Identification of Substances Produced by Cercospora brachiata in Absence of Light and Evaluation of Antibacterial Activity

Cercospora brachiata is a phytopathogenic fungus. To know more about the metabolites produced by this fungus, the objective of this work was to identify, isolate and characterize substances present in extracts of the growth broth and mycelium, using gas chromatography with mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR). It was also objective to evaluate the antibacterial activity of the extracts. Among the compounds identified, fatty acids, esters, and steroids can be highlighted. The main compounds identified are 9-hexadecenoic, hexadecenoic, oleic, octadecanoic, lauric, myristic, palmitic, doceno-13-enoic, stearic, linoleic, and nonadecanoic acids present in almost all extracts. For the antibacterial activity, the broth microdilution method was used. The ethyl acetate extract of the mycelium presented inhibitory concentrations (MICs) against the bacterium Actinomyces naeslundii (100 μg mL−1) and Streptococcus sanguinis (200 μg mL−1). Finally, two steroids were isolated and identified in the hexane extract of mycelium: ergosta-6,22-dien-3β,5α,8α-triol and brassicasterol.


Introduction
Fungi can be classified according to the type of host. Fungi that use host plants are called endophytic plants which do not cause plant diseases, while phytopathogens cause many types of diseases in theirs hosts [1], which is the case of some Cercospora species [2].
Phytopathogenic fungi can produce toxic substances. The toxins, therefore, are secondary metabolites produced by fungi, usually of low molecular mass (<1000 Da), that may be related to the infection process in their hosts [3].
Some fungi are also harmful to plants for being necrotrophic causing plant cells death during plant tissue colonization. One of the genera of necrothophic fungi is Cercospora, which produces toxins such as cercosporin involved in the pathogenicity of these fungi, that can be activated by light [4,5], causing lesions in leaves, stems, and fruits. One of the plants parasitized by one species of Cercospora, known as Cercospora coffeicola, is coffee, whose productivity can be reduced by up to 30% for this pathogen [6,7].
Fungi may also produce compounds capable of preventing the growth of bacteria. This activity is associated with the fact that fungi produce secondary metabolites capable of inhibiting the action of bacteria and even of other fungi, as in the case of extracts from cultures of Cercospora spp. isolates [8]. Bacteria used to test the antibacterial activity herein reported belonging to gram-positive and gram-negative types of the oral cavity. For Kumar (2013), there are more than 1000 species of bacteria in the oral cavity of a human being, and these bacteria are displaced to other parts of the body causing other diseases such as inflammation of the prostate, asthma, pneumonia, endocarditis, diabetes, etc. [9]. Thus, it is necessary to investigate and use new compounds that are capable of inhibiting the growth of these bacteria.
Thus, the objective of this work was to identify the substances present in extracts of the growth broth and mycelium extract of the fungus Cercospora brachiata, a foliar pathogen of Amaranthus viridis, and also to test the antibacterial activity of these extracts against aerobic and anaerobic bacteria of the human oral cavity.

Instrumentation
The composition of extracts and isolated compounds were identified by a gas chromatograph coupled to a mass spectrometer (Shimadzu, QP2010) using a DB-5 capillary column (J&W, 30 m × 0.25 mm × 0.25 m). The conditions used were: helium as a carrier gas with a constant flow of 1.0 mL min −1 ; injector temperature of 220 • C, split 1:20; the temperature of the oven increased from 60 to 246 • C at 3 • C min −1 and maintained for 24 min at 246 • C; the ionizing potential of 70 eV; the range of m/z from 40 to 650. The identification of the compounds by this technique was based on the arithmetic index (AI) calculated and compared to that of Adams (2007) [10]. The similarity index (IS) obtained by the software (LabSolution-GCMS Solution) was also used by comparison with spectra present in the Nist27 libraries, Nist147, Wiley7, Wiley229, and Shim2205. The NMR analyses of 1 H and 13 C were carried out using a Bruker (AscendTM 400 Avance III HD model) equipment with 9.4 Tesla and 400 MHz.

Fungal Culture
Isolation and identification of the fungus C. brachiata were carried out at the Institute of Agricultural Sciences of the Federal University of Uberlândia (Monte Carmelo, MG-Brazil) by Dr. Bruno S. Vieira and collaborators [11], who reported this fungus for the first time in Brazil. The fungus was isolated from infected leaves of Amaranthus viridis L. (Caruru, voucher specimen VIC 47,138 deposited in the Herbarium at the Federal University of Viçosa, MG-Brazil). The fungus was grown in Petri dishes containing the potato-dextroseagar medium (PDA) at 25 • C for 7 days. Mycelium discs (Ø = 10 mm) from the periphery of actively growing cultures were aseptically transferred to erlenmeyer containing 250 mL of the modified Jenkins-Prior liquid culture medium [12]. The erlenmeyers flasks were transferred to an orbital shaker at 150 rpm in the dark at 25 • C for 21 days. The cultures obtained were filtered using Buchner's vacuum funnel to separate the mycelium from the growth broth [13].

Isolation and Purification of Compounds
Extract of growth broth from 5 L of C. brachiata was prepared by liquid-liquid partition using ethyl acetate (5 × 200 mL) at pH 5. After removal of solvent, 0.505 g of extract was obtained.
Extract of mycelia from 174 g of C. brachiata was prepared by maceration using methanol (600 mL) for 5 days at room temperature [14]. After removal of solvent, 100 mL of distilled water was added to the crude extract in a decantation flask and the liquid-liquid fraction was made using hexane (3 × 200 mL) and ethyl acetate (3 × 200 mL). After removal of solvent, 1.33 g of extract in hexane and 0.122 g of extract in ethyl acetate were obtained.
The hexane fraction of the mycelium was used for chromatography. Silica 60 G of 40-270 mesh was used as the stationary phase and ethyl acetate/hexane as mobile gradient phase, starting at a ratio of 1:4. From the first column, 24 fractions were obtained, of which fraction 8 was analyzed in NMR, and ergosta-6,22-dien-3β,5α,8α-triol was identified. The fractions 3 to 7 were pooled to make the second column. From this column, three fractions were obtained, and the compounds from fraction 1 were separated in the third column. From this column, four fractions were obtained, and fraction 3 was analyzed in NMR, and brassicasterol was identified. NMR spectra are presented as supplementary files (Figures S1-S7).

Antibacterial Activity
Biological assays to verify antimicrobial activity were performed by determining MIC (minimum inhibition concentration). The oral bacteria were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The following aerobic bacteria were used: Streptococcus mutans (ATCC 25175), S. mitis (ATCC 49456), S. sanguinis (ATCC 10556), Aggregatibacter actinomycetemcomitans (ATCC 43717). The following anaerobic bacteria were used: Porphyromonas gingivalis (ATCC 33277), Fusobacterium nucleatum (ATCC 25586) and Actinomyces naeslundii (ATCC 19039). The analysis of the antibacterial activity was performed in microplates containing 96 wells. The inoculum, the broth, and the sample were added to each well, yielding a final volume of 100 µL for aerobic bacteria and 200 µL for anaerobic bacteria. The volume of inoculum added in the microplate wells was 20 µL for the aerobic bacteria tests and 40 µL for the anaerobic bacteria tests. The concentrations of the samples tested against aerobic and anaerobic bacteria were from 0.195 µg mL −1 to 400 µg mL −1 . Chlorhexidine (0.115 to 59.0 µg mL −1 ) was used as positive control for aerobic and anaerobic bacteria. To control the technique, metronidazole (0.0115 to 5.9 µg mL −1 ) was used as control for anaerobic bacteria against Bacteroides fragilis and B. thetaiotaomicron. Dimethylsulfoxide (DMSO) (5.0 to 1.0%, v/v concentration) was used as the solvent control. Sterility controls of the positive control, culture medium, and samples were also performed.
For aerobic bacteria, the microplates were incubated in microaerophilia by the flame/candle system at 37 • C for 24 h. After the incubation period, 30.0 µL of the resazurin indicator (0.02% m/v) was added to each well [15]. For anaerobic bacteria were incubated for 72 h in an anaerobic chamber (atmosphere containing: 5-10% H 2 , 10% CO 2 , 80-85% N 2 ) at 36 • C and revealed with the same indicator [16].
Fatty acids play important cellular roles in biological systems. This is because these molecules make up the constitution of the cell walls, regulate the activity of enzymes and inflammatory processes [19]. Fatty acids are long-chain carboxylic acids (hydrocarbons) and are the components of many lipids, including glycerides. As they are one of the building blocks used in the production of complex lipids, they have considerable medicinal and nutritional value. For example, gamma linolenic acid (GLA, an omega-6 fatty acid) in the form of evening primrose oil (EPO) has been used to treat rheumatoid arthritis, multiple sclerosis, schizophrenia, and premenstrual syndrome. Eicosapentaenoic acid acetic acid (EPA) and docosahexaenoic acid (DHA, both omega-3 fatty acids) have physiological effects in areas such as heart, circulation, inflammation, and cancer [20].
According to Blacklock et al. [21], there is a diversity in the synthesis of fungal fatty acids, and their identification by GC-MS can be facilitated when they are esterified, for example, methyl hexadecanoate, (Z) -9-octadecenoate, methyl octadecenoate, methyl palmitate, methyl oleate, and methyl linoleate esters were identified in fungal extracts.
Other compounds identified were steroids, which are substances found in almost every living organism, whether in plants, fungi, bacteria, or in human beings. According to Dewick [22], steroids are modified triterpenoids and their biosynthesis is the route of mevalonic acid (MVA), which is formed by three molecules of acetyl-CoA. Table 1 shows the compounds identified by GC-MS in the ethyl acetate extract of the fungus growth broth and the extracts in hexane and ethyl acetate from the mycelium of the fungus. The structures of the identified compounds are shown in Figure 1.

Isolated Compounds
The hexane fraction was fractionated through column chromatography. Fraction 8 from the first column and fraction 3 from the third column were analyzed by NMR and the compounds ergosta-6,22-dien-3β,5α,8α-triol (31), and brassicasterol (17) were characterized, respectively ( Figure S1). Ergosta-6,22-dien-3β,5α,8α-triol (31) from fraction 8 of first column 3 was analyzed by the 13 C-NMR and 28 carbons were found in the spectrum, which matched the possible structures of the Ergosta-like molecules identified by GC-MS at retention times 75.8 to 93.9 min, with respective similarity indexes of 60 and 80, shown in Table 1. Compound 31 was not identified by GC-MS, probably because the presence of three hydroxyl groups results in a compound not volatile enough to be analyzed in the conditions used in this analysis.
Of the 28 signals observed in the 13 C-NMR spectrum ( Figure S6)   (EPA) and docosahexaenoic acid (DHA, both omega-3 fatty acids) have physiological effects in areas such as heart, circulation, inflammation, and cancer [20]. According to Blacklock et al. [21], there is a diversity in the synthesis of fungal fatty acids, and their identification by GC-MS can be facilitated when they are esterified, for example, methyl hexadecanoate, (Z) -9-octadecenoate, methyl octadecenoate, methyl palmitate, methyl oleate, and methyl linoleate esters were identified in fungal extracts.
Other compounds identified were steroids, which are substances found in almost every living organism, whether in plants, fungi, bacteria, or in human beings. According to Dewick [22], steroids are modified triterpenoids and their biosynthesis is the route of mevalonic acid (MVA), which is formed by three molecules of acetyl-CoA. Table 1 shows the compounds identified by GC-MS in the ethyl acetate extract of the fungus growth broth and the extracts in hexane and ethyl acetate from the mycelium of the fungus. The structures of the identified compounds are shown in Figure 1.   The 1 H-NMR spectrum ( Figure S5) shows the signals of the methynic hydrogens H-6 (δ: 6.5, d, 1H, J: 8. Brassicasterol (17) from fraction 3 of third column 3 was analyzes by the 13 C-NMR and 28 carbons were found in the spectrum that matched the structure identified by GC-MS, at the retention time of 88.4 minutes, with a similarity index of 75% as shown in Table 1. The compound was identified in the fraction extracted in ethyl acetate from the mycelium, however, it was isolated from the fraction in hexane, indicating that not all compounds were extracted by the liquid-liquid partition. The GC-MS data were compared with the NMR data.

Antibacterial Activity
The antibacterial activity was evaluated to test the "Minimum inhibitory concentration" (MIC), that is, the minimum concentration capable of inhibiting bacterial growth. The results are shown in Table 4. The results that are considered present good activity against bacteria are those whose minimum inhibitory concentrations are close to 100 µg mL −1 , whereas concentrations between 200 and 500 µg mL −1 are considered to be moderate activity [25]. Regarding aerobic bacteria, the values found for antibacterial activity are considered moderate for the hexane extract of mycelium against S.mitis and S. sanguinis; for the ethyl acetate extract of mycelium against S. mitis, S. sanguinis, and A. actinomycetemcomitans. The ethyl acetate extract from the broth showed no activity within the range of the concentrations tested (0.195 to 400 µg mL −1 ).
Regarding the anaerobic bacteria, the results showed that the ethyl acetate extract of the mycelium inhibited the growth of all tested bacteria with promising activity to A. naeslundii (100 µg mL −1 ). The hexane extract of the mycelium and ethyl acetate extract from the broth showed moderate activity against the bacteria P. gingivalis and A. naeslundii with MIC values from 200 to 400 µg mL −1 .
Previous work has already reported the antibacterial activity of the metabolites of the fungus Cercospora sp. against the bacteria Bacillus subtilis and Staphylococcus aureus [8]. However, the results were given in a zone of inhibition of halo, presenting values of 15.0 ± 0.3 and 25.0 ± 0.4 mm, respectively. This antibacterial activity observed, even in a qualitative way, corroborates with the data of this work, in which the extracts of the fungus Cercospora sp. inhibited the growth of different oral bacteria with concentrations between 100 and 400 µg mL −1 . Therefore, the antibacterial activity of the metabolites of this fungus is demonstrated.
Fatty acids and steroids were one of the main classes of metabolites found in the extracts, and the results found may be related to the presence of these constituents.
The antibacterial properties of fatty acids are well known, the intensity of activity can be influenced by the length and shape of the chain size and also by the presence or absence of unsaturations. Hydrophobic and hydrophilic interactions of fatty acids with the bacterial cell membrane generate structural changes in the bacterial cell. This process can affect the energy production, inhibit enzymatic activity, impair nutrient absorption, generate degradation products (auto-oxidation and peroxidation), or promote lysis of the cell membrane contributing to growth inhibition or death of the bacterium [26].
Steroids were another class of compounds identified and isolated from the extracts of the fungus Cercospora. Steroids can also be considered to contribute to the antimicrobial action of extracts. Other works have already demonstrated the potential of these compounds against different Gram-negative and Gram-positive bacteria [27][28][29][30]. They have an action similar to fatty acids on cell membranes, as they adhere to the lipid layers of the membranes, favoring the passage of nutrients and cell lysis [31]. Thus, fatty acids and steroids can be the bioactive compounds responsible for the activity of the extracts.

Conclusions
The chemistry of natural products continues to be an important component in science because it is in contact with the various studies related to biota, fauna, and flora. The fungi are part of this biological system and their chemical and biological analyses bring important contributions to clarify the chemical composition of the compounds produced by these fungi and correlations of chemical profiles between genera and species can be obtained.
Among the compounds identified, fatty acids, esters, and steroids were found, which have structural functions in the cell membrane, so it is reasonable that they are found in fungus extracts.
In this work, the antibacterial activity of Cercospora brachiata extracts was evaluated. It was observed that the extracts from extractions with ethyl acetate presented promising values against anaerobic bacteria of the oral cavity.