Phaeophleospora vochysiae Savi & Glienke sp. nov. Isolated from Vochysia divergens Found in the Pantanal, Brazil, Produces Bioactive Secondary Metabolites

Microorganisms associated with plants are highly diverse and can produce a large number of secondary metabolites, with antimicrobial, anti-parasitic and cytotoxic activities. We are particularly interested in exploring endophytes from medicinal plants found in the Pantanal, a unique and widely unexplored wetland in Brazil. In a bio-prospecting study, strains LGMF1213 and LGMF1215 were isolated as endophytes from Vochysia divergens, and by morphological and molecular phylogenetic analyses were characterized as Phaeophleospora vochysiae sp. nov. The chemical assessment of this species reveals three major compounds with high biological activity, cercoscosporin (1), isocercosporin (2) and the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone (3). Besides the isolation of P. vochysiae as endophyte, the production of cercosporin compounds suggest that under specific conditions this species causes leaf spots, and may turn into a pathogen, since leaf spots are commonly caused by species of Cercospora that produce related compounds. In addition, the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone showed considerable antimicrobial activity and low cytotoxicity, which needs further exploration.

species, P. stramenti and P. eucalypticola, were found as endophytes from Eucalyptus sp 11 and Eugenia uniflora 15 , respectively. Despite the fact that the genus Phaeophleospora was described as early as 1916, there were no reports of any bioactive secondary metabolites, and its metabolic potential remained unknown.
Based on morphological and phylogenetic analyses we describe here Phaeophleospora vochysiae as a new species within the genus Phaeophleospora, studied its secondary metabolites, and linked these to biological activities.
Culture characteristics: Colonies spreading, erumpent, lobed margins and moderate aerial mycelium, reaching 27 mm after 14 days at 28 °C. On Potato Dextrose Agar (PDA) medium, the surface is olivaceus-grey with vinaceus margin, the reverse side red due to a diffuse pigment. On oatmeal agar (OA) surface olivaceus-grey with yellow center, and on malt extract agar (MEA) green-grey and reverse iron-grey (Fig. 2).
Antibacterial and Antifungal Activities. The antimicrobial activity of the three crude extracts produced in the PD, Czapeck and MEA culture media were evaluated in order to select the best culture conditions to produce bioactive secondary metabolites. Extracts from PD showed the best results, with potent activity against the phytopathogen Phyllosticta citricarpa, moderate activity against methicillin sensitive and resistant Staphylococcus (+)-Cercosporin (1) (+)-Isocercosporin (2) Compound 3  aureus, and low activity against the phytopathogen Colletotrichum abscissum ( Table 3). The isolated compounds 1-3 (from cultures in PD medium), also displayed both, antibacterial activity against sensitive and resistant S. aureus and antifungal activity against the citrus phytopathogen Phyllosticta citricarpa (Table 3).

Discussion
Endophytic fungi are an important resource of secondary metabolites 5,21 . To better access this important source of bioactive molecules it is essential to explore the diversity of endophytes and to catalogue their species occurrence in different ecosystems 5 . Strains LGMF1215 and LGMF1213 were isolated from healthy leaves of a medicinal plant, Vochysia divergens, commonly found in flooding areas of the Pantanal (Brazil), and were characterized as a new species of Phaeophleospora genus, namely P. vochysiae. Characterization of Phaeophleospora species traditionally was based on morphology 15 in comparison with the type species P. eugeniae 10 . In the past, the asexual morphs of Phaeophleospora were characterized as pycnidial 22 , however, the recently described P. pteridivora has a sporodochial asexual morph 23 . Guatimosim et al. 23 suggested that the genus Phaeophleospora should be defined based on phylogeny analysis instead, due to its high morphological diversity. So far, 19 species of Phaeophleospora were described, however, only 15 were accepted and validated based on ITS phylogenetic analysis (Table S1), but sufficient information to distinguish species within the genus Phaeophleospora was revealed 11,12,23,24 . We suggest a multilocus analysis as valuable tool to better understand evolutionary factors and intra-and inter-specific relationships of the Phaeophleospora species. We also suggest TEF1 and ACT as candidate genes for this analysis, in view of the number of informative sites compared to ITS and LSU sequences (Fig. 1, S-38-40). In the phylogenetic analysis, P. vochysiae is in the same clade (1) as P. gregaria, P. scytaliddi, P. stramenti and P. eugeniicola, but in a single branch, supported by high probability value (Fig. 1). Besides the phylogenetic support, P. vochysiae also showed difference in conidia size and growth rates from the P. scytaliddi (Table S2), the closest related species (99% of similarity in ITS sequence). P. scytaliddi has an elliptic conidium 25 , longer and narrowed compared to P. vochysiae and, different from the others species in this clade, only P. vochysiae produced a red pigment associated with the hypha (Fig. 3). P. gregaria also produced a red  Table 3. Inhibition zones (in millimeters) of LGMF1215 crude extracts of different culture media and compounds 1~3 tested antibacterial and antifungal assays at 100 μg/disc. -denotes no measurable halo, antibacterial control: Ampicillin (1 mg/disc), Antifungal control: Derosal (1 mg/disc), ne: not evaluated. pigment in culture media, however it is not hypha associated 26 . The other species in the clade were associated with leaf spot diseases (Table S1), and, except for P. gregaria, which was described in Australia, they were all isolated in Brazil, from plants belonging to the Myrtaceae family 11,26 . This is the first isolation of a Phaeophleospora species from plants belonging to the family Vochyseaceae (Vochysia divergens). In contrast to those other species, the two strains of P. vochysiae were isolated from asymptomatic plants.
Phaeophleospora is an anamorph of the Mycosphaerellaceae 26 , and although other genera from the family of Mycosphaerella were recognized as producers of various secondary metabolites with antibacterial, antifungal and anti-parasitic activity [27][28][29] , there has been no report of secondary metabolites isolated from the species Phaeophleospora. Therefore, as a new species belonging to a genus without metabolite studies, and isolated from a region exposed to high hydric stress, we hoped for an interesting spectrum of secondary metabolites from P. vochysiae, including new metabolites. Two known compounds, cercosporin and isocercosporin, and a new compound, 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone, were produced in large amounts by the strain LGMF1215 (Fig. 3, S1). These compounds showed antibacterial activity against MSSA and MRSA, and high antifungal activity. Since MRSA strains have acquired resistance to many antibiotics and are associated with higher human death rate than those caused by HIV and influenza combined 30 , the search for new compounds with activity against this pathogen has great importance. In addition to the activity against MRSA, the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone (3) showed complete inhibition of mycelial growth of the phytopathogen P. citricarpa. On the other hand, compound 3 showed no cytotoxic activity against the human tumor cells evaluated, which may suggest a more selective, and less toxic antimicrobial activity (Table 3, Fig. 4). Therefore, compound 3 may be used for the control of the phytopathogen P. citricarpa, the causative agent of citrus black spot (CBS), a disease subjected to phytosanitary regulations in the European Union 31 .
The phytotoxic compounds cercosporin and isocercosporin are perylenequinones isolated for the first time in 1981 and 1991, from Cercospora beticola and Scolecotrichum graminis, respectively 16,17 , while 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone (3) was never found before, only similar cyclopentenone derivatives, i.e. ring-contracted analogues compared to 3, such as similin A (4, produced by the fungus Sporormiella similis) 18 , phomapentenone A (5, produced by the fungus Phoma sp. NRRL 25697) 19 and boydone B (6, produced by the plant associated Pseudallescheria boydii NTOU2362) 20 ( Figure S41). The cercosporins are photosensitizing compounds that interact with molecular oxygen to produce highly toxic singlet oxygen 32 . Nearly all bacteria and fungi are susceptible to cercosporin cell damage, however, the species of Cercospora 16,33 and Phaeophleospora vochysiae are resistant. The resistance against cercosporins by P. vochysiae and the host Vochysia divergens, can be associated with different factors, the most common are the production of enzymes or compounds that quench or block the formation of oxygen species, such as carotenoids 34 or antioxidants 35 . In addition, some studies suggest that cercosporin associated with the Cercospora hyphae is present in the reduced form, which makes the compound nontoxic, or not photoactived 36 . The association of this compound with the hyphae was also observed here in P. vochysiae (Fig. 3), and can be one explanation for the isolation of this species from healthy tissues from V. divergens. However, we cannot exclude the possibility of P. vochysiae to cause disease in certain unfavorable conditions.
In conclusion, the species P. vochysiae, isolated from healthy tissues of the medicinal plant Vochysia divergens, was described by morphological and phylogenetic analyses, and was characterized as a source of highly active secondary metabolites, including the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone (3). This new compound showed activity against MRSA and P. citricarpa, but no cytotoxicity. P. vochysiae also produced the phytotoxic compounds cercosporin (1) and isocercosporin (2) in culture conditions, suggesting the potential of this species to cause diseases, under specific conditions or in different host plants. More studies are necessary to find out whether the cercosporins are associated with the pathological mechanisms involved in the leaf spot diseases caused by other Phaeophleospora species, e.g., in Eucalyptus spp.

Material and Methods
Taxonomy. Strains LGMF1213 and LGMF1215 were isolated from V. divergens leaves collected in the Pantanal, in the region of Nhecolândia (S18°10.07′, W57°23.03′) in Brazil. For the isolation of endophytes, leaves with no marks, scratches or wounds were collected. To eliminate epiphytic microorganisms, a purification protocol of six steps was used 37 . The leaves were fragmented and inoculated in Petri dishes with medium PDA (Potato Dextrose Agar). The plates were incubated at 28 °C for 30 days, and the growth was verified daily. The cultures were deposited in the Laboratory of Genetics of Microorganisms (LabGeM) culture collection, Federal University of Paraná, Curitiba, Paraná, Brazil (http://www.labgem.ufpr.br/).
For the macro-and microscopic analysis, strain LGMF1215 was grown in plates in Potato Dextrose Agar (IBI Scientific), Oatmeal Agar (IBI Scientific) and Malt Extract Agar (Bacto Difco) culture medium at 23 °C and 28 °C, respectively, for 40 days, as described by Crous et al. 12 . Culture characteristics were studied from cultures 21 days after inoculation. Microscopic preparations were performed in distilled water, with 50 measurements per structure in microscope Axio Imager Z2 (Carl Zeiss, Jena, DE), equipped with Metafer 4/VSlide software (Metasystems, Altlussheim, DE), using differential phase interference contrast (DIC) illumination with software support, ImageJ.
Genomic DNA extraction was carried out using the UltraClean ™ Microbial DNA Kit (MO Bio, Carlsbad, CA, USA). The internal transcribed spacer region (ITS) 1, 5.8 S, ITS2, LSU, elongation factor (TEF-1) and actin were amplified using the primers ITS4 and V9G 38 , LR0R and LR5 23 , EF-728F and EF-986R 23 , ACT-512F and ACT-783 R 18 , respectively. The PCR product was purified using the enzymes EXO1 and FastAP (ThermoFisher, Waltham, MA USA). The PCR product was sequenced using BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems, Foster City, CA, USA), and sequences were analyzed on an ABI3500 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). The sequence was compared with type specimens sequences available in the Genbank database of NCBI (http://www.ncbi.nlm.nih.gov/) and Mycobank (http://www.mycobank.org/), and aligned using CLUSTAL_X v.1.81 39 . Bayesian inference of the phylogeny was performed in MrBayes version 3.2.1 40 , with permutations allowed until a frequency of division ≤0.01 was reached. The general time-reversible (GTR) substitution model was used. FigTree version 1.4.2 41 was used to edit the phylogenetic trees that were constructed. Sequences obtained in this study were deposited in GenBank with the accession numbers listed in Table S1.

Fermentation, Extraction and Isolation. Phaeophleospora vochysiae
LGMF1215 was cultivated on PDA-agar plates at 28 °C for 7 days. Fragments of agar (5/flask) were used to inoculate two Erlenmeyer flasks (500 mL) containing 250 mL of PD (IBI Scientific), MEA (Bacto Difco) and Czapeck media (Bacto Difco), and cultured for 21 days, at 27 °C and 250 rpm. Cultures were extracted with EtOAc (3 × 500 mL) and then the recovered organics were evaporated in vacuo at 40 °C. The antibacterial activity of crude extracts was determined and the best conditions were selected for a large-scale culture.
Large-scale fermentation (10 L) was performed using Potato Dextrose broth (IBI Scientific). The obtained red/ black culture broth was separated by filtration using Whatman paper n 4. The biomass (mycelium) was extracted with MeOH/EtOAc (5 × 1000 mL) and then the recovered organics were evaporated in vacuo at 40 °C to yield 6.1 g of crude extract. The aqueous fraction was extracted with EtOAc (5 × 500 mL) and the recovered organics were evaporated in vacuo at 40 °C to yield 3.7 g of broth (culture filtrate) extract. The mycelium crude extract was subjected to Sephadex LH-20 (MeOH; 1 × 20 cm), and further purified by HPLC to afford compound 1 (3 mg) (Fig. S2). The EtOAc fraction was also subjected to Sephadex LH-20 (MeOH; 1 × 20 cm) and purified by HPLC to yield compounds 1 (2.6 mg), 2 (1.3 mg) and 3 (7.0 mg) in pure forms (Fig. S1).
Antimicrobial and Antifungal Activity. The Gram-positive bacteria Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (MRSA) (BACHC-MRSA) were maintained in lysogeny broth (LB) liquid media and Mueller-Hinton agar (Bacto Difco, USA). A loopful of each organism was inoculated into a 7 mL culture of LB broth and incubated in a 37 °C orbital shaker at 200 rpm for 10 hours. Each test organism was streaked on a sterile Mueller-Hinton agar plate with a sterile cotton swab. Compounds 1-3 were dissolved in methanol and aliquoted in 100 μg amounts per each 6 mm sterile filter disc and were allowed to dry in a laminar flow hood. The discs were placed on the plates, which were then incubated for 24 hours at 37 °C. The resulting inhibition zones were measured in millimeters.
The fungal strains Phyllosticta citricarpa LGMF06 and Colletotrichum abscissum LGMF1268 were used in disc diffusion assays. Solutions of amphotericin B (positive control) and tested compounds were dissolved in MeOH. Each sterile paper disc was loaded with 10 µL solution and was allowed to dry in the biosafety cabinet for 4 h. The dried discs were then placed on the PDA plate following the homogeneous distribution of fungus. MeOH was used as a negative control. The plates were incubated at 24 °C for 7 and 21 days, when the inhibition zones were measured.
Cytotoxic activity. To assess the viability of human lung non-small cell carcinoma A549 and prostate adenocarcinoma PC3 cell against compounds 1-3 the conversion of resazurin (7-hydroxy-10-oxido-phenoxazin-10-ium-3-one) to its fluorescent product resorufin was monitored. DMEM/F-12 Kaighn's modification media (Life Technologies, NY, USA) with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/ mL, streptomycin, 2 mM L-glutamine was used to grow A549 and PC3 cells (ATCC, Manassas, VA, USA). Cells were seeded at a density of 5 × 10 3 cells per well in 96-well clear bottom culture plates (Corning, NY, USA), incubated 24 hours at 37 °C in a humidified atmosphere containing 5% CO 2 and subsequently exposed to known toxins (1.5 mM hydrogen peroxide or 10 μg/mL actinomycin D, positive control) and test compounds for 72 hours. To assess cell viability, 150 μM of resazurin (Sigma, St. Louis, MO, USA) was added to each well, plates were shaken briefly for 10 seconds and incubated for another 3 hours at 37 °C to allow viable cells to convert resazurin into resorufin. The fluorescence intensity for resorufin was detected on a scanning microplate spectrofluorometer FLUOstar Omega (BMG Labtech, Cary, NC, USA) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm.