Artursson et al., 2006; Yue et al., 2000). number 181913. The diversity of endophytic fungi that colonizes dif- Samples of roots, stems and leaves of the specimens ferent parts of the plants has promoted a wide variety of were washed with autoclaved distilled water, and stored in questions about the potential of these microorganisms as a sterile plastic bags at 6 °C. For the isolation process, the consequence of the various types of environments, such as samples were washed with detergent under tap water, frag- desert, tropical and arctic, in which these host species are mented into 10x12 cm pieces, and subjected to a sequence present (Rosa et al., 2009; Wali et al., 2008). The biotech- of submersions in different solutions in the following order nological potential of endophytic fungi is emphasized by and time: (i) for the leaves, 70% alcohol for 1 min; sodium the amount of scientific investigations in this area, showing hypochlorite 3% for 2.5 min, 70% alcohol for 30 seconds, that these microorganisms can produce a very large number and sterile distilled water for 2 min; (ii) for the roots and of compounds, many of which have biological activities of stems, 70% alcohol for 1 min; sodium hypochlorite 4% for interest, such as phytohormones (Vidal and Jaber 2009), 3 min, 70% alcohol for 30 seconds, and sterile distilled wa- antimalarial, antiviral (Isaka et al., 2007; Lehtonen et al., ter for 2 min (Souza et al., 2004). In this specific situation, 2006; Suryanarayanan et al., 2009), antioxidant, antileish- the water was plated and incubated at 26 °C as a control of manial (Cubilla-Rios et al., 2008; Khidir et al., 2010; sterilization procedure. Since the roots and stems fragments Schulz and Boyle, 2005), cytotoxic (Bashyal et al., 2005; were divided into cortex and bark for subsequent inocula- Davis et al., 2008), and antimicrobial (Aly et al., 2008; tion, the endophytes were isolated from five parts of the Schulz et al., 2002; Zhou et al., 2008) compounds. plants: leaves (L), stem cortex (S), stem bark (Sb), root cor- Thus, this type of research contributes to the elucida- tex (R) and root bark (Rb). tion of new fungal compounds that have potential applica- The plant material was cut into pieces of approxi- tions in the pharmaceutical industry. Furthermore, due to mately 5x5 mm and inoculated in Petri dishes (2 plates with the increase in poor soil and most adverse environment, 9 fragments for each tissue) containing PDA medium Amazonian plants can host fungi that may also contribute -1 added with chloramphenicol 50 g.mL . A total of 270 to the development of agribusiness sectors, through the dis- fragments were inoculated (45 fragments from each part of covery of new compounds for development of cultivars and the plant of the three specimens, in duplicate) and incubated to combat of pests, such as bacteria and pathogenic fungi. at 18 °C for 8 days, following by 4 days, at 26 °C. Accord- To this day, only a small number of studies on this ing to the cultivable endophytes that had been raised, they theme, focusing on species belonging to the Myrtaceae were transferred to test tubes containing inclined PDA me- family, was published, although this family has numerous dium. In order to identify the morphological characteristics species throughout Brazil, many of which are present in the of each isolate, they were inoculated individually into Petri Brazilian Amazon (Landrum and Kawasaki, 1997). The dishes and analyzed. choice of the host plant species is important for the isolation of endophytes of interest from the standpoint of the anti- The isolates were stored in duplicate into mineral oil microbial activity. In this sense, the family Myrtaceae is for the anamorphic fungi (Castellani, 1939); into 2 mL noted for producing a variety of compounds, some with microtubes containing 20% glycerol; and in Petri dishes proven antimicrobial activity (Cruz, 1982). Hence, the aim containing PDA medium, in duplicate, for sterile mycelia of this study was to isolate and evaluate the antimicrobial and Ascomycetes (telemorphic fungi). All isolated endo- activity of the endophytic fungi obtained from the roots, phytic fungi were deposited into the Collection of the Labo- stems and leafs of Myrcia guianensis, a common plant spe- ratory of the School of Health Sciences, State University of cies of the Brazilian Amazon, in order to contribute to the Amazonas (ESA/UEA). Endophytic fungi from Myrcia guianensis 155 Diversity analysis ing results was fractionated in an open column with normal phase silica (70-230 mesh) in a gradient mode. The mobile For the analyze of the diversity, the isolates were phases were: dichloromethane:ethyl acetate 1:1 (FA1), grouped according to their macro and micromorphological ethyl acetate 100% (FA2); ethyl acetate:acetone 1:1 (FA3), characteristics. They were identified by their macroscopic acetone 100% (FA4), 100% methanol (FA5) and metha- vegetative characteristics, which were color, texture, to- nol:water 8:2 (FA6). pography, diffuse pigmentation, color, and topography of the back of the colony, and well as by their microscopic re- Preliminary characterization of fungal metabolites productive structures, using the microculture technique and Chemical prospecting in ME and FBE extracts were comparing the obtained results with taxonomic keys (Bar- performed to observe the presence of four groups of com- nett and Hunter, 1972; Hanlin, 1996). pounds already found as having antimicrobial activity, The colonization rates (CR) of the fungi isolated were which can be present in the secondary metabolism of endo- achieved by the ratio between the total number of isolates phytic fungi (Yu et al., 2010). Phenols and tannis (reaction and the number of fragments within the sample, and the rel- with FeCl ), alkaloids (Hager, Mayer and Dragendorff re- ative frequency (RF) was based on the ratio of the total agents) and quinones (reaction with NH OH) were evalu- number of isolates of a group and the total number of iso- ated according to the method proposed by Matos (2009). lates. The microhabitats occupied by the isolated fungi (Azevedo, 1999) were also evaluated. For the statistical Qualitative analysis of the antimicrobial activity analysis of colonization rates, the Tukey test with p  0.05 The evaluation of the antimicrobial activity of the was used. crude fermentation broth was performed by the agar diffu- sion method (Souza et al., 2004). The fungal metabolites Fungal metabolites production were tested against standard strains obtained from the Col- For the fungal metabolic production, 20% of the iso- lection of Amazon Bacteria (CBAM) and from the Collec- lates from each morphological group were selected. These tion of Amazon Fungi (CFAM) of the Oswaldo Cruz Foun- strains were inoculated in triplicate into 50 mL Erlenmeyer dation, Manaus, Amazonas (FIOCRUZ-AM). The strains flasks containing 10 mL of potato dextrose liquid medium used were Staphylococcus aureus (CBAM-026), Bacillus (PD) added with 0.2% of yeast extract under sterile condi- cereus (CBAM-289), Enterococcus faecalis (CBAM-309) tions (Souza et al., 2004). The flasks were incubated into a Pseudomonas aeuriginosa (CBAM-024) and the fungal shaker during 8 to 11 days, according to the growth of each strains of Candida albicans (CFAM-1132) and Penicillium group, at 26 °C and 120 rpm. As negative control, flasks avellaneum. The fungus P. avellaneum is an species which containing only culture media were incubated under the has been used by many researchers as a target of anticancer same conditions. After the cultivation period, the crude fer- and antifungal compounds (Floss et al., 2004; Hanka and mentation broth was separated from the mycelium by vac- Barnett 1974; Kittakoop et al., 2009). uum filtration and sterilized by filtration through a 22 m Cell suspensions were prepared using the same media Millipore membrane. The crude fermentation broth was the microorganisms were inoculated in: Müller-Hinton for stored at 4 °C. bacteria and Sabouraud for the fungi strains. For the prepa- The fungal strain that had presented the most promis- ration of the inoculum suspension, a concentration of 10 ing results in the antimicrobial activity tests was grown for cfu for bacteria, and 10 cfu for fungi (fungal spore suspen- a preparative scale, in order to obtain a sufficient amount of sion) were standardized. An amount of 100 L from the cell the crude fermentation broth that would ensure the assess- suspension was applied and spread using a Drigalski spat- ment of the bioactive compound. At this stage, 75 Erlen- ula through a Petri dish (20x150 mm) containing specific meyer flasks (capacity 250 mL) containing 100 mL of PD solid medium: brain heart infusion (BHI) for bacteria and liquid medium were used. The methodology to obtain the Sabouraud for fungi. After that, 26 wells with 5 mm in di- metabolic medium was the same described above. ameter were placed on the culture medium of each plate and filled with 100 L of the crude fermentation broth. All Fungal metabolites extraction and fractioning plates were incubated under aerobic conditions for 24 and 72 h, at 28 °C for fungi and 37 °C for bacteria. After that, The mycelium was extracted three times with etha- the inhibition zones were measured. As positive control, nol, and after filtration the ethanolic mixture was concen- antibiotics were used: amoxicillin for bacteria, and keto- trated under vacuum, resulting in the mycelial extract -1 conazole for fungi, both at 2.0 mg.mL . As the negative (ME). The crude fermentation broth was partitioned with control, only the crude fermentation broth was used. ethyl acetate, and the organic extract was concentrated un- der reduced pressure, resulting in the fermentation broth Antimicrobial activities of extracts and fractions extract (FBE). The dried extracts (ME and FBE) were weighed and stored at 4 °C. After the assessment of anti- Extracts (ME and FBE) and fractions (FA1-FA6) microbial activity, the extract that showed the most promis- were tested against the same pathogenic strains as previ- 156 Banhos et al. ously described. On the case of positive results, new assays plants, the larger numbers of isolates were obtained from were done in order to determine the Minimum Inhibitory stem barks, 50 (32.1%) and leaves, 47 (30.1%). Root corti- Concentration (MIC), in accordance with the predetermi- ces provided the lowest number, with five fungi, totaling nation of the National Committee for Clinical Laboratory 3.2% of the isolates (Table 1). Standards (NCCLS), and using the broth microdilution Endophytic fungi diversity method, according to Cos and coworkers (2006). For these tests, the extracts and fractions were dissolved to All isolated fungi were assembled in 14 morphogroups, -1 2.0 mg.mL in DMSO:water (1:9), and 100 L of the solu- seven of known and seven of unknown genera. The genera tion were applied in ELISA plates in the serial dilution identified and their relative frequencies were: Pestalotiopsis mode. In each well, 20 L of the pathogenic organism sus- (33.3%), Phomopsis (25.0%), Aspergillus (11.5%), Xylaria pensions were inoculated. All tests were performed in trip- (5.1%), Penicillium (2.5%), Nectria (1.9%), Fusarium (0.6%) licate. The plates were incubated at 32 °C and after 24 and and Guignardia (0.6%). The relative frequencies of unidenti- 48 h the inhibition zone was measured in mm. fied groups were: Unidentified group 5 (5.1%), Unidentified Minimum Bactericidal Concentration (MBC) and group 6 (6.4%), Unidentified group 7 (2.5%), Unidentified Minimum Fungicidal Concentration (MFC) tests were also group 8 (1.9%), Unidentified group 10 (1.9%) and Unidenti- performed. Aliquots of the positive results observed for fied group 11 (1.2%) (Table 2). MIC tests were inoculated into Petri dishes containing ap- For strain MgRe2 3.3 that showed the best results in propriated culture media (Cos et al., 2006). The plates were the antimicrobial assays it was obtained DNA sequences, incubated at 37 °C for 24-48 h. The MBC or MFC was con- with 539 base pairs, which were compared with the NCBI sidered the lowest concentration of the fraction in which database. The pairwise comparisons revealed trustfully was observed no cell growth on the surface of the inocu- identity with 99% of Nectria haematococca. This result lated culture medium. was confronted with the morphological analyzes, and con- firmed using specialized bibliography (Grafenhan et al., Identification of the most promising strain 2011; Hanlin, 1996). The identification of the most promising strain was Antimicrobial activity confirmed by sequencing of the fungus ITS-1 and ITS-2 rDNA and compared with sequences from the GenBank. From the crude fermentation broth of the 46 endo- The strain was stirred for 6 days (120 rpm) on potato- phytic fungi selected for the tests against the pathogenic dextrose (PD) medium at room temperature. From the my- strains, three were positive against S. aureus, and E. celium separated by filtration, the genomic DNA was ex- faecalis, one against C. albicans and and two against P. tracted by the CTAB method (White et al., 1990) as avellaneum. None of the isolated fungi was active against adaptation of Souza and coworkers (2008). P. aeruginosa B. cereus (Table 3). 42 did not show any ac- tivity against the pathogens tested. Results Nectria haematococca: Extracts, fractions, MIC and MBC Isolation of endophytic fungi From the 270 inoculated fragments of M. guianensis, Antibiosis sensitivity tests performed with the myce- 156 endophytic fungi were isolated. Of these, 53 were iso- lia - ME (0.76 g) - and with the fermentation broth extracts - lated from the specimen 1, 54 from the specimen 2 and 49 FBE (3.58 g) - of this strain confirmed the presence of from the specimen 3 (Table 1). Considering the parts of the bioactive substances with inhibitory effect against S. Table 1 - Total of fungi isolated and colonization rates in each tissue of Myrcia guianensis. Plant parts Specimen 1 Specimen 2 Specimen 3 Averages n° of isolates CR n° of isolates CR n° of isolates CR n° of isolates CR L 15 0.166 19 0.211 13 0.144 15.66 0.173 R _ _ 3 0.033 2 0.022 1.66 0.018 Rb 10 0.111 9 0.100 11 0.122 10.00 0.111 S 11 0.122 7 0.077 6 0.066 8.00 0.088 Sb 17 0.188 16 0.177 17 0.188 16.66 0.184 Total 53 0.196 54 0.200 49 0.181 52.00 0.192 For the isolation it was used a total of 90 fragments from each specimen of M. guianensis. L – Leaves; R – Root Cortex; Rb – Root Bark; S – Stem Cortex; Sb – Stem Bark; CR – colonization rate. Averages with a same letter indicate no significant difference according to the Tukey test (p < 0.05). Endophytic fungi from Myrcia guianensis 157 Table 2 - Morphological groups and relative frequencies of endophytic fungi isolated from Myrcia guianensis. Morphogroup Specimen 1 Specimen 2 Specimen 3 Total RF (%) LR S T LR S T LR S T Pestalotiopsis 64 11 21 1258 869 23 52 33.3 Phomopsis 367 16 546 15 2248 39 25.0 Aspergillus 2125 2136 –167 18 11.5 Xylaria 1–12 1–34 11–28 5.1 Unknown 6 1–23 1–12 2–138 5.1 Unknown 7 –––– 4–48 1–12 10 6.4 Unknown 8 –––– 1124 ––––4 2.5 Unknown 9 1––1 1–12 ––––3 1.9 Penicillium ––11 ––22 ––114 2.5 Unknown 10 2––2 –1–1 ––––3 1.9 Unknown 11 –––– –1–1 –1–12 1.2 Nectria ––11 ––11 ––113 1.9 Fusarium –1–1 –––– ––––1 0.6 Guignardia –––– –––– 1––11 0.6 Total 16 12 24 53 16 10 28 54 15 11 24 49 156 53.3 L – leaves; R – root; S – stem; T – total of each specimen. Table 3 - Antibiosis results of the crude fermentation broth from endophytic fungi isolated from of M. guianensis which presented some activity against the pathogenic strains. Endophytic fungi Genera Tested microorganisms Strains Pa Sa Ef Bc Ca Pv MgF2.1.2 Pestalotiopsis –++ ++– – – MgF1.2.1 Phomopsis –++ ++– – – MgR2.1.1 Unknown 10 –––– ++ ++ MgRe2.2.3B Nectria – ++ + + – – +++ Negative control amoxicilin ketoconazole –––––– +++ ++ +++ +++ NT NT NT NT NT NT + + + + + + Pa - Pseudomonas auriginosa;Sa- Staphylococcus aureus;Ef - Enterococcus faecalis;Bc- Bacillus cereus;Ca- Candida albicans;Pv - Penicillium avellaneum; NT – Not tested. + Inhibition zone between 10 and 15 mm; + + Inhibition zone between 15 and 30 mm;+++ Inhibition zone between 30 and 45 mm. aureus, E. faecalis and P. avellaneum only in the FBE. Discussion Negative results were observed for ME (Table 4). The frac- Isolation of endophytic fungi tions FA1 (0.35 g), FA2 (0.52 g), FA3 (0.11 g), FA4 (0.09 g), FA5 (1.32 g) and FA6 (0.19 g) obtained from the The results showed that M. guianensis is a good chromatographic fractioning of FBE were tested against source of endophytic fungi, since only one type of culture the same pathogenic strains. FA1 and FA2 showed positive medium was used for the isolation process, and this unique result against the tested strains (Table 4). method allowed the acquirement of a considerable number of endophytes (totaling 57.7% of the inoculated frag- ments). No microorganism had appeared from the last Preliminary characterization of fungal metabolites washing water, so the surface disinfection method was con- Qualitative analysis of the compounds present in the sidered efficient. ME and FBE showed the presence of phenols in both extracts. The three specimens of M. guianensis presented simi- The presence of tannins was found only in the ME, and the lar relative distribution of isolated fungi among all parts of presence of quinones was observed only in the FBE. It was not the plant. While someone could expect the higher numbers observed the presence of alkaloids in any of the extracts. of isolates in the leaves and stem barks (Table 1), a smaller 158 Banhos et al. amount of fungi in the roots was somewhat unexpected, since as this tissue is in contact with the ground, which is a source of numerous microorganisms. However, several works have presented the leaves and stems as main parts where endophytes are found (Gazis and Chaverri, 2010; Suryanarayanan et al., 2009), which may be related to how the endophyte penetrates the plant (by its aerial interac- tions), and also to the favorable conditions of certain tissues for the fungus protection (Ahlholm, 2002; Brand and Gow, 2009). On the other hand, the similarity among the results for M. guianensis specimens suggests that the isolation pro- cess is expressing the real number of cultivable endophytic fungi present in the healthy tissues. Besides, almost every isolated genus presented a relative frequency greater than 1, except Guignardia, known as an orange’s pathogen (Spó- sito et al., 2011) and Fusarium, the agent that causes the addlement of the roots (Yu et al., 2004), both cases recog- nized as a systemic colonization (Table 2). In a study performed by Pinto (2011), in the isolation of endophytic fungi from leaves and stems of M. sellowiana, a lower frequency of isolates (12.7%) was ob- served when compared to the findings presented here (53.3%), as the treatment leaves in the disinfection step was the same surface, and the medium used for isolation also (BDA), the discrepancy in results may be related to season- ality of these microorganisms in the host, considering that the collection of plant material for the isolation occurred at different times in the year 2009, during the rainy season and the other during the dry season (Pinto, 2011). The statistical analyses of the averages of the fungi isolated in each tissue showed that there is no significant difference (p  0.05) between the number of isolates from the stem barks (Sb) and from the leaves (L). The same was observed for the number of isolates from the root barks (Rb) and from the stem cortices (S). On the other hand, there is a notable difference between the averages of colo- nization rate (CR) in the roots cortices (0.018), and in the stem barks (0.184). Clearly, for the conditions applied, the amount of cultivable isolates in the stem barks is ten times greater than in the root cortices, showing the importance of the plant fragmentation for the fungal isolation. Since many fungal species are not cultivable, in order to analyze the ef- ficiency of the endophyte isolation, it is necessary to re- member that these numbers just express the frequency of cultivable endophytes under determined isolation condi- tions, such as superficial disinfection, culture medium and growth temperature. Endophytic fungal diversity It was found a greater diversity of genera in the speci- men 2, than in 1 and 3. In contrast, specimens 1 and 3 pre- sented more isolates of the genus Pestalotiopsis (Table 2). Some of the most frequent isolates of M. guianensis,as Pestalotiopsis and Phomopsis are known as endophytes of other plant species from tropical climate, and some have Table 4 - Antibiosis activities of the extracts and fractions from the fermentation broth of the fungus Nectria haematococca cultivated in preparative scale. Tested strains Extracts Fractions Control FA1 FA2 ME FBE FA1 FA2 FA3-6 CON AMO KET MIC MBC MIC MBC -1 -1 -1 -1 (g.mL ) (g.mL ) (g.mL ) (g.mL ) S. aureus – –– NT 23.3  1.1 12.3  0.5 12.3  0.5 28.1  0.28  25  50  25  50 E. faecalis – –– NT – – 24.3  2.0 11.3  0.5 11.6  0.5 46.0  1.00  50  50 P. avellaneum – –– NT 22.3  2.0 23.3  1.1 21.3  0.5 43.3  0.6  12.5  100  12.5  100 ME – Ethanolic extract from the mycelium; FBE – ethyl acetate extract from the fermentation broth; FA1 – fraction from FBE obtained in dichloromethane:ethyl acetate (1:1); FA2 – fraction from FBE obtained in ethyl acetate 100%; FA3 – fraction from FBE obtained in ethyl acetate: acetone (1:1); FA4 – fraction from FBE obtained in acetone 100%; FA5 – fraction from FBE obtained in methanol 100%; FA6 – fraction -1 -1 from FBE obtained in methanol:water (8:2); AMO – Amoxicilin 2.0 mg.mL ; KET – ketoconazole 2.0 mg.mL ; CON – negative control DMSO: water (1:9); MIC – minimum inhibitory concentration; MBC – minimum bactericidal concentration; NT – not tested. Endophytic fungi from Myrcia guianensis 159 demonstrated significant potential for the production of Antimicrobial activity useful biotechnological compounds, such as isopestacin Even though only some of the isolated endophytes isolated from P. microspora. This compound showed anti- have been used in the analyses of the antimicrobial activity, fungal activity against Pythium ultimum, an oomycete that which reduced the possibility of finding strains that pro- causes the addlement of the roots in plants of agricultural duce bioactive compounds, and considering that they have importance (Strobel et al., 2002). been cultivated in only one culture medium, which cannot be suitable for gene expression that led to the production of Another example is the lactones extracted from the some bioactive metabolites, it was gratifying that the strain crude fermentation broth of Phomopsis sp., an endophytic MgRe2.2.3B, belonging to the genus Nectria, had shown fungus from Azadirachta indica. A recent work it was de- activity against S. aureus, E. faecalis and P. avellaneum,a scribed the antifungal action of these lactones against the signal of its great potential as a source of antibacterial and important plant pathogen, Ophiostoma minus, with a mini- -1 antifungal compounds. It should be pointed out that the re- mum inhibitory concentration (MIC) of 31.25 g.mL (Wu sult of the antibiosis test of the crude fermented broth of this et al., 2008). fungus against P. avellaneum was similar to the positive Regarding the diversity of endophytes, it is impor- control ketoconazole. This fact supported the selection of tant to note that the identified groups at the genus level this strain for subsequent assays, as well as for its molecular were also found in other studies concerning tropical identification. plants from the Amazon region (Hanada et al., 2010; Extracts, fractions, MIC and MBC Souza et al., 2008). This is the case of the genera Pestalotiopsis, Phomopsis, Aspergillus, Penicillium and The test results indicate that the active compounds are Xylaria, which demonstrates the versatility of these among the substances present in the fractions 1 and 2. A endophytic microorganisms in colonize diverse host spe- -1 MIC of 25 g.mL was obtained for FA1 and FA2 against cies. In this sense, it is important to consider that fre- -1 S. aureus and of 12 g.mL against P. avellaneum. Against quencies under 1% indicate genera not common in the -1 E. faecalis the obtained MIC was 50 g.mL for fraction 1 plant’s tissues. In some cases, those indicate that these -1 and 100 g.mL for fraction 2 (Table 4). The minimum fungi may not be a natural endophyte, but an epiphyte or bactericidal concentration (MBC) results were found at even a plant pathogen trying to colonize the plant tissue -1 50 g.mL for both FA1 and FA2 against S. aureus. The transitorily. In this work, it was possible to observe the fractions did not present bactericidal capacity against E. presence of two plant’s pathogen genera, Fusarium and faecalis in any of the tested concentrations. FA1 and FA2 Guignardia (Spósito et al., 2011; Yu, et al., 2004), both showed fungicidal ability against P. avellaneum, but this found at a frequency of 0.6%, indicating its momentarily -1 capacity is only observed at a dose of 100 g.mL .Al- colonization. though the most promising results for the antimicrobial ac- Plants of Myrcia genus appear as a promising source tivity were obtained against the P. avellaneum strain, of endophytic microorganisms, which produce metabolites different results were observed during the fungicidal tests. with proven antimicrobial activity. Pinto (2011) evaluated These findings demonstrate the importance of such tests, the antimicrobial activity of extracts from endophytic fungi since this evaluation confirms or denies the effective action isolated from M. sellowiana, and found 62 extracts active of the investigated compound against pathogenic microor- against S. aureus, 20 against E. coli and 11 against both ganisms. pathogens (Pinto, 2011). In addition to that, the author found 35 endophytic fungi strains that produced metabo- Preliminary characterization of fungal metabolites lites which are active against the pathogenic fungus The production of phenols and quinones by endo- Colletotrichum gloeosporioides. These findings justify the phytic fungi and antimicrobial activity of these compounds investigation among this vegetal genus as a source of anti- are fairly known. In a study carried out by Li and coworkers microbial producing endophytic fungi and corroborate the (2008), two new antimicrobial substances (pestalachloride results presented in this work. A and B) were isolated, which are phenolic compounds produced by the endophytic fungus Pestalotiopsis adusta In this work it was also possible to verify the isolation (Li et al., 2008). In another work, it was observed the pro- of a fungi from the genus Nectria, a known producer of pig- duction of quinones by Ampelomyces sp., an endophytic ments such as anthraquinone and naphthoquinone (Barbier fungus isolated from Urospermum picroides, with signifi- et al., 1988; Barbier, et al., 1990). The genus Nectria had cant activity against the pathogenic bacteria S. aureus, S. not been previously described as endophytic fungi from epidermidis and E. faecalis (Aly et al., 2008). Amazon hosts, or as a producer of antimicrobial com- pounds. 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