New 1,3-benzodioxin-4-ones from Synnemapestaloides ericacearum sp. nov., a biosynthetic link to remarkable compounds within the Xylariales

Surveys of foliar endophytes from the Acadian forest region over the past three decades have identified numerous phylogenetically diverse fungi producing natural products toxic to forest pests and diseases. The life histories of some conifer endophytes can be restricted to plant foliage or may include saprotrophic phases on other plants tissues or even alternate hosts. Considering the potentially broad host preferences of conifer endophytes we explored fungi isolated from understory species and their metabolites as part of an ongoing investigation of fungal biodiversity from the Acadian forest. We report a hitherto unidentified Xylariomycetidae species isolated from symptomatic Labrador tea (Rhododendron groenlandicum) leaves and mountain laurel (Kalmia latifolia) collected in coastal southern New Brunswick, Canada. Morphological and phylogenetic evidence demonstrated the unknown species was a novel Synnemapestaloides (Sporocadaceae) species, described here as Syn. ericacearum. A preliminary screening assay indicated that the culture filtrate extract of the new species was potently antifungal towards the biotrophic pathogen Microbotryum violaceum, warranting an investigation of its natural products. Two natural products possessing a rare 1,3-benzodioxin-4-one scaffold, synnemadoxins A-B (1–2), and their postulated precursor, synnemadiacid A (3), were characterized as new structures and assessed for antimicrobial activity. All isolated compounds elicited in vitro inhibitory antifungal activity towards M. violaceum at 2.3 μg mL-1 and moderate antibiotic activity. Further, the characterization of synnemadoxins A-B provided a perspective on the biosynthesis of some related 1,3-benzodioxin-4-ones produced by other fungi within the Xylariales.

Surveys of foliar endophytes from the Acadian forest region over the past three decades have identified numerous phylogenetically diverse fungi producing natural products toxic to forest pests and diseases. The life histories of some conifer endophytes can be restricted to plant foliage or may include saprotrophic phases on other plants tissues or even alternate hosts. Considering the potentially broad host preferences of conifer endophytes we explored fungi isolated from understory species and their metabolites as part of an ongoing investigation of fungal biodiversity from the Acadian forest. We report a hitherto unidentified Xylariomycetidae species isolated from symptomatic Labrador tea (Rhododendron groenlandicum) leaves and mountain laurel (Kalmia latifolia) collected in coastal southern New Brunswick, Canada. Morphological and phylogenetic evidence demonstrated the unknown species was a novel Synnemapestaloides (Sporocadaceae) species, described here as Syn. ericacearum. A preliminary screening assay indicated that the culture filtrate extract of the new species was potently antifungal towards the biotrophic pathogen Microbotryum violaceum, warranting an investigation of its natural products. Two natural products possessing a rare 1,3-benzodioxin-4-one scaffold, synnemadoxins A-B (1-2), and their postulated precursor, synnemadiacid A (3), were characterized as new structures and assessed for antimicrobial activity. All isolated compounds elicited in vitro inhibitory antifungal activity towards M. violaceum at 2.3 μg mL -1 and moderate antibiotic activity. Further, the characterization of synnemadoxins A-B provided a perspective on the biosynthesis of some related 1,3-benzodioxin-4-ones produced by other fungi within the Xylariales. PLOS

Introduction
Foliar endophytes of conifers in the Acadian forest of Eastern Canada have proven an exceptionally rich source of structurally diverse biologically active natural products [1,2]. Our studies have revealed that the life histories of conifer endophytes can be restricted to plant foliage or may include saprotrophic phases on other host tissues or even alternate hosts. For example, Phialocephala scopiformis is a common Picea endophyte that produces its sexual reproductive structures (apothecia) on decaying Picea wood and fallen branches [3]. In contrast, apothecia of Phialocephala piceae, a foliar endophyte of Picea, are reported only from decaying hardwood [3][4][5]. A griseofulvin-producing Xylaria endophyte of Pinus strobus was also isolated as an endophyte of Vaccinium angustifolium, indicating a plurivorous life cycle involving both overstory and understory species [6]. While considering the potentially broad host preferences of conifer endophytes, an unidentified species in Xylariomycetidae was isolated from symptomatic Rhododendron groenlandicum leaves. The resulting strain, tentatively identified as a Seimatosporium species, generated a potently antifungal culture filtrate extract, prompting further investigation. Morphological and phylogenetic analyses indicated this strain, as well as a conspecific strain isolated from symptomatic Kalmia latifolia leaves, represented a novel Synnemapestaloides (Sporocadaceae) species. Herein, we describe the new species as Synnemapestaloides ericacearum, providing morphological and phylogenetic evidence supporting its distinction from Synnemapestaloides and other morphologically similar species within the Sporocadaceae. From the culture filtrate extract of Syn. ericacearum DAOMC 250336, three new natural products, including two metabolites possessing a rare 1,3-benzodioxin-4-one scaffold, were structurally characterized and assessed for antimicrobial activity.

Results
Strains from symptomatic leaves of Rhododendron groenlandicum (collected on Taylors Island, Saint John, NB, 45.212018, -66.136745) and Kalmia latifolia (see below) were conspecific based on morphology and identical ITS and LSU sequences. Both isolates were sterile on all media tested; however, sporulation by one strain (DAOMC 250336) was induced when malt extract agar (MEA) blocks containing mycelia were floated in sterile water. This species is morphologically similar to Seimatosporium (Sporocadaceae), a plurivorous genus including several species described from hosts in the Ericaceae, for example Seim. arbuti, Seim. ledi, and Seim. rhododendri on Rhododendron [7,8]. The ITS-LSU phylogeny places both strains in a strongly supported monophyletic clade sister to Synnemapestaloides rhododendri and Syn. foliicola ( Fig  1). Based on morphological and phylogenetic evidence, we consider these two strains to represent a new species, described herewith as Synnemapestaloides ericacearum.

Natural products
The incubation of Syn. ericacearum DAOMC 250336 in 2% malt extract broth yielded ethyl acetate culture filtrate extracts that exhibited strong in vitro antifungal activity using a modified Oxford diffusion assay. Three new natural products including two rare 1,3-benzodioxin-4-ones were structurally characterized and assessed for antimicrobial activity (Fig 3). The first metabolite (1) purified was isolated as a dull brown oil with the molecular formula C 15 H 18 O 6 determined by an [M-H]-ion at m/z 293.1033 indicative of seven units of unsaturation. Its UV spectrum showed absorption maxima at 224, 268, and 306 nm suggesting the presence of a conjugated structure. Its 1 H NMR spectrum displayed a single aromatic methine at δ 6.51 (s), deshielded methine at δ 3.18 (q, 7.0), and five methyl groups at δ 2.58 (s), 2.11 (s), 1.73 (s), and 1.28 (d, 7.0) as well as a methoxy group at δ 3.87 (s). Interpretation of the HSQC and 13 C NMR spectra of 1 revealed eight of the fifteen carbon signals were quaternary. These signals were resultant of two carbonyls at δ 175.4 and 161.9, five sp 2 carbons at δ 165.0, 157.7, 143.2, 122.7, and 106.2 and one sp 3 carbon at δ 107.2 ( Table 1). The presence of eight highly deshielded quaternary carbons and four singlet methyl groups supported a highly substituted, conjugated structure. As expected from the 1 H spectrum, only one COSY cross-peak was observed for compound 1, between δ 1.28 (9-Me) and δ 3.18 (H-9; Fig  4). HMBC correlations from the singlet methyl at δ 1.73 (2-Me) to δ 107.2 (C-2) and 47.8 (C-9), the cross peak from 9-Me to the carbonyl at δ 175.4 (C-10) and the chemical shift of C-2 suggested the presence of a 3,3-dioxy-2-methylbutanoic acid moiety in 1 (Fig 4). The presence of a single carboxylic acid (C-10) was supported by methylation of 1 with excess diazomethane that yielded a hexamethyl derivative (1a), identifying one exchangeable acidic proton within the structure ( Figure J and Table E in S1 File). The high number of deshielded quaternary carbons and extensive HMBC coupling for 1 suggested a pentasubstituted aromatic ring. A lactone or other ring system fused to the aromatic moiety possessing a carbonyl at δ 161.9 (C-4) was theorized to constitute the remaining degrees of unsaturation. HMBC correlations from the singlet methyl at δ 2.11 (6-Me) to δ 143.2 (C-5), 122.7 (C-6) and (C-7), and from δ 3.87 (7-OMe) to δ 165.0 (C-7) and 98.1 (C-8) revealed a methoxy functionality vicinal to the only aromatic methine proton. HMBC cross-peaks were also observed from δ 2.58 (5-Me) to δ 161.9 (C-4), 106.2 (C-4a) and C-5, and from δ 6.51 (H-8) to C-4a, C-6, C-7 and C-8a (δ 157.7; Fig 4). These data and the chemical shift of C-8a revealed that compound 1 possess a modified 6-hydroxy-4-methoxy-2,3-dimethylbenzoic acid core structure. Together, the chemical shift of C-2, lack of other exchangeable acidic protons and an HMBC correlation from 2-Me to C-4 fuses the previously characterized 3,3-dioxy-2-methylbutanoic acid moiety to the aromatic functionality yielding a rare 1,3-benzodioxin-4-one structure (Figs 3 and 4). The 1,3-dioxin-4-one ring satisfies the seventh unit of unsaturation dictated by the molecular formula and NMR data.
The planar 1,3-benzodioxin-4-one structure of 1 was elucidated as a new natural product; however, the configurations of the adjacent chiral centers at C-2 and C-9 remained elusive. Due to the lability of the 3,3-dioxy-2-methylbutanoic acid moiety, conventional NOESY  3 ). To determine the relative configurations of C-2 and C-9, the in silico approach described by Willoughby et al. 2017 [17] was used. Briefly, molecular dynamics were calculated with AMBER (University of California) to generate a library of all possible conformers for each stereochemical configuration. Density function theory was used to calculate frequency and free energy values for each conformation to generate in silico computed chemical shifts. Comparisons of mean absolute error (MAE) between experimental and computed chemical shifts of all reported 1 H and 13 C resonances for 1 and its methyl ester 1a revealed the relative configurations of the enantiomeric mixture of 1 as 2R,9R and 2S,9S (Tables A-D in S1 File). This in silico approach is not able to differentiate between enantiomers of 1; however, is discriminatory of diastereomers. The HRMS and spectroscopic data support 1 as a new rare 1,3-benzodioxin-4-one structure reported here as synnemadoxin A (Fig 3).
The second metabolite characterized from Syn. ericacearum DAOMC 250336 was also isolated as dull brown oil with the same UV absorption maxima as synnemadoxin A (1). Metabolite 2 was more polar and assigned the molecular formula C 15 H 18 O 7 based on an HRMS peak at m/z 309.0981 [M-H]corresponding to the addition of an oxygen atom compared to synnemadoxin A (1). The NMR spectroscopic data for 2 were very similar to synnemadoxin A (1), indicating 2 was a structurally similar 1,3-benzodioxin-4-one metabolite ( Table 1). The primary difference between the two 1,3-benzodioxin-4-ones NMR data was the substitution of a methyl group in synnemadoxin A (1) with an oxygenated methylene in 2 revealed by 1 H and 13 C signals at δ 4.70 (s) and δ 55.0, respectively (Table 1). Together, the differences in NMR chemical shifts, increase in molecular weight, and HMBC correlations from δ 4.70 (6-CH 2 OH) to δ 145.7 (C-5), 125.4 (C-6) and 165.6 (C-7), are indicative of C-6 bearing a hydroxymethyl moiety in 2 instead of a methyl group (Fig 3).  (Fig 3). An additional carbonyl carboxylic acid chemical shift at δ 171.8 (C-1") and trans-double bond together with the HRMS data, and similar NMR spectra suggested a reduction of the 1,3-dioxin-4-one ring in 3 (Fig 4). Here were report compound 3 as a new structure, (E)-6-((3-carboxybut-2-en-2-yl)oxy)-4-methoxy-2,3-dimethylbenzoic acid, or synnemadiacid A (Fig 3).
Natural products 1-3 were individually tested for in vitro antifungal activity against Microbotryum violaceum and Saccharomyces cerevisiae, and antibiotic activity towards Bacillus subtilis and Escherichia coli (Table 3). Synnemadoxins A-B (1-2) and synnemadiacid A (3) were potently inhibitory to M. violaceum (formerly Ustilago violacea), a proxy for rust needle diseases [18], each with a MIC of 2.3 μg mL -1 . However, no activity was observed for each metabolite at the highest concentration tested (150 μg mL -1 ) against S. cerevisiae despite the crude culture filtrate extract showing activity at 50 mg mL -1 using a modified Oxford diffusion assay. Moderate antibiotic activity for compounds 1-3 against B. subtilis were observed at 9.3 μg mL -1 ; and 1 and 3 had MIC values of 18.8 μg mL -1 against E. coli.

Discussion
Synnemapestaloides was established to describe Syn. rhododendri, the causal agent of a leaf and twig blight disease of Rhododendron brachycarpum in Japan [10]. Handa et al. [10] provided morphological evidence distinguishing the monotypic Synnemapestaloides from other pestaloid genera, namely the presence of columnar synnemata. A later phylogenetic study confirmed the distinction of Synnemapestaloides from Seimatosporium and other Sporocadaceae genera, while also establishing the new combination Syn. foliicola for a presumably saprotrophic species known from Chamaecyparis and Juniperus, previously placed in Seimatosporium [19,20]. The unappendaged three-septate conidia of Synnemapestaloides ericacearum distinguish it from the five-septate conidia of both Syn. foliicola and Syn. rhododendri. Synnemapestaloides rhododendri conidia are fusiform and more-or-less pestaloid, with unbranched or dichotomously branched apical appendages and unbranched or irregularly branched basal appendages, while Syn. foliicola conidia are broadly ovate with unbranched apical and basal appendages [10,20]. Watanabe et al. [19] described similar conidiomata ontogeny for both Syn. rhododendri, which produces up to ca. 500 μm tall synnemata, and Syn. foliicola, which produces short synnemata (sporodochia). The authors hypothesized that this conidiomata ontogeny might be a synapomorphy for Synnemapestaloides. We interpret the conidiomata of Syn. ericacearum as acervular, with the conidiogenous cells and conidia observed under the ruptured leaf cuticle (Fig 2G and 2H). However, detailed time-lapse observations would be necessary to elucidate the conidiomata ontogeny of Syn. ericacearum. There are no reports of sexual states in Synnemapestaloides. Currently, Syn. ericacearum is only known as an associate of leaf spots on the Ericaceae hosts Kalmia latifolia and Rhododendron groenlandicum. However, it is conceivable that the life cycle of Syn. ericacearum contains an endophytic phase, which is common for species from the Sporocadaceae (e.g., [13,21,22]). The association of Syn. ericacearum with leaf spots characteristic of those caused by Syn. rhododendri, Seimatosporium, and other Sporocadaceae species indicate it is likely a pathogen, although its pathogenicity was not tested.
Many strains of fungi within Sporocadaceae representing the genera Pestalotiopsis, Bartalinia, Monochaetia, Robillarda, Seimatosporium, and Strickeria have been critically evaluated for natural products [23]. This interest resulted from the putative identification of the plant anticancer compound paclitaxel (taxol) from Taxomyces andreanae, collected as an endophyte of Taxus brevifolia, and subsequently from several related species including endophytic strains of Bartalinia, Monochaetia, and Pestalotiopsis [24]. Horizontal transfer of biosynthetic genes from plants to fungi was postulated to explain paclitaxel production by these fungi. However, more recent evidence shows the genomes of the putative endophyte producers' do not possess any significant homology to plant paclitaxel biosynthetic genes, questioning its biosynthesis by these fungi [25].
Strains of species within Sporocadaceae collected as endophytic or plant-associates have proved to be an abundant source of structurally diverse natural products. The polyhydroylated macrolides seimatopolides A and B with peroxisome proliferative activity were characterized from a Seimatosporium discosioides (GenBank: EF600969) strain isolated from Rosa multiflora [26,27]. A Seimatosporium endophyte of Salsola oppositifolia synthesized several aromatic compounds acid together with metabolites structurally similar to terreic acid, an antiinsectan metabolite isolated from a Picea endophyte [1,28]. From the same plant host, the acaranoic acids, seimatoporic acid A-B were reported together with (R)-mellein and five of its derivatives [29]. The simple isocoumarin (R)-mellein and its derivatives have been reported from phylogenetically diverse plant-associated fungi including Pezicula, Diaporthe and Rhytismatales species [30][31][32][33][34]. Pestalotiopsis, Robillarda and other related but unidentified species likely associated with woody plants (collected from three-toed sloth hair) generated cytotoxic and antibiotic culture filtrate extracts; however, metabolite structures were not elucidated [35].
The biogenesis of these highly functionalized P. fici metabolites are proposed to originate with the oxidation of dihydromaldoxin (= pestheic acid) to maladoxin, which possesses a reactive diene functionality. Diels-Alder cascades from the reaction of the maladoxin diene and terminal alkene of (+)-iso-A82775C generate the observed structural diversity for this strain [37]. Due to the reactivity of the maladoxin diene moiety, the Diels-Alder reaction in the presence of the terminal diene of (+)-iso-A82775C (Fig 5B) is speculated to occur almost spontaneously, without major enzymatic involvement. Maladoxin was not isolated from rice culture extracts of P. fici, whereas the less reactive dechloro congener was [41]. The recovery of maladoxin from the mycelium of the previously noted Xylariales (unidentified Xylaria sp.) species together with dihydromaldoxin (= pestheic acid) and other related diphenyl ether intermediates suggests a similar biosynthetic pathway, absent the presence of a reactive diene moiety or additional enzymatic input [36]. Since 1,3-benzodioxin-4-one containing natural products are rarely reported, we speculate synnemadoxins A-B (1-2) arise from the oxidation of synnemadiacid A (3) similar to the oxidation of diphenyl ether intermediates to maladoxin (Fig 5A-5C) by related fungi within the Xylariales [36,39,41]. Oxidative cyclization of the diphenyl ether dihydromaldoxin is reported to yield enantiomeric mixtures of the 1,3-benzodioxin-4-ones maldoxin and dechloromaldoxin [39,48]. A similar oxidative scheme using synnemadiacid A (3) as a precursor would help explain the observation of enantiomeric synnemadoxins A-B (1-2).
In summary, we have described a new species, Syn. ericacearum (Sporocadaceae) from the ericaceous hosts R. groenlandicum and Kalmia latifolia. The culture filtrate extract from Syn. ericacearum DAOMC 250336 was potently antifungal. Two natural products possessing a rare 1,3-benzodioxin-4-one scaffold, synnemadoxin A-B (1-2), and their postulated precursor, synnemadiacid A (3), were characterized as new structures and assessed for biological activity. All isolated compounds elicited in vitro inhibitory antifungal activity towards M. violaceum at 2.3 μg mL -1 and moderate antibiotic activity. This study helps to link rare 1,3-benzodioxin-4-ones produced by Syn. ericacearum and other fungi within the Xylariales.

General experimental section
NMR spectra of purified metabolites were recorded with a Bruker Avance 400 Spectrometer (Milton, Ontario) at 400.1 ( 1 H) and 100 MHz ( 13 C) using a 5 mm auto-tuning broadband probe with a Z-gradient. Secondary metabolites were dissolved in CD 3 OD (CDN Isotopes, Point Claire, Quebec) and referenced to the solvent peak (δ H 3.31 and δ C 49.1). LC-HRMS spectra were acquired with a Thermo Q-Exactive Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA), coupled to an Agilent 1290 HPLC system. Extracts and metabolites were separated by an Agilent Zorbax Eclipse Plus, RRHD C 18 (2.1 x 50 mm, 1.8 μm) column and a mobile phase consisting of acetonitrile (ACN) -ddH 2 O with 0.1% formic acid at a flow rate of 0.3 mL min -1 . Normal phase and semi-preparative fractions were screened by LC-UV-MS with a Waters 2795 separation module, Waters 996 diode array detector, and Micromass Quatro LC mass spectrometer. Fractions were separated by a Phenomenex Kinetix C 18 (100 × 4.60 mm, 2.6 μm) column (Torrance, CA, USA) using a mobile phase also consisting of ACN-ddH 2 O with 0.1% formic acid at a flow rate of 1.0 mL min -1 . Semi-preparative HPLC was achieved with an Agilent 1100 HPLC system equipped with a diode array detector, Phenomnex Luna C 18 (250 x 10.00 mm, 5 μm) column, and a mobile phase consisting of ACN-ddH 2 O. Gradients were programmed accordingly for each purified compounds with a flow rate of 4 mL min -1 . Silica gel (Silicycle; 40-63 μm) was utilized for normal phase flash chromatography. Optical rotations were acquired with an Autopol IV polarimeter (Rudolph Analytical, Hackettown) and UV spectra were recorded with a Varian Cary 3 UV-VIS spectrophotometer scanning from 190-800 nm.

Isolation and identification of fungal strains
Two collections of symptomatic leaves of Kalmia latifolia and Rhododendron groenlandicum were made during an ongoing survey of endophytes and other plant-associated fungi in Eastern Canada. Conidia from acervuli associated with leaf spots were transferred to Petri dishes containing 2% malt extract agar (MEA; 20 g Bacto malt extract, Difco Laboratories, Sparks, Maryland; 15 g agar, EMD Chemicals Inc., New Jersey; 1 L distilled water) and incubated at 20˚C. To induce sporulation, DAOMC 250336 was also grown on cornmeal agar (CMA; Acumedia Manufacturers Inc., Lansing, MI), oatmeal agar [49], and water agar (1.5% water agar (WA, with 1 mL trace metal solution; [50]) under 12:12 h fluorescent light and ambient natural light conditions. Additionally, MEA blocks (ca. 1 cm 3 ) containing mycelia were placed in 9 cm Petri dishes containing ca. 25 μL sterile autoclaved tap water and exposed to ambient light.
Total genomic DNA was extracted from MEA cultures using the Ultraclean Microbial DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA) following the manufacturer's protocol. The ITS region was amplified and sequenced using the primer pair V9G and LS266 [51,52]. Partial LSU was amplified using the primer pair LROR and LR5 and sequenced using LROR, LR3, LR3R, and LR5 [53]. PCR and sequencing protocol were carried out according to McMullin et al. [33]. Sequence contigs were assembled and trimmed using Geneious R8 v. 8.1.5 (Biomatters, Auckland, New Zealand).
ITS and LSU sequences of related Sporocadaceae species were obtained from GenBank by conducting BLAST searches using DAOMC 250336 and DAOMC 251621 as query sequences. A concatenated ITS and LSU dataset containing 59 sequences was aligned using MAFFT v. 7 [54] and visually examined in Geneious. MrModeltest v. 2.2.6 was used to determine the most suitable sequence evolution model (GTR+I+G) using the Akaike information criterion [55]. The ex-type culture of Phlogicylindrium uniforme (CBS 131312; Phlogicylindriaceae) was selected as outgroup because of its phylogenetic position outside of Sporocadaceae [19]. MrBayes v. 3.2 was used to perform Bayesian inference (BI) phylogenetic reconstruction [56]. Three independent Metropolis-coupled Markov chain Monte Carlo (MCMCMC) samplings were performed with 11 heated chains and one cold chain. Sampling occurred every 500 generations until the standard deviation of split frequencies reached a value < 0.01 (commands: stoprule = yes stopval = 0.01). The first 25% of trees were discarded as burn-in and the remaining trees were retained and combined into one 50% majority rule consensus tree. A maximum likelihood (ML) analysis was performed using RAxML v. 8.2.4 [57] in PAUP Ã v. 4.0b10 [58] starting from a random starting tree with 1000 bootstrap replicates. Trees were visualized in FigureTree 1.4.2 (available at http://tree.bio.ed.ac.uk/software/Figuretree/) and exported as SVG vector graphics for assembly in Adobe Illustrator v10 (Adobe Systems, San Jose, CA). Novel sequences used in this study were accessioned in GenBank and the novel species and associated metadata were deposited in MycoBank (www.mycobank.org). Collecting on Campobello Island was part of the 2016 Campobello Island Mycological Foray hosted by the New Brunswick Museum and J.D. Irving, Limited gave permission to conduct fungal collections on their sites.

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.

Fermentation and extraction
For inoculation into culture medium, Syn. ericacearum DAOMC 250336 growing on a 2% malt extract agar (MEA; Difco Laboratories) was excised and macerated in sterile ddH 2 O under aseptic conditions. Aliquots were used to inoculate (5% v/v) fifteen 250 mL Erlenmeyer flasks each containing 50 mL of 2% malt extract (Difco Laboratories) broth. First stage cultures were incubated for one week on a rotary shaker (100 RPM) in the dark at 25˚C. Resulting mycelia were macerated under aseptic conditions, and individually transferred to Glaxo bottles containing 1 L of the same culture medium. Second stage cultures were incubated stationary as described above for eight weeks.
After the incubation period, Syn. ericacearum mycelia were separated from the culture filtrate by suction through Whatman #4 (Whatman GE Healthcare, UK) filter papers. Mycelia were lyophilized and stored at -20˚C. The culture filtrate was saturated with NaCl and extracted with ACS grade EtOAc. The aqueous culture filtrate was discarded and the organic layer was filtered through anhydrous Na 2 SO 4 and Whatman #1 filter papers prior to drying by rotary evaporation. The resulting culture filtrate extracts were dissolved in HPLC grade MeOH, passed through 0.2μm PTFE (25 mm) syringe filters (Tisch Scientific, USA), dried under a gentle stream of nitrogen gas and weighed. The resulting culture filtrate extract (1.20 g) was stored dry in an amber vial in a freezer at -20˚C.

Metabolite isolation
The culture filtrate extract of Syn. ericacearum DAOMC 250336 was assessed for in vitro antifungal activity with a modified Oxford disc assay and subsequently chromatographed using a bioassay-guided approach. Flash column chromatography implementing a short Silica column and step gradient elution system of hexanes-EtOAc (0-100% v/v) in 10 percent increments followed by 5%, 10%, 25% and 50% EtOAc-MeOH (v/v) yielded 14 fractions screened for antifungal activity. Fractions 5-8, and 12-14 all exhibited antifungal activity with the former group showing a greater inhibitory effect in vitro. Based on LC-UV-MS, fractions with similar LC-UV-MS spectra were combined and metabolites were purified by reverse phase semi-preparative HPLC. Fractions 6-7 which eluted with 60-70% hexanes-EtOAc (v/v) were combined (48 mg) and further separated by semi-preparative HPLC with a linear ACN-ddH 2 O gradient programmed from 5-100% ACN over 25 minutes to afford compound 1 (14.9 mg). Compound 3 (46.0 mg) and additional compound 1 used for chemical derivatization were purified using the same semi-preparative HPLC method from fraction 8 (149.0 mg) that eluted with 80% hexanes-EtOAc. Fraction 12 (39.0 mg) eluted with 10% MeOH-EtOAc and provided compound 2 (6.9 mg) prior to further purification with a linear semi-preparative HPLC method programmed from 5-100% ACN over 20 minutes. 1 H, 13 C and HMBC spectra for natural products (1-3) can be found in the supporting information ( Figures A-I

In silico computed chemical shifts
Theoretical chemical shifts were calculated for the four possible stereoisomers of compounds 1, and 1a based on a computational method described by Willoughby et al. [17], with some minor modifications. Briefly, the AMBER12 program suite [59] using the Generalized AMBER Force Field (GAFF) was used to explore the conformational space of each stereoisomer [60]. Minimized structures were heated to 500K over 5 ps (1 fs time step) and equilibrated at 500K for 1000, 100 ps cycles (0.5 fs time step). Following each cycle, structures were cooled to 0K over 5 ps (1 fs time step). Unique conformations were identified manually based on the C-2-Me, C-2, C-9, C-9-Me dihedral angles, and potential energy recorded following each cycle. Up to 20 conformations for each stereoisomer were removed for geometric optimization and frequency calculations using the B3LYP functional with a 6-31+G(d,p) basis set [61]. NMR shielding tensors were subsequently calculated from the geometry optimized structures with the B3LYP functional with the 6-311G (2d,p) basis set. Duplicate conformations with identical shielding tensors and zero point energies were removed and the chemical shift contribution for each conformation were averaged based on Boltzmann distributions. The scaling factor and intercept were determined using a linear fit of the theoretical tensors of all protons not predicted to be affected by the stereocenters of interest (positions 8, 5-Me, 6-Me and 7-OMe). The scaling factor and intercept were -0.99972 and 31.8085, respectively.

Antimicrobial assays
Initial antifungal activity of the Syn. ericacearum DAOMC 250336 culture filtrate extract (50 mg mL -1 ) and flash chromatography fractions were screened using a modified Oxford diffusion assay against Microbotryum violaceum and Saccharomyces cerevisiae homogeneously spread on 2% MEA as previously described by McMullin et al. [33]. Purified natural products 1-3 were tested for in vitro antimicrobial activity against M. violaceum, S. cerevisiae, Bacillus subtilis (ATCC 23857) and Escherichia coli (ATCC 67878). M. violaceum was grown in 20 g L -1 malt extract (Bacto), 2.5 g L -1 peptone (Bacto) and 2.5 g L -1 yeast extract (Sigma, St. Louis, MO, U.S.A.) whereas S. cerevisiae was inoculated and grown in 1 g L -1 yeast extract supplemented with 10 g L -1 glucose. Bacteria were inoculated and grown in 5 g L -1 yeast extract, 10 g L -1 peptone, and 10 g L -1 NaCl. Nystatin was the positive control for antifungal assays and chloramphenicol was the antibacterial positive control. DMSO was the negative control for all in vitro assays. Isolated metabolites and positive controls were individually tested at 150, 75, 37. Supporting information S1 File. Supporting Information: Figure    Ltd.) facilitated collection trips. Dr. Mark Sumarah is thanked for providing helpful comments on the manuscript.