Introduction

Endophytic organisms are those that live inside host plants without causing apparent injury, since they are in harmonic environmental conditions (Higginbotham et al., 2013; Ikram, Ali, Jan, Guljan, & Khan, 2019). According to the plant species, its age, geographic location and the environmental conditions where the plants live, there is a significant variation in the diversity of endophytic fungi (Bandara et al., 2006; Arnold, 2007; Song, Otkur, Zhang, & Tang, 2007). It is known that the greatest biodiversity of endophytic fungi is present in the leaves of tropical plants, which may contain species not yet found in other biomes (Arnold & Lutzoni, 2007; Arora et al., 2019) and which can produce several new compounds (Li et al., 2018). During the evolution, endophytic microorganisms have developed a number of characteristics and functions to survive in their specific ecological niches (Kusari, Hertweck, & Spiteller, 2012). The endophytes are known as a rich source of biomolecules, as alkaloids, terpenoids, flavonoids and steroids, with structural diversity and biotechnological applicability (Nair & Padmavathy, 2014; Li et al., 2018).

Due to the potential of endophytes to assist their hosts in the protection against pathogens, it is interesting to study these fungi aiming for bioactive substances. Moreover, new fungicides are required for the control of diseases that attack the important crops (Leadbeater, 2015), e. g. anthracnose and the pod and stem blight, diseases that cause great damages in crops worldwide. The pod and stem blight are caused by fungi in the genus Diaporthe Nitschke (=Phomopsis (Sacc.) Bubák). At least four Diaporthe species occur in soybeans and are responsible for serious diseases and yield losses (Pereira, Pereira, & Fraga, 2000; Santos, Vrandecic, Cosic, Duvnjak, & Phillips, 2011). The fungus Diaporthe phaseolorum (Cooke & Ellis) Sacc. (=Phomopsis phaseoli (Desm.) Sacc.) causes seed decay (Hepperly & Sinclair, 1978; Pereira, Pereira, & Fraga., 2000), pod and stem blight (Pedersen & Grau, 2010) and stem canker in soybean (Glycine max (L.) Merrill), leading to considerable crop production losses worldwide (Udayanga, Castlebury, Rossman, Chukeatirote, & Hyde, 2015).

The fungus Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. is one of the plant pathogens responsible for anthracnose, a disease that attacks various crops, such as avocado, strawberry, eggplant, coffee, cashew, citrus, etc. (Freeman, Katan, & Shabi, 1998). In view of the relevance of these diseases, it is important to seek new alternatives that may act in its control, such as fungicides (Leadbeater, 2015). The use of synthetic fungicides is progressively restricted because of harmful effects on the environment and human health, and the emergence of highly resistant fungal strains. Therefore, there is a great demand for new natural fungicides (Ribera & Zuñiga, 2012). Due to the potential of endophytes to protect their hosts against pathogens, the search in these environments becomes promising.

Here, we worked with several endophytic fungi from plants in the genus Begonia Plumier found in the Atlantic Rain Forest (Correia, Lira, Assis, & Rodrigues, 2018), to assess their potential to produce active extracts against plant pathogens and identify the compounds.

Material and methods

General procedures

The ¹H nuclear magnetic resonance (NMR) was recorded on a Bruker 14.1 Tesla, AVANCE III model spectrometer (operating at 600.23 MHz for hydrogen frequency) with cryoprobe™. The chemical shifts are given on a δ (ppm) scale. High-resolution mass spectrometry (UPLC-qTOF) was acquired on a UPLC Xevo G2-XS Q-TOF (Waters, Milford, USA), quadrupole time-of-flight mass spectrometry operating in electrospray positive mode in the following conditions: Column Acquity UPLC BEH C₁₈ (2.1 x 100 mm, ¹.7 µm, Waters, Milford, USA). The chromatographic conditions were a gradient of H2O/ACN (98:2, v v^-1, 9 min. 100% ACN) during 10 min., flow rate 0.5 mL min.^-1. The parameters for the mass spectrometry detection were: MS^E continuum, m z^-1 100-1200 Da range and ramp collision energy of 30V. Data acquisition was performed using the software MassLynx

High-performance liquid chromatography (HPLC) was conducted using an Agilent 1100 Series UV/Vis with a quaternary pump, coupled with a UV detector MWD (Multiple Wavelength Detector), using a reversed phase column C₁₈ (4.60 x 250 mm, 5 μm; Phenomenex Kinetex), with a mobile phase in gradient of H₂O/ACN (90:10, v v-1, 30 min. 100% ACN), flow rate 1 mL min.^-1 and λ = 220 nm. HPLC-grade solvents were utilized (LiChrosolv, Merck, Germany). Solid-phase extraction was carried out using silica gel, cyano and C18 cartridges of different dimensions (Phenomenex, USA and Supelco, Sigma-Aldrich, Germany) and P.A grade solvents (Synth, Brazil). Silica gel₂₅₄ (Macherey-Nagel) was used for TLC. Spots were detected under UV light (254 and 365 nm).

Optical absorbances were measured on a TECAN reader, model SUNRISE, operated by Magellan v.7.1 software, at 620 nm.

Biological material

The endophytic fungi used in this study (n= 88) were isolated from Begonia fischeri Schrank (HRCB 64226), B. venosa Skan ex Hook (HRCB 64227) and B. olsoniae L.B. Sm. & B.G. Schub (HRCB 64229) and identified by morphological and molecular techniques in a previous study (Correia, Lira, Assis, & Rodrigues, 2018).

Cultivation, extraction and isolation

For the screening of extract with anti-fungal activity, each fungal isolate was cultured on malt extract agar 2% medium (20 g L^-1 of malt extract - Kasvi and 15 g L^-1 of agar - Acumedia) at 28°C for 7 days. Then, three mycelium fragments (7 mm2 in diameter) were transferred to Erlenmeyer flasks with malt extract 2% broth, pH 6.0, and incubated at 28°C under agitation (150 rpm) for seven days (150 mL batch in 250 mL Erlenmeyer flasks for each fungal strain). The mycelium was separated from the supernatant using vacuum filtration and the liquid was extracted three times with EtOAc. Evaporation of the solvent in vacuum gave the ethyl acetate extracts, which were tested against the fungi C. gloeosporioides and D. phaseolorum in the paper disk assay.

The selected fungal strain was cultured under the same conditions (28 x 250 mL Erlenmeyer flasks, each containing 150 mL of malt medium, pH 6.0). The flask cultures were incubated at 28°C on a rotary shaker at 150 rpm for 9 days. The liquid cultured medium was extracted with EtOAc three times, and the organic solvent was evaporated to dryness under vacuum to afford 1.0 g of the ethyl acetate extract.

The ethyl acetate extract was separated on a Sep-pak cyan column (10 g) using a gradient elution of CH2Cl2-MeOH (100: 0 to 0: 100), resulting in 5 fractions (1-5). Fraction 1 (877 mg) was separated on Sep-pak silica gel column (10 g) with a gradient of hexane- CH₂Cl₂-MeOH, yielding 5 fractions (1A-1E). Fraction 1B (60 mg) was fractionated on a C₁₈ reverse phase Sep-pak column (1 g) using a H₂O-MeOH gradient, yielding 3 fractions (1B1-1B3). Fraction 1B3 (8.3 mg) was purified by HPLC with a H₂O-ACN gradient to give 1.2 mg of active compound 1B3P1 (1).

Fumiquinone B (1). Purple crystals, UV λ_max (MeOH) nm (ε) 226 (2,400), 269 (3,700), 292 (3,200); ¹H NMR (DMSO_d6, 600.23 MHz) δ 1.74 (s, 3H, CH₃-5), 3.72 (s, 3H, OCH₃-3); HRESIMS [M+H]⁺ m/z 185.0438 (calc. for C₈H₉O₅+ 185.0450); HRESIMS [2M+H]+ m z-1 367.0654 (calc. for C₁₆H₁₅O₁₀⁺ 367.0665).

Antifungal assays

Paper disk assay

The paper disk assay (Heatley, 1944) was used to determine antifungal activity during the screening and the separation, and purification procedures. The fractions were applied to 6 mm diameter sterile paper disks, Filter Macherey Nagel, MN 640 m, 7 cm (1 mg for AcOEt extract, 0.5 mg for fractions and 0.2 mg for pure compounds) and placed on the edge of Petri dishes with 2% malt medium. An inoculum of the pathogen (a 7 mm diameter mycelium fragment removed from a fresh culture) was added to the opposite border of the Petri dish. Areas of inhibition of mycelial growth around the paper disks were measured after incubation for 5 days at 28°C. Inhibition percentages were calculated in comparison with the control containing only the pathogen using the Image J program (Image Processing and Analysis in Java). This experiment was conducted with one replicate. The fungi C. gloeosporioides (deposit number CMAA 1688) and D. phaseolorum (deposit number CMAA 1695) used in this study were isolated from guarana plant and soybean, respectively, obtained and identified by Prof. Dr. João Lúcio de Azevedo, from the Laboratory of Genetics of Microorganisms, Department of Genetics, ESALQ-USP.

Microdilution assay

The microdilution assay was performed on a 96-well microplate according to the guidelines of the National Committee on Clinical and Laboratory Standards (Clinical and Laboratory Standards Institute [CLSI], 2008) with modifications. For the assay, the pure compound was diluted in DMSO and 2% malt medium to final concentrations of 2500; 1250; 625; 312; 156; 78.1; 39; 19.5; 7.7; 4.8 and 2.4 μg mL^-1 in each well. A suspension of inoculum of D. phaseolorum (10⁵ spores mL^-1, 60 μL) was added to each well, totalizing a final volume in the well of 100 μL. As negative control, wells containing medium, inoculum and DMSO were evaluated, but without the compound. As a positive control, the commercial fungicide Score was used, whose active principle is difenoconazole. The microplate was incubated in B.O.D. at 28°C and analyzed in a microplate reader at 620 nm at 12 hours intervals for 120 hours.

The minimum inhibitory concentration (MIC) of the compound was determined spectrophotometrically after the above-mentioned incubation periods, according to the (CLSI, 2008) guidelines, with modifications. For both the isolated compound and the commercial fungicide, MIC was determined as the lowest concentration showing absence of growth or equal to the initial growth as compared to growth (up to 12 hours) in the well free of compounds. The commercial fungicide used was the Score (Syngenta, Switzerland).

Results

The bioassays against the plant pathogens D. phaseolorum and C. gloeosporioides were carried out with the ethyl acetate extracts obtained from 88 endophytic fungi, being 36 from B. fischeri, 24 from B. olsoniae and 28 from B. venosa. From the 88 fractions evaluated, 14.7% were active against C. gloeosporioides, 11.3% against D. phaseolorum and 6.81% inhibited both plant pathogens, varying in inhibition indexes. In relation to the host plants, 27.7% of the selected endophytes from B. fischeri inhibited the growth of at least one of the plant pathogens evaluated, against 50% of B. olsoniae endophytes and 7.14% of B. venosa (D. phaseolorum inhibition only). The 24 active fractions were reevaluated in a new bioassay (in triplicate). The results are shown in Table 1.

The strain AM29, identified as Neopestalotiopsis sp. (deposited under accession CRM 1920 at CRM- UNESP, Microbial Resource Center (WDCM 1043), was selected by its activity against D. phaseolorum for a chemical investigation of its secondary metabolites.

After culturing the Neopestalotiopsis sp. AM29 in liquid medium, filtration and partitioning with ethyl acetate, the AcOEt extract (1.0 g) was obtained. This extract showed 23% activity against the mycelial growth of D. phaseolorum and was fractioned in a biomonitoring way until it obtained the pure active compound 1B3P1 (1.2 mg), with 58% of activity against D. phaseolorum. The molecular mass of the pure compound was searched in the Dictionary of Natural Products database and identified by spectroscopic analysis (MS and NMR) by comparison with Hayashi et al. (2007). With the data obtained in the ¹H NMR spectrum, it was possible to correlate the hydrogen shifts (δ_H) of 1B3P1 (1) with the compound fumiquinone B (2,6-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, CAS 1011259-29-2), a quinone derivative, Figure 1 (Hayashi et al., 2007).

The isolated compound fumiquinone B was tested against spores of D. phaseolorum using the microdilution assay (Figure 2).

The assay was carried out after 120h of incubation. The compound fumiquinone B was active against spores of D. phaseolorum, with a MIC of 312 µg mL-1 (1,695.3 µM). The MIC of the commercial fungicide difenoconazole (positive control, not shown) was 2.4 µg mL-1 (5.9 µM).

Discussion

Probably the ethyl acetate extracts from the endophytic fungi that showed inhibition against the evaluated plant pathogens have different compounds, since the best inhibitors for D. phaseolorum were not the same for C. gloeosporioides. Regarding the inhibition of the former, the extracts obtained from the endophytes Cuvularia sp1, Curvularia sp2, Diaporthe sp5 and Neopestalotiopsis sp. presented the best results. For C. gloeosporioides, the extracts of Diaporthe sp1 and Epicoccum nigrum presented the higher inhibition scores.

In the literature, there are few studies with secondary metabolites from Neopestalotipsis species. This is due to the fact that the genus Neopestalotiopsis was recently described by Maharachchikumbura, Hyde, Groenewald, Xu, and Crous (2014). Initially, De Notaris, in 1839, described the genus Pestalotia De Not, which was divided by Steyaert, in 1955 in Pestalotia, Pestalotiopsis Steyaert and Truncatella Steyaert. Until the 1990s, the taxonomy of Pestalotiopsis and related genera was based on conidial characteristics, mainly pigmentation. However, this method of identification was controversial, due to the variation of other morphological characteristics, such as colony color, texture and shape (Hu, Jeewon, Zhou, Zhou, & Hyde, 2007; Solarte, Muñoz, Maharachchikumbura, & Álvarez, 2018). Then, by morphological and DNA analyzes (ITS, β-tubulin and 1-alpha elongation factor) the genus Pestalotiopsis was segregated into two new genera: Neopestalotiopsis Maharachch., K.D. Hyde & Crous and Pseudopestalotiopsis Maharachch., K.D. Hyde & Crous. Eleven new species were introduced in Neopestalotiopsis, twenty-four in Pestalotiopsis and two in Pseudopestalotiopsis (Maharachchikumbura et al., 2014). Due to this recent review of the genus, most of the available information is on the genus Pestalotiopsis, which is commonly isolated as endophyte (Wei et al., 2007; Figueiredo et al., 2007). It has been isolated from the families Podocarpaceae, Theaceae and Taxaceae (Wei et al., 2007), of the species Rhizophora apiculata DC., Cocos nucifera L., Taxus wallichiana Zucc., Camellia sasanqua Thunb., Fragraea bodenii Wernh., Cordemoya integrifolia (Willd.) Baill. (Wei & Tong, 2003), Maytenus ilicifolia Mart. Ex Reissek (Figueiredo et al., 2007), R. mucronata Lam. (Zhou et al., 2017), among others. This fungus is known for the production of compounds with medicinal applications (Tejesvi, Kini, Prakash, Subbiah, & Shetty, 2008), as antioxidant, antihypertensive, antimicrobial (Tejesvi et al., 2008; Zhao et al., 2015) and antifungal activity (Strobel et al., 2002); agricultural application, as anti-oomycete activity (Li & Strobel, 2001); and antifungal against Fusarium oxysporum Schltdl., F. fujikuroi Nirenberg (= F. verticillioides (Sacc.) Nirenberg), Macrophomina phaseolina (Tassi) Goid., Epicoccum sorghinum (Sacc.) Aveskamp, Gruyter & Verkley (= Phoma sorghina (Sacc.) Boerema, Dorenb. & Kesteren) and Sclerotinia sclerotiorum (Lib.) de Bary (Tejesvi, Kini, Prakash, Subbiah, & Shetty, 2007); in addition to producing compounds with industrial applications (Xu, Ebada, & Proksch 2010; Maharachchikumbura, Guo, Chukeatirote, Bahkali, & Hyde, 2011).

Quinones are a group of natural compounds found in fungi, bacteria and plants (Monks & Jones, 2002). They are generally colored and semi-volatile, belonging to the class of polycyclic aromatic oxygenated hydrocarbons (HPAO). The structure of the quinones presents two carbonyl groups in an unsaturated ring of six carbon atoms, and they are classified as benzoquinones, naphthoquinones, anthraquinones or phenanthraquinones, according to the type of aromatic system (Sousa, Lopes, & Andrade, 2016).

Quinones are synthesized essentially via the polyketide pathway or the shikimate pathway, biosynthetic pathways absent in animals (Thompson, 1971). The biological reactivity of quinones in biological systems can be attributed to the fact that they are oxidizing and electrophilic. In relation to toxicity, this is influenced by the chemical structure, mainly by substitution effects (Monks & Jones, 2002). They have a role in photosynthesis, like electron transporters. Several quinones are vitamins or have antioxidant activity. Quinone derivatives are common in biologically active molecules. Many of the drugs clinically approved or still in clinical trials against cancer are quinone-related compounds (El-Najjar et al., 2011). Quinones, such as 1,4-naphthoquinone, 1,2-naphthoquinone, 1,4-benzoquinone, and anthraquinone have moderate antifungal activity to Colletotrichum spp. (Meazza, Dayan, & Wedge, 2003).

The quinone fumiquinone B has been isolated from the fungi Aspergillus fumigatus Fresen. (Hayashi et al., 2007) and Xylaria feejeensis (Berk.) Fr. (García-Mendez, Macías-Ruvalcaba, Lappe-Oliveiras, Hernández-Ortega, & Macías-Rubalcava, 2016), and is known for its nematicidal activity against Bursaphelenchus xylophilus Steiner & Buhrer, 1934 (Hayashi et al., 2007) and for its phytotoxic properties (García-Mendez et al., 2016).

Although the MIC of fumiquinone B against D. phaseolorum is higher than the one of the commercial fungicide difenoconazole, the isolated compound may present a different mode of action, as it pertains to the quinones class, and difenoconazole belongs to the group of triazoles that act on the cell membrane of fungi (Odds, Brown, & Gow, 2003).

Here, we emphasize the ability of the endophytes in producing compounds that may be used to control plant pathogens such as C. gloeosporioides and D. phaseolorum. The endophyte Neopestalotiopsis sp. AM29 produced fumiquinone B, which showed activity against the latter pathogen. This is the first report of the production of the compound fumiquinone B by fungi in the genus Neopestalotiopsis, as well as it is the first report of its activity against D. phaseolorum. Further studies need to be performed to evaluate the toxicity of the compound, additionally in vivo tests are also necessary.

Eletronic supplementary material

The mass spectra (positive and negative mode), high-resolution mass spectrum (positive mode) and ¹H NMR of the compound fumiquinone B in DMSO_d6 were shown in the supplementary material (Figures S1, 2, 3, 4 and 5).

Conclusion

Our study showed that endophytic fungi from Begonia plant species produce antifungal compounds. One of these metabolites, fumiquinone B, found in a isolate of Neopestalotiopsis, inhibited the in vitro growth of plant pathogens.

Acknowledgements

The authors thank Dr. Roberto Gomes de Souza Berlinck for the support, Prof. Ludovic Jean Charles Kollmann and Prof. Eliane Jacques for their help in collecting and identification of the plants. Financial support was provided by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grant (2014/15760-3) and BIOTA/BIOprospecTA FAPESP grant (2013/50228-8). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, scholarships to D. F. G. (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Conselho Nacional de Desenvolvimento Científico e Tecnológico 142079/2016-2) and A. M. L. C. (FAPESP: 2014/12021-5) are also gratefully acknowledged.

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